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User Manual - Hitachi Solutions America

1. Draw Setting 5 88 FMBIO Image Analysis Tools Zoom In Vertically Zoom Out Vertically trle S a Overlay Trace Setting Drawing Tool Tool Description Zoom In Vertically Magnify in vertical direction Zoom Out Vertically Reduce in vertical direction Drawing Use drawing tools for Overlay Trace window Overlay Trace Setting Displays Overlay Trace Setting window Vertical Zoom tools You can proportionally increase or decrease the vertical range of all traces in the window simultaneously Note Use the Image Zoom tools to control the vertical magnification See page 5 7 Drawing tools You can draw and type text on your overlay trace 1 Click the Drawing Tool button L4 or choose Draw from the Trace menu The Drawing Tool bars become active and the Draw menu appears in the menu bar 2 Usethe Drawing tools to draw and type text on the overlay trace See Chapter 8 Drawing Tools 3 When you have finished choose Save Project from the File menu An Overlay Trace icon is added to the 1D Gel Document tree 5 89 Image Analysis Tools FMBIO Overlay Trace Setting Window To display the Overlay Trace Setting dialog box click the Overlay Trace Setting button Ei or choose Setting from the Trace menu Overlay Trace Setting M Test Channel 0 M Test Channel 1 M Test Channel 2 Item Description Channel Place a checkmark in the box next to each channel you want to display You can a
2. On Locks selected figure in position Unlocks selected figure f xI Edit Polygon Drag apex handles to change polygon shape 8 9 Drawing Tools FMBIO Tool Draw 3 Function Paint Color Transparency o Paint Color Set color of shape fill Color 2 x Basic colors mI ey Bee ee en ieee f ie i ie NN NENNEN Se ee 8888 BEER EE mr Custom colors ip pep pe ip pp M Define Custom Colors gt gt Cerca Transparency Makes paint color transparent FMBIO Drawing Tools Tool Draw 3 Function Line Color Line Width Select Line Type No Line Line Color Set line color Color x Basic colors NI Ce Bee mI rr mr eee mimi i imi im ie NNN At EEE HN Custom colors ip pe pp eg ip pe po M Define Custom Colors gt gt mea e Line Width Set thickness of line No Line Makes line transparent Line Type Choose solid line dashed line or dotted line 8 11 Drawing Tools FMBIO Tool Draw 3 Function Polyline Polygon Polyline Draw lines connected at different angles Polygon Draw closed polyline Straight Line Arrow Double headed Arrow Diamond Circle Line marker styles Select arrows circles or diamonds to mark line endpoints 8 12 FMBIO Drawing Tools Tool Text Attributes Function Font Rotate Text Text Color Bold Align Right Italic azr uj ees 5 Align
3. Background and High Signal cutoff thresholds The values in the High Signal Percent and High Signal Number fields change to reflect the selected region In the following example 1 85 of the pixels have a value between 4 081 and 65 535 These pixels will appear on the monitor as black FMBIO Image Analysis Tools Background 90 44 1537 High Signal 1 85 4081 All regions of the image with gray level values below the background cutoff threshold value will appear white As shown 90 4496 of the pixels have a value between 0 and 1 537 These pixels will appear on the monitor as white All other elements of the image that fall within the range between the high signal and background cutoff thresholds appear as shades of gray Histogram The Gray Level Histogram shows the 16 bit gray level gradations 0 to 65 535 on the X axis and the number of pixels in the image on the Y axis of pixels 20000 40000 50000 60000 gray level value Boundary lines indicate the high signal cutoff threshold and background cutoff threshold The red line slider indicates the background cutoff threshold value The blue line slider shows the high signal cutoff threshold value The green line on the histogram displays the current mapping curve By default the mapping curve is displayed 5 17 Image Analysis Tools FMBIO Mapping Curve Background Threshold High Signal Threshold Show Selected Range This option magnifies the hi
4. 3 The boundary color changes to yellow FMBIO Array Analysis Registering Markers You can assign known amounts to spots and use these spots as markers to quantify unknown samples Standard Curve Type To set the type of standard curve 1 Click the Preferences button amp or choose Preferences from the Array menu 2 In the Preferences dialog box click the Quantification tab Preferences 3 Select the desired standard curve in the list box then select a straight or Logarithmic scale for the X axis 4 Click OK Spot Markers To set any combination of spots as markers Array Analysis FMBIO Select any spot or group of spots then click the Set Selected Spot s to Marker button MI or choose Set Selected to Marker from the Array menu The boundary color of each spot changes to violet Click the Open Spreadsheet button E or choose Open Spreadsheet from the Array menu Enter a quantity value for each marker spot in the column labeled Mol If necessary use the bottom scroll bar to view the right side of the Channel table ff Channel 1 Bl x Grid Cell Markers To set cells in a grid as markers 1 Double click on the grid containing the marker cells The Grid Marker dialog box is displayed In the Grid Marker table enter the amount of each marker corre sponding table cell Any cell containing a value greater than zero is set as a marker 7 14 FMBIO Array
5. 30807 CSFIPO 23902 TPOX 10 854 227 10 TPOX 310 36 CSFIPO 243 46 TPOX 11 16957 23131 TPOX 31429 CSFIPO 31822 CSFIPO 196 72 Amelogenin X 20769 322 14 CSFIPO 201 97 Amelogenin y 6750 181 55 THOT 30621 CSFIPO 185 69 THOT 22321 TPOX 15871 THO 5 24311 227 34 TPOX 181 55 THO 231 54 TPOX 235 51 TPOX J 239 48 TPOX J 273 12 D165539 276 85 D168539 SITES helal 10 18 FMBIO Allele Calling with STaR Call bp Genotype bp Genotype 306 07 CSF1PO 301 79 CSF1PO 231 54 TPOX 30572 CSF1PO 4 2796 243 46 TPOX 226 40 TPOX 231 54 TPOX 196 58 Amelogenin 238 02 TPOX 158 58 THO1 243 46 TPOX 3 43 185 81 THO1 93 8 14157 4 2596 196 58 Amelogenin 276 83 D155539 9 158 58 THO1 292 99 D163539 13 181 54 THO1 226 40 D75820 9 185 64 THO1 h 15241 2 60 238 78 D75820 12 192 65 THO1 144 138 14 D55818 12 1396 142 20 D55818 13 1387 272 85 D16S539 154 429 89 F13AD1 6 538 276 54 D155539 4792 6 80 394 13 FESFPS 10 697 288 89 D168538 12 266 319 11 LPL 1 1170 292 91 D165539 13 3913 3 2196 You can edit values for Not in range alleles e g microvariants in the merged worksheet Click on the cell containing the data you want to edit 2 Type the value 3 Click on another cell in the sheet After you have finished editing the worksheets choose Created Merged Landscape from the STaR Call menu The new merged landscape worksheet will reflect the current values in each workshee
6. 2 Click the Marker Setting button M or choose Set Marker from the 1D Gel menu 3 Highlight the lanes containing the marker by holding down the Shift key while clicking on them with the mouse Select the correct marker from the Template list then click Adopt The selected marker appears next to each lane in the list 6 14 FMBIO 1D Gel Analysis Marker Setting mee were ee Calculate Base Pairs page 5 77 1 Click the Volume Calculation button FA or choose Volume Calculation from the 1D Gel menu to calculate base pair sizes in reference to the assigned marker Click the Open Spreadsheet button E or choose Open Spreadsheet from the 1D Gel menu to view the table of base pair values for each layer Click the tabs at the bottom of the window to switch among channels Choose Export from the SpreadSheet menu to save the spreadsheets as comma delimited text files that can be exported to STaR Call TM 1D Gel Analysis F MBIO Multi Color Experiment Flowchart Open Project Create 1D Gel Analysis y Poor Color Separation Adjust Gray Level N y Perform Color Separation 6 16 Good Color Separation Set Migration Lines y N Autoband Set Marker Calculate Base Pairs Export Spreadsheet to STaR Call FMBIO 1D Gel Analysis Multi Color Experiment Procedure The following section is a summary of steps
7. 410976 2820010 109373 420438 lt 3801136 1164813 14520003 Sample 2 2824674 1153 69 473598 1 lt 2830695 107930 745589 3638482 1004977 9307893 2816880 110700 328373 2820244 120738 336133 Exporting a Spreadsheet You can save the spreadsheet as a tab delimited text file for export to other programs such as Microsoft Excel and Microsoft Word To export a spreadsheet 1 Choose Export from the SpreadSheet menu SpreadSheet V 2 Inthe Save As dialog box select a location for the file and then enter a unique name 3 Click Save 7 20 FMBIO Array Analysis Comparing Grids Image Analysis allows you to compare grid data You can compare data from two channels in the same grid or two different grids including grids in dif ferent projects For example you can conduct data mining for gene expression by concentration or volume The resulting images and spread sheets can be pasted into other applications To compare grid data 1 Choose Send Data to Grid Comparison from the Array menu Fiata The Grid Comparison window is displayed The list box contains all open projects with grid data EB Project Array Experiment 96641774 183645861 181 10654103737 96E 1241763921 27930 15E1 2527626 1044304 820488301 1233247 11203294641 1881 45821 198675851 160204161 1505826C1 2046 14 76543650 84851472 90461732 9621178E1 15139363 9352691C1 120282511 212663 10344573
8. Any damage caused by an improper use of the equipment or use not described in this manual Any damage caused by disasters of fire flood storm earthquake thunder and other natural causes Any damage caused by moving the equipment after it has been installed at the customer site Any damaged caused by addition modification reverse engineering and or are not permitted or any other articles Any damage caused by setting up the Scanning Unit and FMBIO System improperly Any damage caused by improper control or mismanagement of the equipment Any damage caused by improper uses faulty operation abnormal uses or mistakes of application of the equipment Expendable supplies Any damage caused by the peripheral devices Any damage caused by products purchased from other than Hitachi Software Engineering Co Ltd or its authorized dealers Exclusions Hitachi Software Engineering Co Ltd s FMBIO is to be used FOR RESEARCH PURPOSES ONLY and is NOT to be used for diagnostic purposes Hitachi Software Engineering shall not in any case be held liable for special incidental consequential indirect or other similar damages ensuing from the use of this product This product is designed to operate safely and reliably Hitachi Software Engineering Co Ltd will not be held responsible for any complications resulting from the use of this product For more information about the software license and limited warranty of this system please refe
9. C use an air conditioner to lower the ambient temperature The minimum operating temperature is 10 C 50 F Moving the Scanning Unit The unit s optical scanning device is a precision instrument Do not relocate or move the scanning unit without prior authorization from Hitachi Software Engineering or risk voiding your warranty Shipping The FMBIO unit is a precision device that weighs approximately 50 kg When transporting it you must protect it from impact and torque Pack it ina sturdy shipping container with a rigid structure and ample internal cushion ing Transport it on a sturdy cart The equipment must always stay upright never tip it on its side or on its end 9 2 FMBIO Maintenance Shutdown When the FMBIO unit is not to be used for an extended time turn off the Main switch on the rear panel and disconnect the power cord Troubleshooting the Scanning Unit At the first sign of a problem with the operation of the scanning unit check the red Error light on the front panel Red Error Light Flashing 1 Turn off the Power switch and wait 5 seconds 2 Turn on the Power switch and wait for the FMBIO self diagnostic routine to end 3 Ifthe Error light continues to flash call Hitachi Software Engineering service personnel Responding to Problems WARNING Shutdown the Scanning Unit at the first sign of trouble If the Scanning Unit emits an unusual smell smoke noise or excessive heat immedi ate
10. Ii Nj x Rap provided I N1 gt 0 3 The operations explained above for Channel 1 are the applied to Channel 2 Channel 2 Image pixel value Pa Disappeared Electrophoresis distance mm FMBIO Image Analysis Tools Individual Band Color Separation To separate the colors in an image using individual bands you must select at least one non overlapping medium intensity representative band of each color Ada eren xL 1 Click the Color Separation button Ed or choose Color Separation from the Image menu SHOW ESTE GULITIT The Color Separation window is displayed Image Analysis Tools FMBIO Color Separation Basic channel C Background 854 p Target Channel v o CTTv 3CH 0 ea DDDD 2CH 9 96 o inlane 4CH 0 m a al 4 Mode Y Origine jE Preview Template cance 2 Choose a channel from the Selected Fluorophore drop down list box and select a color Note In the Blend mode the color associated with each channel appears in the box adjacent to it 3 Inthe Whole Image window move the Zoom Cursor to an area with medium intensity bands of that color 4 Inthe Zoomed Area window pick a band of medium intensity that matches the color but does not overlap any other band Surround the band with the Selection Cursor Make the Selection Cursor larger than the band to include a portion of the image background 5 32 10 FMBIO Ima
11. Long pass filters reject shorter wavelength light while allowing longer wave lengths to pass through Long pass filters are named for the wavelength at the midpoint of the transition range for example OG 610 The midpoint of the sigmoid curve that describes the transition from 0 to maximum transmission The narrower this transition range is the better especially when the two flu orophore emission frequencies are close The name of the long pass filter may also include letters such as OG orange glass RG red glass E emission LP long pass EFLP edge filter long pass OG and RG are colored glass absorption filters whereas E LP and EFLP filters are coated interference filters Colored glass filters are cheaper and have broader transition slopes than coated interference filters Match filters to fluorophores in such a way as to maximize the amount of signal separation This multi color image contains red green and blue signals which were created with FITC TMR and CXR dyes matched with 505 585 and 650 filters respectively 4 25 Read Image Software FMBIO 9 PPlex11 4 26 FMBIO Read Image Software Example Scanning Process This section describes the process of scanning using as an example the detection of fluorophore labeled str alleles WARNING This is only an example These procedures are not meant to describe the only or optimal method to carry out the scanning of any material It is included only to ill
12. Straight Line 677048 00 0 OBand2 267924 00 D 0 0 00 ClBand3 33689200 0 00 EAE DlBand4 73963200 0 00 DlBand5 267471200 0 00 OBand6 1904868 00 0 00 OBand7 2258960 00 0 00 OBand8 190709200 0 00 OBand9 2094980 00 0 00 O Band 10 1930660 00 0 00 LlBand 11 199792400 0 00 O Band 12 2654736 00 0 00 O Band 13 203103200 0 00 2 Select a channel from the drop down list box Select a lane number containing standard markers from the drop down list box The defined bands in the lane appear in the list box 4 Click on a marker band to highlight it then enter a concentration value 5 79 Image Analysis Tools FMBIO in the field below the list box 5 Click Enter The value appears in the Concentration column 6 Continue to enter concentration values for all marker bands O Band 6 34143 0 OBand 119222 0 0 0 LlBand8 1469974 00 OBand9 506150 00 OBand 10 431650 00 OBand 11 502399 00 LlBand 12 488807 00 O Band 13 519335 00 O Band 14 2107995 00 OBand 15 407016 00 OBand 16 377121 00 LlBand17 25429 00 OBand 18 877367 00 IE Enter Place a checkmark in the box next to each marker band you want to use cOooouUocoooorioco Ooocomocooooon0 Select a standard curve fit in the drop down list Linear straight line least squares regression Second Order second order curve least squares regression Logarithmic natural logarithms least squares regression 9 Sel
13. Volume Calculation Types 5 78 Setting Quantification Markers 5 79 Volume Calculation 5 81 Displaying Results 5 81 Updating Spreadsheet Values 5 85 Exporting the Spreadsheet 5 86 Overlay Trace 5 87 Overlay Trace Window 5 87 Overlay Trace Setting Window 5 90 Exporting Lane Trace Data 5 91 Printing 5 94 Batch Analysis 5 96 Procedure 5 96 1D Gel Analysis 6 1 1D Gel Menu and Tools 6 2 Analyzing 1D Gels 6 6 Summary of Analysis Steps 6 6 Single Color Experiment Flowchart 6 7 Single Color Experiment Procedure 6 8 Multi Color Experiment Flowchart 6 16 Multi Color Experiment Procedure 6 17 Array Analysis 7 1 Array Menu and Tools 7 2 FMBIO Creating an Array Analysis 7 3 Analyzing Spots in an Image 1 5 Creating a Grid 7 6 Modifying a Grid 7 9 Grouping Spots and Grids 7 9 Creating a Template 7 10 Background Calculation 7 11 Registering the Background Value 7 12 Registering Markers 7 13 Setting a Partial Quantity 7 16 Mol Calculation 7 18 Displaying a Spreadsheet 7 18 Exporting a Spreadsheet 7 20 Comparing Grids 7 21 Data Sheet Filecard 7 22 Overlay View Filecard 7 23 Grid Comparison Filecard 7 28 Scatter Plot Filecard 7 30 8 Drawing Tools 8 1 Draw Menu and Tools 8 2 Right click Menu 8 3 Setting the Margins 8 3 Rotating Text 8 4 Draw Tools 8 7 Working with Draw Objects 8 14 9 Maintenance 9 1 Cleaning 9 1 Service 9 1 Laser Head 9 2 Photomultiplier Tube 9 2 vi 10 FMBIO Operating Temperature 9 2 Moving the Scanning Un
14. agarose gel acrylamide gel membrane and TLC thin layer chromatography Each parameter name is linked to a pre defined set of default scanning parameters Agarose Acrylamide Membrane TLC Horiz dpi 150 150 150 150 Vert dpi 150 150 150 150 Repeat 256 256 256 256 1Ch 80 80 45 45 2 Ch 100 100 55 55 3 Ch 80 80 45 45 4 Ch 100 100 55 55 You can modify any of these parameters as needed to accommodate your particular gel conditions You may also specify and save a customized set of parameters for repeated use See page 4 20 4 1 Read Image Software FMBIO Selecting SCSI ID The SCSI ID of the FMBIO scanning unit is set to 6 at the factory If you have any other peripherals attached to the SCSI port of the computer make sure they do not share the same SCSI ID number e Verify that the SCSI ID is set to Auto or 6 in the Read Image software Setting the Reading Resolution The higher the resolution or dots per inch of the scanned image the more information is collected during the scan The resulting file for a scan col lected at a higher resolution requires more storage space You can specify resolution for both vertical and horizontal scan directions Each parameter name has a limited number of resolution choices for each direction e Select horizontal and vertical scan resolutions Reading Sensitivity The intensity of the charging voltage generated by the photomultiplier tube PMT influences how much of the co
15. gt button e f you want to load a previously saved Multi Band Separation template click Template Select a template and then click Apply The detected bands appear in the Status Edit Sheet Status Edit Tools msjes mlm pe ies Es imn Detected Band Numbers SOOM SON 7 Review each band in the Status Edit Sheet verifying that the assigned colors are representative of the colors viewed in the gel image Typically the Multi Band Color Separator accurately selects bands and assigns colors to them In a rare instance you might want to edit the bands displayed 8 Ifyou want to change the color or status of a band click its row in the Status Edit Sheet The Status Edit Sheet toolbar is activated 5 47 Image Analysis Tools FMBIO Not Band p Skip mum mu B es Eo Overlap Band Color None Tool Description Band Colors Designates band color for the selected band None No color is assigned Excludes band from multi band color separation Overlap Overlapped band Excludes band from multi band color separation Not Band Band is not listed in template Color list is shifted down Skip The Detected Band numbering skips the band e fthe band was not assigned the appropriate color click the appropriate color The new color is displayed e Ifthe band is overlapped by another band click Overlap The band is grayed out e f you do not want to use the band click None The
16. take from five to ninety minutes See page 3 13 for Guidelines to Scanning with the FMBIO Scanner software 3 1 Operating the Scanning Unit FMBIO Starting the FMBIO Scanner Turning on the power to the scanning unit first then turn on the computer and start the FMBIO Scanner software e To start the Scanner program double click the Scanner icon The FMBIO scanning unit has two electrical switches e The Main switch located on the rear panel next to the SCSI cable port e A Power switch located inside the front panel For both electrical switches the Off position is marked with a circle O and the On position is marked with a vertical line The locations of the power switches are shown in the following two figures Power Ready and Error lights Power Figure 3 1 Inside front panel with Power switch 3 2 FMBIO Operating the Scanning Unit SCSI cable ports AC power Power cord female terminal SCSI cable Figure 3 2 Rear view of FMBIO scanning unit Turn on the computer that is connected to the scanner by a SCSI cable 2 Set the Main switch to the On position 3 Open the front panel of the scanning unit and turn on the Power switch When the scanning unit is first turned on it automatically performs a self diagnostic routine During the routine the Ready light on the front panel blinks Self diagnosis Normal When the Ready light shines steadily without blinking the self dia
17. 3 software requirements 2 3 specifications 1 5 focusing point 4 9 FMBIO Index G gamma level 5 12 Genotype 10 12 genotyping 10 1 to glass plates cleaning 3 9 loading 3 10 o 3 11 orientation 3 10 specifications 3 9 Gradient End 5 62 Gradient Start 5 62 gray level adjustment 4 10 5 13 histogram 5 17 showing range 5 18 template 5 23 visually defining 5 21 Gray Level Adjustment window 5 16 grid cell marker 7 14 modifying 7 9 templates 7 15 grid options 7 6 grouping spots and grids 7 9 H help STaR Call 10 10 high signal cutoff threshold 4 11 5 13 histogram 5 17 5 20 I icons ImageAnalysis A 2 Index 3 Index FMBIO image adding 5 4 diagnostic guide 5 24 export 5 7 exporting A 4 icon 5 4 information 5 5 A 4 multi color 5 9 setting 5 10 Image tools 5 4 A 3 imaging system organization 10 22 system ORI 10 22 system utilized 10 22 importing STR files 10 14 10 20 installing STaR Call 10 2 10 4 K keyboard shortcuts A 1 L laboratory ORI 10 22 lane boundary lines 5 50 5 53 editing 5 53 copying and deleting 5 54 list 10 24 selecting 5 54 template 5 55 Lane window 5 46 magnification 5 46 lane marker name 10 24 Index 4 laser beam 3 4 head 9 2 Line Selection 5 6 long pass filters 4 25 M Main switch 3 2 3 3 manual band input 5 64 mapping curve 5 17 mode selector 5 18 type 5 18 marker 5 68 adding 5 72 7 13 registering 5 68 template 5 72 media type 4 7 Medium G
18. 62517539 E Spot73 Spot73 106379898 70319071 Spot85 Spot85 118364568 57411895 Spot2 Spot2 112332471 55658839 Spot 4 Spott4 84851472 65221922 t Spot26 Spot26 111256720 64628890 Spot38 Spot38 110558639 55677439 1 Spots SpotSQ 113266345 24836703 T Spot62 Spot62 1 10948473 63499039 Spot74 Spot74 58737835 1 Spot86 Spot86 49871391 Spot3 Spot3 26526550 Spot15 Spot 85839102 Spot27 Spot27 95101378 70305471 Spot39 px 114542829 56815107 Spot51 Spot5 mj 8 To return to the original magnification click the Reset Magnification button 9 Tocopy the image to the clipboard click the Capture Image button 7 35 8 Drawing Tools You may use the Drawing tools before or after analysis functions to create text and graphics elements for inclusion any document or overlay trace With these tools you can do the following e Call attention to faint or targeted bands e Include comments with the scanned image e Package experimental results with a professional appearance CTTv Loci plus Amelogenin EHI ATT T I IIN EELLLLLUE x we ama e oe ee 24 a n E 8 1 Drawing Tools FMBIO Draw Menu and Tools 1 To use the drawing tools click the Drawing Tool button kd or choose Drawing from the Tools menu The Draw menu appears in the menu bar and the Drawing tool buttons a
19. 7 Show Mapping Curve In the Full Image Display drag the Zoom Cursor to a region containing average intensity bands background If necessary adjust the size of the Zoom Cursor to include the desired area The area appears in the Zoom Display In the Zoom Display surround a band with the Selection Cursor Whole Image Zoomed Area Ce PROUT EELS BATELA LE UUUUL Move the Selection Cursor to a background area away from the band then click the Background button The calculated Background cutoff threshold is applied to the Full Image and Zoom windows and the values appear in the Background Percent and Background Value boxes Use the High Signal and Background threshold marker lines in the histogram to adjust the gray scale range FMBIO 1D Gel Analysis 6 When you have finished click OK Set Lanes page 5 50 1 Click the Multiple Lane Selection Tool button HER or choose 1D Gel gt Tools gt Lane Selection 2 Create a rectangle surrounding the group Position the tool above the top migration line at the corner of the first lane in a sequence of lanes 3 Drag the tool below the bottom migration line to the opposite corner of the last lane in the sequence 4 Release the mouse The Multiple Lane Setting dialog box is displayed Multiple Lane Setting x r Options Auto Iv Check Regularity on Lanes C Manual Input Number of Lanes 1 255 fi Lane Width v Anta Deternined Uber D
20. Call 3 0 test Files CTT J PowerPlex CTTv Am 9 3 Locate the file you want to evaluate Select a STR Lookup Table in the drop down list box Repeat the process to add more files Select a conversion method STR plain Uses base pair sizes to call alleles Deletes all columns except for the bp column STR with OD values Uses base pair sizes to call alleles and OD values to calculate n 4 stutter percentages Deletes all columns except for the bp column and the OD column STR with IOD values Uses base pair sizes to call alleles and IOD values to calculate n 4 stutter percentages Deletes all columns except bp column and the OD column Click OK to import the files The Evaluate Select Allelic Ladders dialog box is displayed Note Do not choose recalculate No recalculation of values can occur until the STR files are imported A dialog box displays lanes along with the number of bands in each lane You are prompted to choose the lanes containing allelic ladders for averaging The default selection is all of the lanes that contain the highest number of bands The default choices are highlighted 10 15 Allele Calling with STaR Call FMBIO STaR Call Evaluate Select Allelic Ladders 2 x r Select Allelic Ladders to Average Lanes Lane6 Note This is a good time to verify that the correct number of bands are in the chosen lanes The number in parentheses preceding
21. Center Align Left Underline Font Sets text font and size Text Color Sets color of text Bold Bold text Italic Italic text Underline Underline text Align Left Align text lines left Align Center Center text lines Align Right Align text lines right Rotate Text Rotate text Drawing Tools FMBIO Working with Draw Objects 8 14 To select an object click on it with the cursor to select it A selected object has rectangles at its four corners To move an selected object drag it to the new location To change the size of a selected object click one of the corner handles and drag it To make the size change proportional hold the Shift key as you drag the corner To delete a selected object press the Delete key 9 Maintenance Cleaning WARNING Always turn the power switch OFF before per forming maintenance To clean the exterior surfaces of the unit use only a clean cloth or sponge moistened with mild detergent solution Wipe dry with a cloth Never use benzene paint thinner or other organic solvents for cleaning purposes and never apply adhesives or other chemicals to the exterior surfaces of the unit Doing so could result in discoloration and deformation of the covers with consequent loss of laser beam protection Never attempt to clean adjust or perform any other maintenance operation on the laser head the photomultiplier tube or any other optical mechanical or electrical component of the unit CA
22. Color Separation Show Image Setting Dialog Image Information Export Image Transparent Background Line Selection Area Selection Show Spectrum See Gray Level Adjustment on page 5 13 See Color Separation on page 5 29 See Image Setting Dialog Box on page 5 10 View sample image and scanning infor mation from theFMBIO Read Image file Save an image as a TIF file for export See Transparent Background on page 5 35 Draw a line to select an area on the image Draw a rectangle to select an area on the image See Spectrum on page 5 25 Appendix A Normalize Baseline Switch Axis m Display Channel Tool Description Normalize Baseline Normalizes vertical range Swich Axis Switches between the horizontal and vertical spectrum views Display Channel Toggles display of spectrum for individual channels Appendix A 1D Gel Tools Select Tool Lane Selection Tool Multiple Lane Selection Tool Lane Alignment Automatic Lane Fitting Lee ea ea ee Band Edit Delete Band Automatic Band Detection Lane Template Tool Description Select Select objects Lane Selection Define a single lane Multiple Lane Selection Define multiple adjacent lanes Lane Alignment Aligns lanes with migration lines Automatic Lane Fitting Automatically adjusts lane boudaries Lane Template Saves all lanes on image Show Hide Lanes Appendix A Show Hide Band Informa
23. Description Set Selected Spots to Background Set Selected Spots to Marker Calculation Open Standard Curve Open Spreadsheet Preferences Array Template Set Grid Marker Used selected spots to represent the background value Uses selected spots as the markers Performs a calculation Displays the Standard Curve window Displays the Spreadsheet window Displays the Preferences window Save or apply spots and grids Displays the Grid Marker Setting window Appendix A Command Description Group Ungroup Group the selected spots grids Grid Comparison Opens the Grid Comparison module Drawing Tools For a description of the Drawing tool icons see Chapter 8 Drawing Tools Tools Index This an index of all tool buttons in Image Analysis Tools A Align Bottom 8 9 Align Center 8 13 Align Horiz Center 8 9 Align Left 8 8 8 13 Align Right 8 8 8 13 Align Top 8 9 Align Vert Center 8 8 Arrow 8 12 Automatic Band Detection 5 44 5 63 6 13 6 23 6 29 B Band Color 5 48 6 25 Band Edit 5 64 6 14 6 29 Band Style 5 40 5 67 6 5 6 13 6 29 Bold 8 13 Bring Forward 8 8 Bring to Front 8 8 C Calculate medium grid 7 18 Calculation 7 18 Circle 8 12 Color Separation 5 5 5 31 5 34 Create Grid Spot 7 2 7 7 Create Oval Spot 7 2 7 5 Create Rectangle Spot 7 2 7 5 D Diamond 8 12 Double headed Arrow 8 12 Drawing 5 89 7 2 8 2 E Edit Polygon 8 9 Ellipse 8 7 F Fit
24. Image Analysis Tools Tool Description Zooming Tool 1 Click the button The cursor transforms into a magnifying glass with a sign 2 Drag the magnifying glass cursor to create a rectangle surrounding a region of the image The image is magnified to show the region Fit Window Once a magnification is selected th eimage fills the entire window Moving Tool Click the button and then drag the image with the hand cursor Saving an Image You can save the entire image or a selected area of an image as a TIFF or BMP file 1 If you want to crop the image click the Select area to draw button or choose Area Selection from the Image menu Place the crosshair pointer on a corner of the rectangular area you want to save and click and drag the mouse to the opposite corner of the area Release the mouse 2 Choose Save Image as from the Image menu The Save Image as 5 7 Image Analysis Tools FMBIO dialog box is displayed Save Image as Parameter Description Image Size Save the entire image or a selected area Select the area using the Area Selection tool Image Type TIFF or BMP Invert Gray Inverts the image gray scale values Image Rotation Bits Channel Color Separation Flip None 180 270 or 360 8 bit or 16 bit Save any of the channels represented by colors 1 2 3 and 4 If color separation has been performed in Images Analysis save the color separated image or the r
25. Quantity 2 Off gog ejeje ejeje Array Analysis FMBIO Type Area Size Grid Area Part Quantity On CC e Vc enm eie ogg V 8 mm Part Quantity On ogg oon ggg V 8 mm ggg Mol Calculation Image Analysis calculates the mean signal intensity standard deviation and volume of each spot If you have specified quantification markers Image Analysis also calculates the quantity of sample in each spot e Click the Calculation button F3 or choose Calculate from the Array menu Note Values are only quantified if they fall within the highest and lowest standard marker values Displaying a Spreadsheet You can select the spot information you want displayed in the Channel spreadsheet l Click the Preferences button Sif or choose Preferences from the Array menu FMBIO Array Analysis 2 In the Preferences dialog box click the Spot Information tab Preferences The calculated values appear in the spreadsheet and the volume of each spot grid cell is displayed on the image e Click the Open Spreadsheet button El or choose Open Spreadsheet from the Medium Grid menu Array Analysis FMBIO ii 2D practice 3605450 11329 10 15303040 2937422 88823 1136588 2828521 101040 855488 29305 51 91166 773985 i 3468884 883136 10510054 Sample 1 2830419 91972 830253 2843357 93154 1265864 3608634 1067821 12471802 2831236 1006 76 756039 2822785 115338
26. Select the channel to show the channel image data To hide a channel s image data remove the checkmark adjacent to the channel Note You cannot turn off the channel display in Mono display mode Changing Channel Color You can change the color associated with a channel in the multi color image The Color Chooser is the colored square next to the channel name No two channels may have the same color assignment e Click the Color Chooser for the channel you want to change A pop up menu of available colors is displayed Click the desired color 5 11 Image Analysis Tools FMBIO Changing Intensity Level You can change the intensity of each color associated with a channel e Drag the gamma level bar to the right to increase intensity Slide the bar to the left to decrease intensity Displaying Detected Bands After you use Image Analysis to define bands on all channels you can choose which channel displays the band definition lines e Click the radio button adjacent to the channel name The black dot in the radio button indicates the active channel In the image Image Analysis displays the band definition lines for the active channel For more information on detecting bands see page 5 60 5 12 FMBIO Image Analysis Tools Gray Level Adjustment The proper setting of the Gray Level Adjustment parameters enhances signal and suppresses noise resulting in clean readable images Conversely improper settings might result in ei
27. Window 5 6 Font 8 13 G Gray Level Adjustment 5 5 5 16 6 9 6 20 I Image Information 5 5 Italic 8 13 L Lane Selection 6 23 Lane Style 5 40 5 53 6 5 6 12 6 27 Lane Template 5 55 5 57 Line 8 7 Line Color 8 11 Line marker style 8 12 Line Type 8 11 Appendix A Line Width 8 11 Lock 8 9 M Marker Setting 5 68 6 14 6 18 6 30 Moving 5 6 Multi Band Color Separation 5 45 6 23 Multiple Lane Selection 5 51 6 11 6 27 N New Overlay Trace 5 87 New Project 5 3 6 8 No Line 8 11 None 5 48 5 49 6 25 Not Band 5 48 6 25 O Open Project 5 3 Open Spreadsheet 1D Gel 5 83 5 86 6 15 6 31 10 8 Medium Grid 7 14 7 19 Open Standard Curve 1D Gel 5 80 Medium Grid 7 15 Oval 8 7 Overlap 5 48 6 25 Overlay Trace Setting 5 89 5 90 P Paint Color 8 10 Polygon 8 7 8 12 Polyline 8 12 A 16 Preferences 1D Gel 5 41 5 61 5 77 5 84 6 22 Medium Grid 7 6 7 11 7 13 7 16 Print 5 95 Q Quantification 7 13 Quantification Setting 5 79 6 29 R Rect 8 7 S Save Project 5 3 Select 1D Gel 5 53 Draw 8 7 Medium Grid 7 2 7 5 7 6 7 9 Select Line Type 8 11 Send Backward 8 8 Send to Back 8 8 Set Selected Spot s to Marker 7 14 Set Selected Spots to Background 7 12 Show Image Setting Dialog 5 5 5 10 Show Hide Band Information 5 40 5 85 6 5 Show Hide Comment 1D Gel 5 40 6 5 Medium Grid 7 2 Show Hide Lanes 5 40 6 5 Show Hide Spot Information 7 2 Skip 5 48 6 2
28. and gamma controls to fit the particular conditions of an image With Image Analysis you can modify the image to reveal a very broad range of signal intensities Just remember that considerably more information is latent in the image than can be displayed with any particular set of display parameters e Many factors contribute to the success of DNA detection Variables include gel type and concentration sample preparation band size dye concentration staining time destaining time gray level settings and laser focusing point Further optimization of protocols may result in higher detection sensitivity 4 23 Read Image Software FMBIO e For best scanning results avoid dust specks by using only powderless gloves rinsing gloves with distilled water thoroughly cleaning all containers used for staining and filtering all buffers and solutions with a 0 45 um filter e Loading buffers containing xylene cyanol and bromophenol blue fluoresce strongly when excited by the FMBIO laser and can interfere with the gel image For best results use loading buffers containing no xylene cyanol and bromophenol blue concentrations decreased 1 10 e Gels can be cast with ethidium bromide to save time after electrophoresis However electrophoretic mobility of linear double stranded DNA is reduced by up to 15 in the presence of ethidium bromide and background staining is not uniform when casting agarose gels with ethidium bromide Therefore post st
29. by the number of times a sequence is repeated STR loci may be detected by polymerase chain reaction amplification using labeled primers Electrophoretic separation is then used to distinguish alleles by size STRs are abundant and widely distributed throughout the human genome They are well characterized and highly polymorphic making them ideal for use in individual human identification The discriminatory power of STR analysis is greatly enhanced by evaluating samples at more than one locus simultaneously multiplexing When com paring forensic samples at eight loci matching probabilities can exceed 1 in 118 000 000 In parentage investigations multiple locus analysis can result in paternity probabilities of 0 9979 and higher STR data are efficiently analyzed using a unique combination of instrumen tation non isotopic chemistry and software that makes processing DNA samples fast safe and easy for human identity applications and databasing 10 6 FMBIO Allele Calling with STaR Call Preparing for Allele Evaluation Before you can use STaR Call Genotyping Software you must use 1D Gel tools in Image Analysis to analyze a gel Perform the following steps to gen erate results for allele evaluation 1 Ina one dye image adjust the gray level see page 5 13 In a multi color project perform a color separation see page 5 50 2 Define the lanes to be analyzed see page 6 11 STaR Call can accept up to 51 lanes for analy
30. coordinates Four values that define the scan area Setting button Opens the FMBIO Parameters window Read button Begins an image scan Changes to Pause during a scan Changes to Resume during a Pause PreRead button Begins a prescan Changes to Cancel during a scan All Area button Sets the scan area to the total scan area Comment field Stores your comments 4 15 Read Image Software FMBIO fi ch4pro1 fb2 FHBIO2 Readlmage su Bid EH Blood Stain 2987934UCO ER Convicted Offender sample Using the Scan Control Window In addition to providing the tools for defining the scan area and performing scans the Scan Control window displays Scan settings specified in the Parameters window See page 4 6 Image file type orientation gray level adjustment settings defined by items in the Command menu See page 4 10 Autofocus and the active channels settings defined by commands in the FMBIO menu See page 4 13 4 16 FMBIO Read Image Software Important Before defining the scan area and beginning the scan review the settings displayed in the text of the Scan Control window Verify the gray level adjust ment and active channels are appropriate for your scan Defining Scan Area The PreRead Display in the Scan Control window represents the total scan area of the FMBIO scanner bed The material you are scanning is positioned within the total scan area but may be smaller than the total area When th
31. field 2 Enter the data Note Enter numeric values in the base pair and concentration fields Enter alphanumeric values in the label field Band Information display In the 1 D Gel Experiment window Image Analysis can display the OD IOD IOD Rf base pair concentration value or a user defined label next to the band To display values in the 1D Gel Experiment window 1 Click the Preferences button q or choose Preferences from the 1D Gel menu then click the Band Information tab Select the item you want to display See page 6 6 for an explanation of these parameters 2 Select the lanes The information is displayed next to the band FMBIO Image Analysis Tools The Show Hide Band Information button or 1D Gel gt View gt Band Information menu item toggles the display on and off Updating Spreadsheet Values Changes you make to your 1D Gel experiment may affect the values displayed in the spreadsheet These changes include e Adding or deleting values e Changing units of measurement e Moving adding or deleting bands or lanes Whenever you make these changes you must be sure the corresponding values in the spreadsheet are updated Image Analysis Tools FMBIO To update spreadsheet values after changes in the 1D Gel Experiment window e Click the Volume Calculation button ES or choose 1D Gel Volume Calculation Exporting the Spreadsheet You can save the spreadsheet as a tab delimited text f
32. last scan area used with each Parameter Set Time The amount of time it takes to perform a scan is a product of the area of the scan the resolution dpi and the number of repeats per scan line Repeats As you increase the number of repeats per scan line the signal to noise background ratio increases Signal At lower resolutions the amount of visible signal may increase but resolution of bands and spots decreases Resolution The resolution of bands and spots improves as you increase both resolution dpi and repeats per line Saving parameters You can save a set of customized scanning parameters as a Read Image file You can open this file and reapply the saved settings to new scans Multiple scans of the same gel If necessary you can remove a gel from the FMBIO scanning unit remount it in the gel box for additional electrophoresis and then rescan the gel 3 13 4 Read Image Software The Read Image software controls the FMBIO scanning unit as it generates digital images of gels membranes blots and microtiter plates Read Image converts the experimental data into a digital file which you can store on the computer hard disk or on a peripheral storage device You can then use Image Analysis software to analyze the image Note This manual does not discuss sample preparation Refer to standard texts such as Ausubel or Maniatis and to the documentation accom panying your fluorophores With Read Image software you c
33. of these parameters might result in either saturated or faint images The Gray Level Adjustment parameters modify the signal intensity of each dot or pixel in the scanned image A pixel is the smallest unit of a scanned image FMBIO Read Image files contain 16 bits per pixel or 65 536 grayscale levels 2 6 colors black and white 65 536 shades of gray Most computer monitors are 8 bit and can therefore only display 256 25 shades of gray 4 10 FMBIO Read Image Software The Gray Level Adjustment tool tells your computer monitor how best to display your image It allows you to optimize your image by assigning these 256 shades of gray to the region of sample signal only All pixels below the sample signal range will be shown as white and all pixels above as black Cutoff Thresholds Cutoff thresholds are applied to the range of 2 6 or 65 536 shades of gray that are generated during a scan Background or noise lies at the low end of this range signal lies near the high end of this range By adjusting the cutoff thresholds you are adjusting the range between the highest and the lowest acceptable gray shades When the high cutoff is too low dark band images may be blurry with indistinct edges When the low cutoff is too high faint images are lost in the background When you change the percentages for high and low cutoff thresholds you change how the image appears on your monitor You do not change how much imaging
34. points to the right and the ball bearing on surface of the holder faces up See Figure 3 3 on page 3 7 5 In this position find the two set screws in the groove on the right edge of the filter holder There is one set screw adjacent to each optical filter position 6 Usea 1 5 mm Allen wrench to loosen the set screw that holds in place the filter you want to change Note One full turn loosens the screw sufficiently do not remove the set screw 3 6 FMBIO Operating the Scanning Unit Alphanumerics or m on edge of filter Filter positions 4 d Set screws 7e m E Ball bearing ra VA Finger hole am E d 9 __ P4 1 5 mm Allen wrench w if Figure 3 3 Changing optical filters 7 After one full turn of the set screw hold the filter holder over a clean lint free tissue and gently tilt the holder upright until the filter falls out Set the filter aside 8 Return the filter holder to a horizontal position and hold the replacement filter over the appropriate hole in the filter holder The alphanumerics on the edge of each filter should be upright If there is an arrow on the edge of the filter it should point up Drop the filter in place 9 Turn the set screw one full turn to tighten the filter Important To avoid breaking the filter do not overtighten the set screw 10 Return the filter holder to its place in the scanning unit Be sure the finger hole in the filter holder is
35. saved Read Image file the Scan Control window is displayed Read Image auto matically displays the scanning parameters You may modify these parameters or apply them unchanged to a new scan e Usethe commands in the File menu to create open and save Read Image files See page 4 20 for a description of this menu 4 3 Read Image Software FMBIO Setting the Parameters To set the FMBIO parameters choose Set Parameters in the Command menu SettingDialog Acrylamide Gel SE I e Haw Id 6 Use the Setting dialog box to specify e Parameter name e Scanning resolution e SCSIID e Experiment type e Channel name sensitivity and filter e Focusing height e Scan line repeats For more information about this window see page 4 6 4 4 FMBIO Read Image Software Additional Scanning Parameters Use items in the Command menu to specify e Gray level adjustment settings including high and low cutoff thresholds e Orientation of the scanned image See page 4 10 for more information about this menu Use items in the FMBIO menu to choose e Autofocus e Active channels See page 4 13 for more information about this menu 4 5 Read Image Software F MBIO Features of the Parameters Window SettingDialog Acrylamide Gel fiso gl gl Haw Id 6 STR allele Name Description Parameter Name Reading Resolution SCSI ID Experiment Type Channel Name Reading Sensitivity Fil
36. the CD for example D setup exe then click OK 6 Follow the instructions on the screen 2 3 Installation FMBIO Understanding Personal Computers Users of the FMBIO system should be familiar with basic IBM compatible computer and Windows 2000 functions New users should familiarize themselves with Windows 2000 operation before attempting to operate the FMBIO system Refer to your manuals and the online help installed with Windows 2000 Setting Color Display 1 In the Windows Taskbar click on the Start button Choose Settings gt Control Panel 2 Inthe Control Panel window double click the Display icon 3 Click the Settings tab then select the 256 Colors setting or higher 2 4 FMBIO Installation Display Properties 256 Color igh Color 16 bit MM True Color 32 bit Inte ths montar ox f emen o am Note When 256 colors are selected only the active window is displayed with sharpness and clarity while background windows may have a rough appearance When High Color 16 bit or True Color 32 bit is selected all windows are displayed with sharpness and clarity but at the cost of slower screen drawing The number of colors you choose depends on the complexity of your images and the speed of your system Printer Images generated by the FMBIO system can be printed on conventional postscript laser printers with 600 dpi resolution While the images rendered by such machines are adequate for many referen
37. the color separation process Image Analysis Tools FMBIO Indicating Lanes To analyze the data in a lane the lane must first be defined with lane bound aries Image Analysis automatically numbers each defined lane Any data outside the lane boundaries are not included in the analysis You can define single lanes or multiple lanes in sequence Adjustment to lane position width and shape can be made after the lanes are set You can also create a lane template for use on other gels nd m m e i nmi izi N _ e EB Zd Ye i id The Project window lists the lanes defined for each channel PPlex111_prj OF x L3 Project E 1D Gel Document 2 CTTv 3cH OOOOOOomm oo d 0 7 OC C nM o iJ DDD 2CH HB inlane 4CH zi EJ ii iB 3 5 50 FMBIO Image Analysis Tools Defining Lanes You can define up to 255 adjacent lanes simultaneously To define multiple sequential lanes 1 Click the Multiple Lane Selection Tool button Ead or choose 1D Gel gt Tool gt Multiple Lane Selection 2 Position the tool above the top migration line at the corner of the first lane in a sequence of lanes 3 Drag the tool below the bottom migration line to the opposite corner of the last lane in the sequence 4 Release the mouse The Multiple Lan
38. the lane number is the number of bands in the lane The allelic ladder dialog box offers three ways to select allele ladders a Click the Auto Select button to let STaR Call choose the lanes with the most bands as in the example shown here The average of these ladders becomes the standard for comparison b Click on one allelic ladder to select it as the standard for comparison c Press and hold down the Ctrl key as you click more than one allelic ladder These ladders will be averaged and the average used as the standard for comparison 8 Click OK to complete allelic ladder selection STaR Call puts the average base pair values of the allelic ladder standards into the STR Lookup table These allelic standard values appear in the Paste Values column Ranges are then generated using the Paste Values for the allelic ladder and the values from the and columns After the STR Lookup table is filled the current table is evaluated Each base pair value is compared to the ranges in the lookup table If the base pair value is within the range for an allele the corresponding allele is entered into the Genotype column Otherwise Not in range is dis 10 16 FMBIO Allele Calling with STaR Call played If you evaluate STR data with OD or IOD values stutter percentages are entered under the Percentages column for all values that fall within the stutter cutoff range assigned for that locus Note Ifa band is no
39. the length width location or angle of each defined lane or copy a selected lane e Click the Select Tool button Rj or choose 1D Gel gt Tool gt Select and then click on the lane Six handles appear in the lane boundary Handles also appear on the center line to adjust the lane boundaries for curved lanes Image Analysis Tools FMBIO There are several methods for editing the lane boundaries e Place the cursor inside the lane then drag the entire lane to the new location e The top center handle moves the position of the top boundary e The bottom center handle moves the position of the bottom boundary e Dragging an interior center handle bends the lane laterally Holding down the Ctrl key while dragging bends only the segment of the lane between the adjacent handles e The side handles change the width of the lane Drag a handle away from the center to make the lane wider drag the handle toward the center to make the lane narrower e The corner handles change the lane width at the top or bottom of the boundary Copying and Deleting Lanes e To make a copy of a selected lane choose Duplicate from the Edit menu or press Ctrl C on the keyboard e To delete a lane boundary select the lane and press the Delete key Selecting Multiple Lanes Hold the Shift key as you click each lane To select all lanes choose Select All from the Edit menu 5 54 FMBIO Image Analysis Tools Lane Templates Image A
40. used the commands in the 1D Gel menu to define bands See Defining Bands on page 5 60 To select the Calculation parameters e Click the Preferences button q or choose Preferences from the 1D Gel menu then click the Calculation tab Band Information Multi Band Color Separation l Automatic Band Detection Calculation m Background Value Channel pred g amp Background of Gray Level Adjustment B User Defined p el m Type C Typel C Type2 C Type3 Typed E E E 5 77 Image Analysis Tools FMBIO Background Value The background value defines a cutoff value in grayscale bits per pixel below which signal will not be calculated Specify the background value for each channel in your image Background of Gray Level Adjustment This option allows you to use the background cutoff threshold value for volume calculations It is automati cally entered User Defined This option allows you to enter a background value Calculating Normalized Volume IOD Image Analysis can subtract the background signal for more accurate volume IOD calculation Use this feature with any of the four background types Volume Calculation Types Image Analysis offers four types of volume calculation Each type offers a different method of defining the area of the selected peak relative to the background Type C Typel E b eed Type 1 The area incorporates any signal that is greater than th
41. used in the analysis of a multi color image While this summary uses diagrams from the Promega 1 1 Powerplex the process is also applicable to other types of multi color images Create a New Project page 5 3 1 In the File menu choose New Project A new Project window is displayed E Projecti Iof x E3 2 Inthe Project menu choose Add Image then select a file that contains a digital image of a one dimensional gel Image Analysis adds an icon representing the image file to the project tree 6 17 1D Gel Analysis FMBIO Samplelmagel 3 The Image menu appears in the menu bar and its corresponding tool buttons become active 4 Click the Marker Setting button MI or choose Set Marker from the 1D Gel menu 6 18 FMBIO 1D Gel Analysis Marker Setting pores ace el 5 If you are using an internal lane standard such as Promega s CXR select the channel used to scan the internal lane standard and then select Layer from the Marker Mode drop down list box 6 If an internal lane standard was not used select the channel used to scan any standard markers on the gel and then select Separate from the Marker Mode drop down list box Create 1D Gel analysis page 5 13 e Choose New 1D Gel Experiment from the Project menu The 1D Gel Document window 1D Gel menu and its 1D Gel tool bar appear 1D Gel Analysis FMBIO Adjust Gray Level page 5 13 1 Inthe Image Setting dialog box se
42. 0 FMBIO 1D Gel Analysis reference to the assigned marker Click the Open Spreadsheet button Fl or choose Open Spreadsheet from the 1D Gel menu to view the table of base pair values for each layer Click the tabs at the bottom of the window to switch among channels Choose Export from the SpreadSheet menu to save the spreadsheets as comma delimited text files that can be exported to STaR Call 7 Array Analysis Image Analysis can extract information from two dimensional 2D objects as small as one pixel by one pixel This capability is especially useful when you need to analyze arrays and other tightly spaced objects Using the analysis tools in the Medium Grid menu you can analyze dot blots slot blots gels or arrays These tools allow you to calculate the fol lowing parameters and display them in a table Spot coordinates Position Spot area Area Average signal intensity of the fluorescence of the pixels in a designated spot area Mean Volume of relevant pixels in a spot area Volume Quantity in spot e g micrograms DNA Mol Standard deviation of signal intensity range in each designated spot area Note For quantification of 1D gels see Setting Quantification Markers on page 5 79 7 1 Array Analysis FMBIO Array Menu and Tools Tar GUI Drawing Tool Select Tool Show Hide Spot Information Show Hide Comment Create Grid Spot Create Oval Spot Create Rectangle Spot 7 2 FMBIO A
43. 01 79 CSFIPO 305 72 CSF1PO 226 40 TPOX 231 54 TPOX 239 02 TPOX 243 48 TPOX 196 58 Amelogenin 158 58 THOT 292 99 D165539 Hi 226 40 0758620 3 238 78 D75820 12 175 97 D138317 E 138 44 D58818 121 142 20 D5S818 13 429 89 FT3ADI B 394 13 FESFPS 10 319 17 LPL 10 181 54 THO1 185 64 THO1 192 65 THO1 272 85 D168539 276 54 D165539 288 89 D168539 282 91 D165539 8 15241 1 144 8 154 9 4792 12 266 13 3913 2 60 5 80 3 21 The CODIS Specimen Information appears in the Merged worksheet 10 26 Appendix A Software Reference Keyboard Shortcuts Keyboard shortcuts are a combination of keys that send a command to the computer Each keyboard shortcut behaves just like one of the commands in the Menu bar Most keyboard shortcuts use the Control key represented by the abbreviation Ctrl and one other key For example to open a project you can either choose Open Project from the File menu or you can use the keyboard shortcut To use the keyboard shortcut to open a project hold down the Control key and then press the O key This shortcut is represented as Ctrl O Menu Command Keyboard Shortcut Close Ctri W Copy Ctrl C Cut Ctrl X Duplicate Ctrl D New Project Ctrl N Open Project Ctrl O Paste Ctrl V Print Ctrl P Save Project Ctril S Select All Ctri A Appendix A Icons Image Analysis Tools File and Edit Tools New Project Open Project Save Projec
44. 1 as the number of cells vertically enter a value in the cell height field Click OK Click the Create Grid Spot button Es Place the crosshair cursor in the center of the spot at the top corner and then drag the cursor through the spots in the top row 7 7 Array Analysis FMBIO 1 Click here 2 Drag cursor across top BE oom 3 3 Release mouse 20 i m ic 4 ay 2 za 54 0 1 7 8 Release the mouse button then move the cursor down until the inner rectangle passes through the spots on the four sides 4 Move tool down 5 Click at corner 9 Then click the mouse button The grid is created according to the grid options set in the Preferences dialog box 7 8 FMBIO Array Analysis Modifying a Grid You can use the Select Tool k to modify the grid size position and rota tional angle To move the grid e Use the up down left and right arrow keys on the keyboard e Place the Select Tool cursor inside the grid and then drag the crosshair to move the shape To enlarge or compress a grid e Place the Select Tool cursor over a transformation handle initially the upper left or lower right handle and then drag the cursor until you have the desired shape To rotate a grid e Place the Select Tool cursor over a rotation handle initially an upper right or lower left handle and then drag the cursor about the midpoint Grouping Spots and Grids You can group
45. 5 Appendix A standard curve type 7 13 Straight Line 8 12 T Text 8 7 Text Color 8 13 Transparency 8 10 U Underline 8 13 Unlock 8 9 V Volume Calculation 5 81 5 86 6 15 6 30 10 7 Z Zoom In 5 6 Zoom In Vertically 5 89 Zoom Out 5 6 5 7 A 3 Zoom Out Vertically 5 89 Zooming 5 6 Index Symbols 1D gel analysis 6 1 6 6 defining bands 5 60 genotyping 10 7 menu 5 37 spreadsheet 5 83 tools 5 37 A 4 605 nm filter 4 13 A about STaR Call 10 10 AC power 3 3 acrylamide gel 4 7 active channel 4 14 adding STR 10 20 agarose gel 4 7 All Area 4 18 allele ladder 10 16 allelic ladder 10 16 10 19 analysis worksheet 10 18 analyzing spots 7 5 Area Selection 5 6 Auto Band 5 60 Auto Focus 3 12 4 13 B background cutoff threshold 4 11 gray level adjustment 5 78 Medium Grid 7 11 percent 5 44 subtraction 5 78 value 5 78 Background Area 1 5 43 Background Area 2 5 43 band adding 5 64 area 5 43 boundary lines 5 67 deleting 5 65 display 5 12 editing 5 64 hiding 5 67 information 5 84 manual input 5 64 merging parameters 5 41 moving 5 65 recognition 5 60 volume 5 77 6 6 volume percent 6 6 basepairs 6 6 Basic channel 5 32 Blend display mode 5 11 5 32 buttons A 2 Index FMBIO C calling alleles 10 1 to 10 3 channel active 4 14 5 12 color 5 11 5 12 displaying 5 11 sensitivity 4 8 cleaning glass plates 3 9 sample stage 3 9 scanning unit 4 22 9 1 closing ReadImage 4 20 CMF head
46. 6 csriPO 11 10 10 310 36 31100 40 30807 30707 csriPO 10 10 100 306 07 307 00 M 3009 30291 csFIPO af 100 100 301 91 30300 42 29706 299 06 csFIPO 8 100 100 298 06 29900 48 29316 295 16 csFIPO 7 100 1 00 294 16 29500 4 28947 29147 csF PO ef 100 100 29047 29100 48 25008 25208 POX 13 10 109 251 08 25200 18 24620 24820 FOX 12 100 100 24720 24800 AZ 24248 24448 POX 11 100 100 243 48 24400 4B 23852 24052 OX 1 100 100 239 52 24000 48 23456 23656 POX af 100 100 235 56 236 00 20 23052 23252 POX 8 100 1 001 231 52 23200 21 226 33 228 33 TPOXK 7 100 100 227 33 228 00 2 22221 22421 IPOX 6 100 1 00 22321 22400 23 199 33 203 33 Amelogenin Y 200 200 201 33 218 00 24 195 43 199 43 Amelogenin X 200 200 197 43 21200 25 192 62 194 62 THO1 11 100 100 193 62 203 00 26 189 19 190 69 THO1 10 0 50 100 189 69 199 00 qu 184 54 186 13 THOl 93 100 049 185 64 198 00 28 180 54 18254 THOl 9 100 1 00 181 54 19500 z L4 LP bIN Summary Z CTTVARENEWSCH PowerPlex CTTv Am 9 3 Merged Z Merge 1 E Ready a _ NUM Seay Title Description Ranges Genotype amp Paste Values Values derived from the and Paste Values columns used to assign the allele names Values determined by the Evaluate STR Data function Variance values used to derive the values in the Ranges column Average values for allelic ladders selected in t
47. 61 11256720 951013761 181 1 16811 181633341 16043591 94394420 987 1800 1043327411 105586361 145428261 20821963 93238022 97078250 93329516 932043 4070711611 132663481 157504881 184363221 164751621 169757851 166234871 188860 8004360 1 10948473 905173221 174637261 152791541 159860301 1 48029361 1654674 10637989E107678560 86381562 86261870 90888428 868717741 123296541 155405 7 21 Array Analysis FMBIO Data Sheet Filecard You can view the numerical data for each grid in the Data Sheet You can also copy the spreadsheet and use it in another application such as Microsoft Excel fm Grid Comparison Window Compare by volume Data Sheet Overtay View Grid Comparison Scatter Plot Selected Spots Set selected grid as T Bp T Project gt Array Experiment gt Channel 1 Gridl 2 988417741 183645861 181 106541037379661 2417839721 279301561 252782801044304 8 8204883C11233247 11203294641 188145821 198875841 160204121 150582801 204614 DE Gridi cm 78543650 84851472 90461732 9621178 1 15139363 93526911 120282511212663 E 1034457361 11256720 9510137 11811158111815333 115043591 94394420 987180t 10433274111055863 11454282 120821983 93238022 97078250 93329516 9320434 4070711611 13266346 167504821 184363271 164751821 169757861 166234821 188688 Channel 3 E Gridi c 800436061 10948473 905173221 174637261 152791541 1598603C1 148029341 185467 HH 10637989107678560 86381562 86261870 90888428 86
48. 8717741 123296541 155405 Grid A7 Data Sheet Buttons Copy Spreadsheet Switch Active Channel I Project gt Array Experiment gt Channel 1 gt Grid1 Item Description Switch Active Channel Switch the active data channel Copy Spreadsheet Copies spreadsheet to clipboard 2 Select the grids to compare by first clicking the first grid to highlight it 7 22 FMBIO Array Analysis 3 Click the Channel 1 button m Compare by Volume Set selected grid as I m 4 Highlight the second grid and click the Channel 2 button rr 5 Inthe Compare by drop down box select a Concentration Volume Standard Mean or Area 6 Click the Switch Active Channel button to display the grid data for each channel 7 If you want to copy the spreadsheet to the clipboard click the Copy Spreadsheet button Overlay View Filecard You can overlay the two grids and to visually screen each spot for differences in the data values You can adjust the gray scale for each channel and enhance the detection of spots with small values The Screening feature allows you to display only spots that exceed a minimum Channel 1 Channel 2 ratio and view the numerical data for those spots The Overlay View is par 7 23 Array Analysis FMBIO ticularly useful for quickly filtering spots for quantitative changes in expression Item Description Spot Radius Adjusts the spot radius Channel Set
49. Analysis Grid Marker Setting E mm EE s 5 exem a 3 If you plan to use these grid markers again save the parameters as a template Entera name in the Parameter Set list box e Click Save 4 Click OK The boundary color of the cells changes to violet Grid Templates To use a Grid Marker Template 1 If you want to use an existing grid marker template select the template from the Parameter Set list box 2 Click Load Displaying the Standard Curve Click the Open Standard Curve button or choose Open Standard Curve from the Array menu The standard curve window is displayed 7 15 Array Analysis FMBIO 2D practice y 1 22072E7Log CO 1 00649E7 Setting a Partial Quantity You can restrict the area of a grid cell that is used for volume calculations For example this feature is useful when the spots in a grid are far apart rel ative to their size To specify a partial quantity 1 Click the Preferences button KI or choose Preferences from the Array menu 2 Inthe Preferences dialog box click the Part Quantity tab FMBIO Array Analysis Preferences 3 Click On Note When Part Quantity is turned off the entire area of the spot grid cell is used 4 Choose the type of partial boundary Rectangle or Oval 5 Enter the horizontal 0 68 100 and vertical 0 68 100 dimensions The following examples illustrate these settings Type Area Size Grid Area Part
50. Box on page 5 10 Magnify the image to view individual bands more closely If you still cannot view a detected band increase the gamma level for the channel If necessary you can manually add move and delete band lines See 5 63 Image Analysis Tools FMBIO Manual Band Input on page 5 64 6 To customize your auto band detection adjust the Automatic Band Detection Preferences on page 5 61 Manual Band Input Bands that are faint partially obscured or highly smeared may not be recog nized by the Automatic Band Detection tool You can manually add bands that have not been detected 1 Double click on the lane you want to edit The lane is selected and the Band Edit button i is activated 2 Magnify the image with the Zoom tools to view the band 3 Align the Band Edit cursor over the center of the band image 4 Click on the band Image Analysis places the band lines onto the band and the cursor transforms into a two headed arrow Initially the active violet band line is the Band End line 5 64 FMBIO Image Analysis Tools 5 Drag the Band End line down to the desired position then release the mouse button 6 Go back and click on the group of band lines The Band Start line becomes active Drag the Band Start line up to the desired position then release the mouse button Moving bands 1 Double click on the lane you want to edit The lane is selected and the Band Edit button ki is automatica
51. Control window Set Parameter Corresponds to the Setting button on the Scan Control window Choose this command to open the FMBIO Parameters window Set Gray Level Adjustment Orientation For a discussion of these commands see page 4 10 4 21 Read Image Software FMBIO FMBIO Menu FMBIO I Help v Auto Focus v 1 Channel v 2Channel v 3Channel v 4Channel Do Cleaning Eject Filter The commands in the FMBIO menu directly manipulate the FMBIO scanning unit These commands only work if your system has been con figured by qualified Hitachi Service personnel Auto Focus See page 4 13 for a description of this command Channels Checkmarks precede the active channels See page 4 13 for a discussion of active channels Do Cleaning Moves the optical unit to allow access to the inside of the scanning unit You cannot change filters when the scanning unit is in the cleaning position See page 9 1 for more information about cleaning the scanning unit Eject Filter Moves the filter holders to make filters accessible for removal Note Use the Eject Filter command only when the optical filter service door is closed 4 22 FMBIO Read Image Software Imaging Basics In the scanning process light signals are converted into a bitstream The FMBIO scanning unit generates 16 bit images Each pixel in the image con tains 216 or 65 536 possible signal intensity levels 16 bit imaging can produce very fine grai
52. DITIONS OF THIS AGREEMENT WHICH INCLUDES THE SOFTWARE LICENSE AND LIMITED WARRANTY IF YOU DO NOT AGREE WITH THESE TERMS AND CONDITIONS YOU SHOULD PROMPTLY RETURN THE PACKAGE UNOPENED TO MIRAIBIO INC MIRAIBIO OR A MIRAIBIO DEALER AND YOUR MONEY WILL BE REFUNDED The enclosed software the Software is licensed not sold to you for useonly upon the terms of this Agreement and MIRAIBIO and or its licensor s reserves any rights not expressly granted to you You are responsible for the selection of the Software to achieve your intended results and for the installation use and results obtained from the Software You own the media on which the Software is originally or subsequently recorded or fixed but MIRAIBIO and or its licensor s retains ownership of all copies of the Software itself LICENSE You may a Use the Software on a single machine at any given time b In no manner engineer or reverse engineer the copy protection hardware or whole or part of the software c Copy the software only for backup or modification purposes or to merge it into other software in support of your uses of the Software on the single machine provided that you reproduce all copyright and other proprietary notices that are on the original copy of the Software provided to you Certain Software however may include mechanisms to limit or inhibit copying Such Software is marked copy protected Any portion of the Software merged into another program wi
53. Detected Band numbers do not change The band is deselected and no color is displayed e Ifyou have applied a template to the Status Edit Sheet and the band is not listed in the template click Not Band The band is deselected and the color list is shifted down e f you do not want to use the band click Skip The Detected Band numbering skips the band Any band that has an Overlap None Not Band or Skip status is not 5 48 10 11 12 13 14 FMBIO Image Analysis Tools used for the multi band color separation mimm a t es Ea If this Multi Band Separation Marker is to be used in the future i e Controls such as K562 9947A etc click the Template button Click Create New and assign it a unique name Once the bands have been verified then click OK The Color Separation window is displayed with the bleed through values that were calculated during the multi band color separation Click Preview Image Analysis performs the multi band separation on the images in the Color Separation windows If the Color Separation parameters are to be used again in the future click the Template button and give the parameter set a unique name Click OK Image Analysis creates a set of color separated images each channel displaying only the signal from a single dye If you want to remove the color separation uncheck the Show Color Separated Images checkbox in the Image Setting dialog box You can now repeat
54. E THE SCOPE OF THE ABOVE WARRANTIES OR CREATE ANY NEW WARRANTIES SOME STATES DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES SO THE ABOVE EXCLUSION MAY NOT APPLY TO YOU IN THAT EVENT ANY IMPLIED WARRANTIES ARE LIMITED IN DURATION TO NINETY 90 DAYS FROM THE DATE OF DELIVERY OF THE SOFTWARE THIS WARANTY GIVES YOU SPECIFIC LEGAL RIGHTS YOU MAY HAVE OTHER RIGHTS WHICH VARY FROM STATE TO STATE LIMITATIONS OF REMEDIES MIRAIBIO s entire liability to you and your exclusive remedy shall be the replacement of the Software media or the refund of your purchase price as set forth above If MIRAIBIO or the MIRAIBIO dealer is unable to deliver replacement media that is free of defects in materials and workmanship you may terminate this Agreement by returning the Software and your money will be refunded REGARDLESS OF WHETHER ANY REMEDY SET FORTH HEREIN FAILS OF ITS ESSENTIAL PURPOSE IN NO EVENT WILL MIRAIBIO BE LIABLE TO YOU FOR ANY DAMAGES INCLUDING ANY LOST PROFITS LOST DATA OR OTHER INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE THE SOFTWARE OR ANY DATA SUPPLIED THEREWITH EVEN IF MIRAIBIO OR AN AUTHORIZED MIRAIBIO DEALER HAS BEEN ADVISED OF THE POSSIBLITY OF SUCH DAMAGES OR FOR ANY CLAIM BY ANY OTHER PARTY SOME STATES DO NOT ALLOW THE LIMITAION OR EXLUSION OR LIBILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES SO THE ABOVE LIMITAION OR EXCLUSION MAY NOT APPLY TO YOU GOVERNMENT LICENSEE If you are acquir
55. Enter a lower number in the value column To avoid this problem in future scans elim inate these objects from your scan area If after these steps you cannot achieve a satisfactory image you may have too much signal Try rescanning the sample at a lower photomultiplier tube PMT sensitivity scan using a different filter or if stains were used destain the gel 5 24 FMBIO Image Analysis Tools Spectrum With the FMBIO system you can accurately signal strength at particular locations on the image This quantification is more accurate than conven tional radiography followed by scanning densitometry because the intermediate film and scanning steps are eliminated The FMBIO spectrum function analyzes the pixel and gray scale data in the digital image and calcu lates the signal strength at a given location You can use two methods to select the location on the image for spectrum analysis e Draw a rectangle on the image The spectrum displays the sig nal strength of the bands within the rectangle e Draw a straight line on the image The spectrum displays the signal strength at each point on the line Area Selection Mehod 1 Click the Area Selection button ril or choose Area Selection from the Image menu 2 Position the tool at a corner of the group of bands you want to select Drag the cursor down to the opposite corner until the bands are con tained in the rectangle 3 Release the mouse button A selec
56. FMBIO Fluorescent Image Scanning Unit and Analytical Software Scanner Software for Windows Image Analysis for Windows v 3 0 Operation Manual Please read this manual carefully before operating the scanning unit Keep this manual near the scanning unit or in a location that is both easily accessible and under your control C 11530 10200 Revised January 18 2002 This product had been tested and complies with the limits for a Class A digital device in accordance with the specifications in Subpart B Part 15 of FCC rules This product has been tested and complies with the limits for a Class A digital device in accordance with the specifications in VDE rules This product has been certified and found to comply with the specifications for a Class I laser device of Japanese Industrial Standards JIS6802 and Title 21 USA code of Federal Regulations Subchapter J of FDA rules This product has been certified and qualified as a strategic object technology as set out in the Foreign Exchange and Foreign Trade Control Law In accordance with the above mentioned law permission from the Japanese government is required for purposes of exportation or delivery outside Japan This product contains devices and technology regulated under USA Export Control Rules and requires additional permission from the American government for exportation to certain countries Exemption From Any Obligation Hitachi Software Engineering Co Ltd shall not be lia
57. IO R Range 10 12 Read button 3 12 4 18 ReadImage opening file 4 20 quitting 4 20 starting 4 2 Reading by 10 23 Reading Date 10 23 reading resolution 4 8 Reading Time 10 23 Ready light 3 3 recalculating results 10 20 red glass filter 4 25 registering background 7 12 markers 7 13 removing STaR Call Macintosh 10 5 STaR Call Windows 10 3 repeats per line 3 13 4 9 requirements STaR Call 10 2 10 3 resolution 4 8 and signal 3 13 results displaying 5 81 Rf 6 6 right click menu 8 3 S sample ID 10 24 sample preparation 4 1 sample stage cleaning 3 9 sample types 3 8 Index 6 saving 10 21 scan settings 4 20 scanned image 4 18 scan duration 3 13 starting 3 12 scan area preview 4 17 Scan Control window 4 3 4 15 to 4 19 scan resolution 4 8 scan settings saving 4 20 scanning unit moving 9 2 shipping 9 2 SCSIID 4 8 self diagnostic routine 3 3 sensitivity 4 8 and background 4 8 setting partial quantity 7 16 shipping scanning unit 9 2 short tandem repeats See STR Show Spectrum 5 6 shutdown 9 3 signal to noise ratio 4 9 spacers in scan area 3 13 specifications FMBIO II 1 5 glass plates 3 9 specimen category 10 25 number 10 24 spot markers 7 13 modifying 7 5 Spreadsheet 7 14 spreadsheet 7 18 1D gel 5 83 entering data 5 84 exporting 5 86 7 19 10 8 updating 5 85 standard curve displaying 7 15 standard marker 5 68 STaR Call evaluating alleles 10 9 installation 10 3 M
58. Image menu A checkmark indi cates the dialog box is displayed SHOW SPECHT The Image Setting dialog box looks like this Image Setting E Eio co dimages imz Gamma iv Ig Level roae Adjustment re El FMBIO Image Analysis Tools Note The Show color separated images option is active only after you have performed a color separation on the image See Color Sep aration on page 5 29 Display Mode The Image Setting dialog box assigns a unique color to each channel in the multi color image The colors available depend on the display mode The Display Mode list box provides three options Blend Over and Mono Use the Display Mode drop down list box to choose how you want a multi color image displayed The colors available in the Image Setting dialog box change as the display mode option changes Mode Colors Available Blend Red Green Blue Colors in each channel blend together when they overlap to make another color Red blends with green to make yellow blue blends with green to make cyan red blends with blue to make magenta Over Red Green Blue Yellow Colors in each channel remains dis tinct when they overlap Use this mode when the multi color project contains a yellow channel Mono All channels display 65 535 shades of gray All channels in the multi color image appear in shades of gray Displaying a Channel You can choose to display or hide the image data from one or more channels e
59. Marker template The band colors appear in the Status Edit Sheet Typically the Multi Band Color Separator accurately selects bands and assigns colors to them In a rare instance you might want to edit the bands displayed Click the row of the band you want to edit The Status Edit buttons are 6 24 FMBIO 1D Gel Analysis activated Status Edit Tools W m k ies Es No Status E 2 Detected Band __ 3 Numbers 5 6 EM H Not Band pz Skip us mm 52 redes Overlap Band Color None e Ifthe band was not assigned the appropriate color click the appro priate color The new color is displayed If the band is overlapped by another band click Overlap The band is grayed out If you do not want to use the band click None The band is de selected and no color is displayed If you have applied a template to the Status Edit Sheet and the band is not listed in the template click Not Band The band is deselected 6 25 1D Gel Analysis F MBIO 12 13 6 26 and the color list is shifted down e If you do not want to use the band click Skip The Detected Band numbering skips the band Any band that has an Overlap None Not Band or Skip status is not used for multi band color separation In this example Band 5 is an overlapping signal so click the band row to select it and then click the None button from the tool bar Lane amm PEREI ENY Template m EEG ess 2 T
60. O Dye or Stain Excitation Emission Filter Vendor nm nm nm Cy2 489 505 505 Amersham Cy3 552 565 585 Amersham Cy3 5 581 596 605 Amersham FluorX 494 520 505 Amersham Alk Phos Substrates Attophos 430 440 560 505 NBL Vector red 605 Vector Labs Use these filters for maximum sensitivity 1 9 2 Installation Preparing for Installation WARNING Failure to follow proper installation procedures could result in physical harm to personnel and damage to the equipment Review the warnings and cautions detailed after the Table of Contents at the beginning of this manual Contents of FMBIO Shipment Before beginning installation check that you received the following items with the FMBIO shipment 1 Scanning Unit 1 Power cord 2 m long and two prong adapter 1 Blower brush for cleaning optical devices 2 Fuses 3A stored inside the AC inlet 1 Optical filters 605nm and 505nm 2 1 Installation FMBIO Installing the Scanning Unit Instrument and software installations are performed by qualified Hitachi Service personnel Any unauthorized attempt to install the scanning unit could void the instrument warranty Prepare a level sturdy non vibrating surface in a well ventilated temperature stable area for the FMBIO scanning unit The unit should not be placed in a dusty or damp location or in an area where it will be subject to vibration or impact A
61. PO 16 File Name Lane Types Lane Info Genotypes E 17 File 1 18 File 2 KIDDI Summar y CTTVASBNew3CH _PowerPlex CTTv Am 9 3 DODDABBNew4CH Z 4 1 ar E Ready 10 17 Allele Calling with STaR Call FMBIO Analysis Worksheet Provides allele evaluation using the selected STR Lookup table El Microsoft Excel fmbi 32581 CSFIPO 15 30607 CSFIPO 10 6194 30572 CSFIPO 10 322 15 CSFIPO 4 24348 TPOXTT 2676 30179 CSFiPO 9 30822 CSFiPO 13 23154 TPOX8 3081 24346 TPOXTT 31429 CSFIPO 12 23802 TPOX 10 310 35 csripo 11 23154 TPOX 8 30607 CSFIPO 10 18581 TH0193 3926 20640 TPOX7 30179 CSFIPOS 158 58 THOS 2535 29815 CSFIPOB 29443 CSFIPO7 THOT 200 43 CSFIPO 6 THO 93 25108 TPOX 13 THO 9 24 19 TPOX 12 THO 8 24345 TPOX11 23949 TPOX 10 29551 TPOX9 TPOX 8 TPOX7 EN E BEBE amt genini Y 4197 43 Amelagenin X 193 51 THO 11 lida gt DIN CTTVAR amp BNew3CH Merged Worksheet Merges data from all analyses Differences in the data among the different analyses are highlighted Excluded alleles are high lighted in red Non agreeing allele calls in overlapping loci between the PowerPlex 1 1 and PowerPlex 2 1 systems are highlighted in yellow 290 44 CSFIPO 302 11 CSFIPO 306 34 CSFIPO 294 13 CSFIPO 30599 CSFIPO 10 31021 CSFIPO 298 15 CSFIPO 31408 CSFIPO 302 14 CSFIPO 23154 TPOX 8 19964 317 96 CSFIPO
62. Rf Relative electrophoresis mobility When the top migration line is 0 and the bottom migration line is 100 it is the ratio of the band peak migration rel ative to the total length bp Fragment length in base pairs determined with standard markers Enter numeric values Concentration Concentration determined with standard markers Enter numeric values Label Enter any alphanumeric label or comment for a band 2 Select the variables to display To save your selections as a template l Click the Template button The Template dialog box is displayed 2 Click the Add button and type in a name for the template The default name for the first template is infol tmp and each additional template is numbered sequentially 3 Click OK and then click Close Spreadsheet Display The results of the analysis are shown in a spreadsheet that can be exported 1 To display a spreadsheet of results click the Open Spreadsheet but ton Fl or choose Open Spreadsheet from the 1D Gel menu The Spreadsheet window is displayed and the SpreadSheet menu appears in the menu bar 5 83 Image Analysis Tools FMBIO 2 Selecta channel by clicking its tab at the bottom of the spreadsheet Each band is listed in order of migration distance Image Analysis displays a set of band information columns for each lane Entering Spreadsheet Data You can enter and edit data in the base pair concentration and label fields 1 Click on the
63. Signal and Background values appear as shades of gray If these gray level adjustment parameters is to be used again in the future click the Template button and assign a unique name Repeat the gray scale adjustment for each channel Switch to a different channel by placing a checkmark next to it in the Image Setting dialog box 6 21 1D Gel Analysis F MBIO 8 Click OK to apply these values to the image Perform Color Separation page 5 29 In this example color separation is performed by using the Multi Band Color Separation window You can also use the color separation window see Individual Band Color Separation on page 5 31 or a previously saved tem plate see Multi Band Color Separation on page 5 41 You must select a lane containing at least one non overlapping medium intensity representative band of each color Select Blend as the display mode 2 Review the Multi Band Separation parameters Click the Preferences button q or choose Preferences from the 1D Gel menu then click the Multi Band Color Separation tab 3 Try these settings and if necessary adjust them for different gels 6 22 Overlap 0 0 mm Band Area 0 1 mm Background Area 1 0 5 mm Background Area 2 0 5 mm Background 100 FMBIO 1D Gel Analysis Preferences Click the Lane Selection Tool button al or choose 1D Gel gt Tool gt Lane Selection Find a lane that contains a minimum of one band of each color Po
64. T TT TT TT TT 1 2 3 4 5 7 767076767 4 5 16 17 ye 100 M Copy gt gt Merge Setting Cancel In addition Band 2 is actually two overlapping bands so click Band 2 to select it and then click the Overlap button If this Multi Band Separation Marker is to be used in the future i e Controls such as K562 99474 etc click the Template button and assign the template a unique name Once the band colors have been verified click OK The Color Separation window is displayed FMBIO 1D Gel Analysis 14 Click OK to perform the color separation Image Analysis creates an image for each channel that displays only the signal from a single dye Set Lanes page 5 50 1 Click the Multiple Lane Selection Tool button E or choose 1D Gel gt Tool gt Multiple Lane Selection 2 Create a rectangle surrounding the group Position the tool above the top migration line at the corner of the first lane in a sequence of lanes 3 Drag the tool below the bottom migration line to the opposite corner of the last lane in the sequence 4 Release the mouse The Multiple Lane Setting dialog box is displayed Multiple Lane Setting x Options Iv Check Regularity on Lanes C Manual Input Number of Lanes 1 255 Lane Width Avito Determined Weer Detned EG 9 mm Cancel 5 Choose either Automatic Lane Detection and Fitting or Manual Input If you choose Automatic Lane Detectio
65. UTION Accumulation of dust in the interior of the scanning unit has been known to cause fires Ask your service representative to conduct regular maintenance at least once a year Service In the event that fire flood earthquake or other adverse environmental inci dent affect the instrument contact your Hitachi Software Engineering repre sentative for inspection and service Do not attempt to reinstall relocate or adjust the FMBIO instrument The FMBIO unit should be serviced annually by Hitachi Software Engineer ing service personnel 9 1 Maintenance FMBIO Laser Head Never attempt to replace the laser head or make any other mechanical repairs or adjustments to the equipment Photomultiplier Tube The photomultiplier tube settings are carefully adjusted before shipping Do not attempt to change or adjust the photomultiplier tube settings If any defective operation occurs such as a noticeable decrease in signal power contact a Hitachi Software Engineering service representative Operating Temperature Do not use the scanning unit in a location where the surrounding air temper ature exceeds 30 C 86 F At ambient room temperatures greater than 30 C the scanning unit may fail to emit the laser beam making it impossible to produce an image Should the laser fail due to excessive room tempera ture place the power switch in the OFF position and allow the scanning unit to cool If the room temperature is close to 30
66. acintosh startup 10 5 Windows startup 10 3 STaR Call menu 10 9 STaR Call installation Macintosh version 10 3 Windows version 10 2 STaR Call results 10 21 starting ReadImage 4 2 scan 4 18 scanning unit 3 2 STaR Call 10 3 10 5 STR cutoff 10 10 import 10 17 Lookup table 10 10 10 11 10 13 10 18 stutter band cutoff range 10 17 percentage 10 17 reporting 10 12 10 12 FMBIO Index summary worksheet 10 17 switch Main 3 2 Power 3 2 T table dyes and stains 1 8 parameter names 4 7 technology 10 23 temperature operating 9 2 template 5 23 gray level adjustment 5 23 marker 5 72 Text Attribute 8 2 time scan 3 13 tissue form 10 25 tissue type 10 25 TLC 4 7 Trace Overlay tools A 10 Trace Overlay window 5 87 Transparent Background 5 6 troubleshooting 9 3 to 9 4 U ungrouping spots and grids 7 10 V View menu 8 2 volume calculation 5 81 band 5 77 peak types 5 78 Z Zoom In 5 6 A 3 Index 7 Zoom Out 5 6 5 7 A 3 Zooming tool 5 7 A 3
67. age Software 15 Click Read Specify a file name and a location for the saved file Note You can terminate a scan before completion by clicking Cancel then save or discard the image data 16 Wait for the instrument to complete all of its scans The FMBIO will autofocus and might scan twice depending on the channels chosen After the scan begins a calculated time for completion is noted in the lower right of the Read Image Window This time is per scan and calculation accuracy improves as the scan progresses 17 To view the results use the Image Analysis software 18 Adjust the gray scale to optimize the image Note See Gray Level Adjustment on page 5 13 for information about gray scale adjustment 19 Ifthe image is satisfactory proceed to Chapter 5 20 Ifthe gray scale adjustment does not produce an adequate image make the adjustments suggested in the Image Troubleshooting Guide at the end of this chapter then rescan the image Table 4 1 Image Troubleshooting Guide Problem Suggested Adjustments Poor band definition increase repeats and or resolution adjust focussing point Too much signal lower the sensitivity Low signal e increase sensitivity adjust focusing point Grainy appearance increase sensitivity increase repeats e adjust focusing point High Background use low fluorescent glass destain gel or membrane adjust focusing point Read Image Software FMBIO Read Image Err
68. aining is highly recommended e Detection sensitivity is higher in acrylamide gels than in agarose gels due to lower background e Ethidium bromide can be used to detect both single and double stranded DNA or RNA The affinity for double stranded DNA is higher than for single stranded DNA or RNA e Longer staining times do not necessarily increase sensitivity e Shortening the ethidium bromide staining time or lengthening the water destaining time may decrease detection sensitivity e Keep all solutions containing ethidium bromide covered by aluminum foil to prevent bleaching of the dye by ambient light e Ethidium bromide is a powerful mutagen Follow usage precautions as described on the MSDS 4 24 FMBIO Read Image Software Multi Color Images Multi wavelength analysis makes it possible to create and analyze signals from multi color gel images Two or more fluorophores of different emission spectra can be run in the same lanes or on the same gel This function is useful for comparative allele analysis or for highly accurate size determi nation by running a marker in the same lane with a sample To scan material with more than one emission wavelength requires that you scan with multiple channels with the appropriate filters in place Multi color analysis depends on the use of appropriate filters Each filter captures one emission wavelength by screening out the others To load filters see page 3 5 Long Pass Filters
69. alog box is displayed 2 Select the STR Lookup Table in the Edit or Delete Lookup Table list box 3 Click the Edit button To delete a STR Lookup table 1 In the STaR Call menu choose STR Lookup Table The STR Lookup Table dialog box is displayed 2 Select the STR Lookup Table in the Edit or Delete Lookup Table list 10 13 Allele Calling with STaR Call FMBIO box 3 Click the Delete button Importing STR files The Import STR function converts an Image Analysis generated table to a STaR Call table format It removes all the unnecessary columns such as the migration distance and Rf columns and adds columns for allele scoring It also adds rows for CODIS export information as follows Row Number Description 1 The Lane name generated by Image Analysis 2 Specimen Number 3 Sample ID 4 Specimen Category 5 Tissue Type 6 Tissue Form 7 Population Group 8 Include Exclude lane 9 Sub Headings 10 Start of bp and Genotype values OD amp Percentages Actual values are used when exporting to CODIS To import an STR file 1 Inthe STaR Call menu choose Import STR Note Always use the Import command to open the Image Analysis results file in STaR Call Errors occur when you use the Open command to open a saved results file 2 Inthe Import STR dialog box click the Browse button 10 14 Oy re as FMBIO Allele Calling with STaR Call STaR Call Import STR zix C HitachiSoft STaR
70. alysis automatically creates a standard curve using the base pair markers as a reference 1 Click the Open BP Curve button or choose Open BP Curve from the 1D Gel menu The curve is fitted to the base pair values Y axis versus Rf X axis Image Analysis Tools FMBIO inlane 4CH You can also choose to use the Least Squares curve fitting 1 Click the Preferences button S or choose Preferences from the 1D Gel menu then click the Calculation tab 2 Click on the Use Lease Squares Method for BP calculation option to 5 74 FMBIO Image Analysis Tools place a checkmark in the box Preferences Channel 1 3 Click OK The calculation is performed automatically 4 Click the Open BP Curve button or choose Open BP Curve from the 1D Gel menu In addition to the base pair curve the correlation Image Analysis Tools FMBIO coefficient is displayed 8 inlane 4CH 79E0 5 76 FMBIO Image Analysis Tools Calculating Volumes and Peak Heights Image Analysis automatically calculates the volume IOD and or peak height OD of each designated band A standard curve is created based on the marker IOD values These reference values are used to determine the concentration of each sample band Once the peak area is defined Image Analysis multiplies the area by the number of grayscale bits in that area to determine the volume Note You cannot compare volume calculations until you have
71. alyzing images The software features analysis functions that include automatic band detection to facilitate data processing quantitation of peak height or peak area and band sizing through comparison to size standards With fluorescent dyes for sample labeling and the FMBIO software the system can analyze e Gels 1D and 2D Membranes Southern Northern Western dot slot TLC low to medium density arrays Multi wavelength analysis makes it possible to create and analyze multi color signal images You can run fluorophores of multiple spectra in the same lanes and simultaneously scan their emission signals This function is useful for comparative allele analysis or when a marker of known size is added to the lane for highly accurate size determination FMBIO software contains spatial analysis functions that accurately estimate fragment migration distances It also contains signal intensity analysis func tions so that it can perform densitometry to estimate band or spot intensity Fluorescent Labels With the FMBIO you can select from a wide range of fluorophores from rhodamine red to fluorescein green and including tetramethylrhodamine X rhodamine beta phycoerythrin ethidium bromide propidium iodide Because of the powerful intensity of the FMBIO laser you can use fluoro phores with emission wavelengths closer to the excitation wavelength FMBIO Table 1 2 shows a list of dyes and stains that can be used with th
72. an Define the type of material being scanned and the scan area e Set scan resolution dpi and number of repeats e Adjust PMT sensitivity e Adjust cutoff thresholds for background and signal e Adjust the focusing point e Set and save an autofocus routine e Add comments to be recorded and saved with the scanned image e Create and save customized scanning preferences The procedures discussed in this chapter can be used with all types of scan nable materials An example which illustrates some of the procedures begins on page 4 27 It is followed by a troubleshooting guide on page 4 31 and a list of error codes on page 4 32 4 1 Read Image Software FMBIO Opening Read Image Read Image provides default scan settings for four types of scannable material agarose gels acrylamide gels membranes and TLC thin layer chromatography You can modify the default settings and save the changes as a custom file e To start the Read Image program double click the Read Image icon When the program opens an untitled Scan Control window is displayed i Untitled FHBIO2 Readlmage eT Use this window to do the following Pre read the scan object e Define the scan area 4 2 FMBIO Read Image Software e Read the scan object Provide a unique name for the image e Enter comments For more information about this window see page 4 15 Read Image Files You can save all the Read Image parameters in a file When you open a
73. angle or oval around each spot of interest a a Modifying the boundary of a spot To move a boundary 1 Click the Select Tool button ki or choose Array gt Tool gt Select 2 Place the cursor inside the boundary until it transforms into a crosshair then drag the crosshair to the desired location To enlarge or compress a boundary l Click the Select Tool button ki or choose Array gt Tool gt Select 2 Place the cursor over a transformation handle initially the upper left or lower right handle and then drag the cursor until you have the desired shape 7 5 Array Analysis FMBIO To rotate a boundary 1 Click the Select Tool button ki or choose Array gt Tool gt Select 2 Place the cursor over a rotation handle initially an upper right or lower left handle and then drag the cursor about the midpoint Lr Duplicating a spot boundary You can duplicate a spot boundary by using the Copy and Paste function in the Edit menu Creating a Grid To create a grid over a group of spots 1 Click the Preferences button q or choose Preferences from the Array menu 2 Inthe Grid Option filecard enter the number of cells in the H hori zontal and V vertical direction By default the grid dimensions are 12 horizontal by 8 vertical boxes 7 6 FMBIO Array Analysis Preferences If you entered 1 as the number of cells horizontally enter a value in the cell width field If you entered
74. anning needs Important Ifthe FMBIO scanning unit has begun a scan or if the unit is in cleaning mode the optical unit does not return to home position when you click the Pause button You cannot change filters when the scanning unit is not in home position See Table 1 2 on page 1 8 for filter information 1 To stop the scan click the Pause button 2 A dialog box opens to display three options Save Don t Save and Cancel e To stop scanning return the optical unit to home position and save the scan data click Save Continue to step 3 e To stop scanning and discard the scan data click Don t Save and continue to step 3 e To continue scanning click Cancel to close the dialog box then click Resume 3 5 Operating the Scanning Unit FMBIO 3 Inthe FMBIO menu turn off Autofocus See the directions for setting an autofocus on page 4 13 4 Replace the 605 nm filter and restart the scan See the directions for changing the optical filters below To insert or change filters 1 Choose Eject Filter in the FMBIO menu 2 Open the front panel then open the optical filter service door Optical filter service door 3 Pull out the appropriate filter holder The filter holder in the top position holds filters for Channel 1 and Channel 3 The filter holder in the bottom position holds filters for Channel 2 and Channel 4 4 Hold the filter holder in your left hand so that the arrow on the edge faces you and
75. ate then click the Apply button 4 Click Close 5 Click OK The selected Color Separation template is applied to the new image Modifying Color Separation Results On some gel images the color separation process can result in over subtrac tion of a dye that has minimal bleed through signal For example a gel image might contain three dyes tetramethyl rhodamine carboxy x rhodamine and fluorescein The two rhodamine derivatives have overlapping emission curves but fluorescein has minimal overlap with the other two dyes The following procedure explains how to exclude the non overlapping dye fluorescein from the color separation process 1 In the Image Setting dialog box turn off the Show color separated images option When the option is off the checkbox is empty 2 Click the Color Separation button or choose Color Separation from the Image menu 3 Choose one of the overlapping dyes carboxy x rhodamine from the Selected Fluorophore drop down list 4 Inthe Target Channel section enter a zero 0 in both the Background and Percent entry fields for the fluorescein channel 5 Inthe Mode section click Preview to examine the new values in the Whole Image window and Zoom window You may see a slight color 5 34 FMBIO Image Analysis Tools change In the Selected Fluorophore list use the drop down list to choose the second overlapping dye tetramethyl rhodamine Enter a zero 0 in both the Back
76. autobanded the lane click the Multi Band Color Separation button 5 or choose Multi Band Color Separation from the 1D Gel menu Using the Band Merging param eters Image Analysis assigns a color to each band and the Multi Band Color Separation window is displayed Lane NENNEN Parameter set Tem Lane Selector mimmm mimm No Status NOOO ER ON Lane Window L g Magnification 100 Copy Merge Setting Selector Carce Image Analysis Tools FMBIO Lane Window The Lane window displays all detected bands and a spec trum trace of the selected lane The color of the Detected Band number indicates the color assigned by Image Analysis Lane Image Lane Trace Detected Band Number Band Area LL Background Area Marker Magnification Selector To view the Lane window at a higher magnifica tion select a percent value from the drop down list box 5 If you are not satisfied with the separation of the colors you can adjust the Multi Band Color Separation preferences Click the Set Multi Band Parameters button and change the parameter values When you click OK the colors are automatically separated according to the new values 5 46 FMBIO Image Analysis Tools 6 On the right side of the window the Status Edit Sheet displays the detected band numbers e Ifyou want to use the colors assigned by the Multi Band Color Separator click the Copy
77. ave insufficient memory you may get various error messages Many error messages occur due to memory allocation problems Allele Calling with STaR Call FMBIO Installing STaR Call Note To install and use the STaR Call Software you must have Excel loaded on your computer If Excel is not already installed on your hard drive install it before attempting to install STaR Call 1 Ifyou have just installed Microsoft Office or Excel run Excel once to allow it to complete some automatic configuration steps 2 Make sure that Excel is closed 3 Insert the STaR Call disk into the drive 4 Select the folder corresponding to the version of Excel that is installed either Excel 5 or Excel 98 5 According to which version of Excel you have e Ifyou have Excel 5 installed double click the INSTALL xla file e Ifyou have Excel 98 installed double click the install xla file 6 Ifa dialog box appears asking you if you want to enable or disable macros click the Enable Macros button The STaR Call Installer dialog box is displayed 7 Click the Install button If you have previously installed STaR Call a window will be displayed asking if you would like to replace the codisdbf xls file e Click the Yes button if you would like to replace it a copy of the old file will be stored in a backup folder located in the Excel startup folder e Click the No button if you want to keep the old version of codisdbf xls file and continue installi
78. aw pre color separated image Flips image horizontally or vertically FMBIO Image Analysis Tools Parameter Description BMP Format Save the image only or include other items such as band and lane markers 3 Select the appropriate parameter settings and click OK The Save As dialog box is displayed 4 Specify a location for the file in the Directory window and enter a filename 5 Click the Save button Viewing a Multi Color Image A multi color image is a combination of two or more scanned image chan nels that share the same scan area and orientation FMBIO Read Image automatically creates a multi color image file whenever two or more chan nels are active during a scan When Image Analysis opens the image file it displays the combined image channels as one multi color image 1 Click the folder expansion h symbol next to the image icon to display the names of each layer in the image PPlex11 m x E3 Project cium BB CTTv 3CH EB DDDD 2CH EB inlane 4CH 2 Inthis example during one multi color scan CTTv DDDD and Inlane CXR were the active channels 5 9 Image Analysis Tools FMBIO Image Setting Dialog Box You use the Image Setting dialog box to change how the image is displayed on the screen e By default the Image Setting dialog box is displayed If it is not dis played click the Show mage Setting Dialog button or choose Show Image Setting Dialog from the
79. ays Marker Setting window Volume Calculation Performs a volume calculation Open Spreadsheet Displays spreadsheet Open BP Standard Curve Displays standard curve Quantification Setting Displays Quantification Setting window Preferences Displays Preferences window Multi Band Displays Multi Band Color Separation Color Separation window 5 38 FMBIO Image Analysis Tools Lane Selection Tool Multiple Lane Selection Tool Lane Template Automatic Band Detection Band Edit he SEa me e s xx Delete Band Automatic Lane Fitting Lane Alignment Select Tool Tool Description Select Tool Selects objects Lane Selection Tool Defines a single lane Multiple Lane Selection Defines multiple adjacent lanes Lane Alignment Tool Aligns lanes with migration lines Automatic Lane Fitting Automatically adjusts lane boundaries Lane Template Creates a template for lane definition Automatic Band Detection Detects selected bands automatically Delete Band Delete all bands in the selected lanes Band Edit Add edit or delete bands 5 39 Image Analysis Tools FMBIO Show Hide Lanes Show Hide Band Information Show Hide Comment sp a Band Style Lane Style Tool Description Show Hide Lanes Toggles display of lane boundary lines Show Hide Toggles display of band information e g Band Information OD MM Bp value Show Hide Toggles display of label information Comments entered in spreadsheet La
80. ble for any damage that occurs as a result of mistakes or omissions in this manual The new duty of guarantee shall not be appended by advise provided by the staff or the representative of Hitachi Software Engineering Co Ltd Hitachi Software Engineering Co Ltd and the manufacturer shall not in any case be liable for the nonperformance of agreement disobedience of guarantee fault responsibility for strict liability special indirect consequential or other similar damages in law These damages include the loss of a profit the loss or revenue the exchange expenses of the instruments the equipment and the services the period of failure the period of the customer the claim by the third party with the customer and the sufferer included the infringement of the property Guarantee For the period of ONE YEAR Hitachi Software Engineering Co Ltd guarantees that there is no defect in the quality of the material and the production of the equipment While the guarantee is valid if the defect or the failure occurs in the equipment Hitachi Software Engineering Co Ltd may repair the equipment If this happens please contact Hitachi Software Engineering service representative while the guarantee is valid The customers shall not make any representative actions and or any guarantees through the manufacturer Hitachi Software Engineering Co Ltd and its licenser The guarantee may apply only in proper use may not apply to the following
81. ble programs as well as specific commands for oper ating the FMBIO scanning unit modifying scan settings and opening the Image Analysis software These additional features are discussed in this section of the manual File Menu File Edit Command FMBIC New Ctrl N Open Ctrl O Close Ctre Save Ctrl S Save As 1 ch prol fb2 Exit Ctl O Most of the commands in the File menu are standard Windows commands New Opens a new Read Image file Open Opens a saved Read Image file Close Closes the active Read Image file Saving Read Image Files Two commands in the File menu provide a way to save Read Image files Save and Save As You can use these commands to assign a unique name to a unique collection of settings that you plan to use repeatedly Recent Files Files that have been opened recently are listed Choose a file to open it Exit Closes only the Read Image software 4 20 FMBIO Read Image Software Command Menu Command FMBIO II Help Read PreRead Set Parameter Set GrayLevel amp djustment Imagerie DE y Orientation The Command menu contains commands to do the following e correspond to buttons on the Scan Control window e modify settings that appear in text on the Scan Control window See page 4 15 for information about the Scan Control window Read Corresponds to the Read button on the Scan Control window PreRead Corresponds to the PreRead button on the Scan
82. bs adjust the lines to encompass only the sample bands Other labs set the markers above the top band in the internal ladder To adjust the migration lines 1 Click on a migration line Each migration line has a handle at each end 5 58 FMBIO Image Analysis Tools Handle Handle we Click the left or right handle and drag it to change the angle of the migration line with respect to the migration direction axis of the gel This is useful for gels that are misaligned as in pulse field gels where migration paths may deviate from the gel axis Click on any other point along the migration line and drag it up and down to move the migration line perpendicular to the gel Image Analysis Tools FMBIO Defining Bands Once lane boundaries are defined you can define bands in each lane Use the Automatic Band Detection Auto Band tool to let Image Analysis automati cally assign bands You can also use the Band Edit tool to manually define bands The Project window lists all defined bands in each channel PPlex111_prj GI x Project 1D Gel Document CTTv 3CH a g Band 1 Band2 Band 3 Band 4 Band 5 Band 6 Band 7 Band 8 Band 9 Band 10 Bandi Automatic Band Detection Band recognition depends on four parameters Gradient Start Gradient End Duration and Noise Level Image Analysis can automatically detect bands by calculating the linear differential c
83. ce and record keeping purposes much information is necessarily left out of images printed on such machines Always remember that images printed on such machines are schematics that do not faithfully represent all the information available in the digital image in the FMBIO file 2 5 Installation FMBIO High quality images that more faithfully represent in hard copy the information present in the FMBIO image file can be created using a high quality image printer Image printer capability should include 256 grayscale 3 color dye sublimation and 300 dpi or higher Peripheral Storage Devices A single digital image file may be 10 MB or larger To conserve hard drive space you may store files on a peripheral storage device 2 6 3 Operating the Scanning Unit This chapter describes the steps you should follow to operate the scanning unit These steps are summarized below Start Scanner software See page 3 2 2 Turn on power to the FMBIO scanning unit and wait for the self diag nosis routine to finish See page 3 2 3 Check that the 605 nm filter is in Channel 1 for autofocusing See page 3 5 4 Clean the sample plates and scanner sample stage See page 3 8 5 Load the sample on the FMBIO sample stage See page 3 10 6 Choose the appropriate parameter set in the Scan Parameter dialog box and if necessary adjust the scanning parameters See Scanner Software 7 Use Scanner software to begin scanning the sample A scan may
84. channel Since the instrument will always autofocus before the first read after power up turn Auto Focus off in the FMBIO menu to save time during subsequent scans The 605 nm filter must be installed in Channel 1 for autofocusing In the Command menu set the gray level adjustment for polyacrylamide gels Examples might be 50 for Low and 1 for High Make any changes needed to Orientation in the Command menu To save the Read Image parameters for future scans choose Save from the File menu assign a filename and location and then click Save Double click on the saved parameter file to launch the Read Image program and return the user to the saved scanning parameters 4 29 Read Image Software FMBIO i ch4pro1 fb2 FHBIO2 Readlmage Blood Stain 2987934UCO Ea Convicted Offender sample 12 Add any comments to an image file by typing those comments in the text box in the lower section of the Scan Control window 13 Click the PreRead button to quickly identify the gel area to scan Note If a small scanning box is already set from a previous scan click on the All Area button before prereading If you need to terminate an in progress prereading click Cancel 14 Designate the scan area by dragging with the mouse from top left to lower right in the viewing window Note DO NOT include SPACERS or other solid objects in this box because they can distort the gray scale balance of the image 4 30 FMBIO Read Im
85. channels as active during the scan FMBIO Read Image creates a multi color image In Image Analysis you create a project to analyze your 1D or array experi ments You can create a project for a single or multi color image You can also display images and data from more than one project simultaneously E InageAnalysis New pri Eie Edt Vie Broject window Help ese ee aaee ele ef es e t wed e este ted pe Tv ferte bc T d entr ns fre EET E Display mode Ema Te Show eolerepreraten images rie rige rec rae C N Z Ready I I dst A SE United Bl0 Resim Ej imagenalsis New pri Pama ma 5 1 Image Analysis Tools FMBIO You can view all the colors in the image simultaneously or select the layers you want to view You can assign a color to each layer in the project and modify the intensity of each color or display all layers in gray scale This chapter describes the tools you can use to modify and analyze the scanned image A22 C x 1D Gel Document li CTTv 3CH xj DDDD 2CH Channel xfi inlane 4CH Lane amp Band Data H Overlay Trace oof CTTv 3CH eK DDDD 2CH Standard Curves tj inlane 4CH E Spread Sheet 3 PPlex11 li CTTv 3CH Channel ff DDDD 2CH inlane 4CH Image Data Note While the following chapter describes each of the analysis tools in the order of use it is not meant to serve as a procedural guide Please refer to Chapter 6 D Gel A
86. ck the Close button Note When a Gray level Adjustment Template is created only the mapping type high signal cutoff threshold value and back ground cutoff threshold value are saved The percent values are not saved Therefore it is imperative that a Gray Level Adjustment template is created for each channel To apply a gray level adjustment template 1 Selecta channel 2 Click the Template button 3 Select the template name then click the Apply button Image Analysis Tools FMBIO Image Diagnostic Guide Image Quality Cutoff threshold Entire image is too dark Entire image is too faint Image signal blurred with black Image background is blank Image is chaotic and unclear due to noise Image and signals unclear Image has too much contrast Scan shows spacers only Increase background and high signal thresholds Decrease background and high signal thresholds Increase high signal threshold Decrease background threshold Decrease background threshold and increase high signal threshold Increase background threshold and decrease high signal threshold If the value settings are too close together then you may be spreading your 256 shades of gray across too small an area Try using the cursor to draw a box that encloses a dark band If highly fluorescent objects such as spacers combs or gel thermometers are included in the image the High Signal value will be set too high
87. color for the selected band None No color is assigned Does not use band for multi band color separation Overlap Overlapped band Does not use band for multi band color separation Not Band Band is not listed in template Color list is shifted down Skip The Detected Band numbering skips the band Appendix A Trace Overlay Tools Zoom In Vertically Zoom Out Vertically bib 2 a Overlay Trace Setting Drawing Tool Tool Description Zoom In Vertically Magnify in vertical direction Zoom Out Vertically Reduce in vertical direction Drawing Use drawing tools for Overlay Trace window Overlay Trace Setting Displays Overlay Trace Setting window Appendix A Array Tools Drawing Tool Select Tool Show Hide Spot Information Show Hide Comment Create Grid Spot Create Oval Spot Create Rectangle Spot Tool Description Drawing Use to draw text and graphics Select Select objects Create Rectangle Spot Define a rectangular shaped spot Create Oval Spot Define an oval shaped spot Create Grid Spot Define a grid over an array of spots Show Hide Toggle display of spot information Spot Information Show Hide Comment Toggle display of label information Appendix A Set Selected Spots to Background Set Selected Spots to Marker Calculation Group Ungroup BIMA GIF si FS Halsa Set Grid Marker Preferences Open Spreadsheet Open Standard Curve Command
88. data is collected Once the image file has been saved you can use Image Analysis software to further experiment with the high and low cutoff thresholds 1 Choose Gray Level Adjustment in the Command menu Command FMBIO II Help Read PreRead Set Parameter Set GrayLevel amp djustment Image Rie DE gt Orientation 2 Enter cutoff threshold values in the fields labeled Low Background and High Signal 4 11 Read Image Software F MBIO Gray Level Adjustment Eo mm 3 Click OK to save the Gray Level Adjustment settings Defining Image Orientation The viewing orientation of the image can be changed in the saved file with the Image Analysis software e Choose Command gt Orientation then choose an orientation 4 12 FMBIO Read Image Software Settings in the FMBIO Menu FMBIO I Help Auto Focus v 1 Channel v 2Channel v 3Channel 4 Channel Do Cleaning Eject Filter The FMBIO menu contains these commands e Requesting an Autofocus before each scan e Determining which channels are active during a scan e Preparing the FMBIO scanning unit for cleaning e Ejecting the filter Performing an Autofocus During an autofocus Read Image adjusts the focusing mirror and other signal tracking and processing elements to find the best optical alignment This procedure takes approximately thirty seconds After the initial auto focus the procedure does not need
89. e FMBIO along with recommended filters Almost all molecular species absorb and then re emit light under certain conditions giving a broad emission spectrum up to 100 nm wide in con densed media When the emission process the excited state takes place at the nanosecond or a slower time scale the phenomenon is called fluorescence FMBIO Table 1 2 Selected Dyes and Stains Detected with the FMBIO Use these filters for maximum sensitivity Dye or Stain Excitation Emission Filter Vendor nm nm nm Protein Stains Sypro Orange 472 570 585 Molecular Probes Sypro Red 530 625 625 Molecular Probes DNA Post Stains Ethidium Bromide 300 511 605 A reliable source Sybr Green I 490 520 505 Molecular Probes High Sensitivity 532 605 605 Molecular Probes Dye Labels FITC Fluorescein 490 520 505 A reliable source FAM 495 535 505 Perkin Elmer JOE 526 548 585 Perkin Elmer HEX 529 560 585 Perkin Elmer TAMRA 559 578 585 Perkin Elmer Tetramethyl rhodamine 546 572 585 605 Molecular Probes Rhodamine X 574 595 605 Molecular Probes Texas red 578 602 605 Molecular Probes ROX 580 605 605 Perkin Elmer Bodipy 563 602 605 Molecular Probes POPRO3 270 593 567 585 Molecular Probes BOPRO3 271 575 600 605 Molecular Probes BOBO3 245 572 602 605 Molecular Probes 1 8 FMBI
90. e Setting dialog box is displayed Image Analysis Tools FMBIO Multiple Lane Setting a Auto aiia User Dennen WET D Jj 5 Choose either Automatic Lane Detection and Fitting or Manual Input If you choose Automatic Lane Detection you can also choose to check the uniformity of the lanes Note We do not advise using automatic lane detection on lanes that are not clearly defined such as lanes produced by sharks tooth combs If you choose Manual Input enter the number of lanes in the sequence then choose to either automatically determine the lane width or specify a width Lane width may range from 1 0 to 100 0 mm in 0 1 mm increments 6 Click OK By default Image Analysis displays a central line down each defined lane with additional lines marking the lane width When the lane is selected square edit handles are displayed on the lane lines 5 52 FMBIO Image Analysis Tools 072501lowmassladderethFMBIOI s E3 B 1D Gel Experiment 605 585 505 625 E 605 H Al Snead Sheet The Project window lists the lanes defined for each channel Displaying Lane Lines spe 0 The Lane Style button Mal toggles among different display options center line with lane borders center lines only lane boundaries only and lane display off You can also choose lane styles from the 1D Gel menu Editing a Lane Boundary You can change
91. e Tool Oval Tool Polygon Tool Text Tool Paper Position Text Tool Polygon Tool Oval Tool Select a figure To select multiple figures click while pressing the Shift key Draws lines Draws rectangles Draws an ellipse rectangle with rounded corners Draws ovals Any shape bordered by straight lines Click to establish the point for the beginning of each straight line Double click at the end of the last point Drag to create a text box and then type text Set the width of the top bottom left and right margins around the image 8 7 Drawing Tools FMBIO Tool Draw 2 Function Bring Forward Send Backward Lj Lig Eas Eha Send to Back Bring to Front Bring Forward Moves selected figure forward one layer Send Backward Moves selected figure back one layer Bring to Front Brings selected figure to top layer Send to Back Sends selected figure to back layer Align Left Align Vert Center Align Right eal Align Left Aligns selected figures left Align Vert Center Centers selected figures vertically Align Right Aligns selected figures right FMBIO Drawing Tools Tool Draw 2 Function Align Top Align Horiz Center Align Bottom Lock Unlock Edit Polygon Align Top Align Horiz Center Align Bottom m oe Aligns selected figures along top edges Centers selected figures horizontally Aligns selected figures along bottom edges Lock Unlock
92. e back ground on either side of the selected peak This gives the largest value of the four calculation types Use Type 1 for bands or spots that are distinctly sepa rated from others or for quantifying isolated faint bands with signal intensities that are close to background value Type 2 The area incorporates any signal that is greater than the spectrum valleys on either side of the designated peak The value of the deeper of the two valleys on either side of the peak is used as the local background value Use Type 2 to quantify individual bands or spots that are packed in with others in a group 5 78 FMBIO Image Analysis Tools Type 3 The peak is split and the lower of the two valleys is defined as the local background value The area is defined as half of the peak between the highest and the lowest signal Use Type 3 volume to quantify border regions of spots or bands where the signal fades into background Type 4 Local background is defined by a line that connects the spectrum valleys at their lowest point on either side of the peak The area incorporates signal above this line and below the peak Use Type 4 calculation to deter mine relative volumes of adjacent bands or spots that are packed in with others in a group Setting Quantification Markers Click the Quantification Setting button E or choose Quantification Setting from the 1D Gel menu Quantification Setting x Standard Curve X Axis
93. e pixel number To obtain greater clarity of an entire image or certain selected bands within it you can modify the range of gray levels in each channel In the Gray Level Adjustment window you can use a histogram to adjust the cutoff threshold values Alternatively you can designate reference background and high signal areas on the scanned image Using these reference areas Image Analysis automatically adjusts all gray levels on the image By adjusting the cutoff thresholds you are adjusting the range between the highest and the lowest acceptable gray shades When the high signal cutoff is too low dark band images may be blurry with indistinct edges When the background cutoff is too high faint images are lost Because signal intensities can vary greatly from one scan to the next it is difficult to assign default values for high signal and background cutoff thresholds Images that contain only faint bands against a clean background can be enhanced with high signal and background cutoff thresholds set to relatively high values Images that contain heavily stained or optically dense regions may suffer substantial loss of content even with low threshold values The diagnostic guide on page 5 24 provides guidelines for threshold adjust ment See also Imaging Basics on page 4 23 5 14 FMBIO Image Analysis Tools Gray Scale Adjustment Options juawysnipy 8487 Aes 10 sanjea ajejduia ayy asn op uojng ejyejduia 4 eu xou5
94. e software See page 4 13 for more information After the initial autofocus routine you may stop the scan and replace the 605 nm filter with a filter of a different wavelength See Autofocus Calibration on page 3 4 for more information Stopping a Scan When you stop a scan in progress you either save or delete the partially scanned image To stop a scan in progress click Pause 2 A dialog box offers three actions after you click Pause Save Don t Save and Cancel e Click Save to save scan data and end the scanning process e Click Don t Save to discard scan data and end the scanning process e Click Cancel to close the dialog box then click Resume to continue 3 12 FMBIO Operating the Scanning Unit the scanning process Scanning Guidelines Following is a list of guidelines for use when scanning with FMBIO Read Image Auto Focus After the FMBIO is turned on it performs an autofocus before it begins the first scan The 605 nm filter must be in Channel position although Channel 1 does not have to be active during the autofocus PreRead Use PreRead to identify the area of interest on the sample and to make sure that spacers and other reflective items are not included in the scan area Scan area The rectangle on the Scan Control window represents the total possible scan area You may specify a smaller rectangle within the total scan area to represent a pre defined scan area By default Read Image displays the
95. ect a straight line or logarithmic X axis scale 10 Click OK 11 Click the Open Standard Curve button or choose Open Standard Curve from the 1D Gel menu The curve is fitted to the marker IOD values Y axis versus concentration X axis 5 80 FMBIO Image Analysis Tools y 1 41833E7Log CO 1 57743E7 aoi 0 0E0 1 0E1 2 0E1 3 0E1 4 DE1 Concentration Volume Calculation After the lanes bands and markers have been established Image Analysis can compare the known markers to unknown sample bands Using the markers as reference points the volume IOD concentration and fragment length of the sample bands is calculated e To initiate the analysis click the Volume Calculation button Es or choose Volume Calculation from the 1D Gel menu Displaying Results You can display the results of the calculation in a spreadsheet and on the image To choose the variables to display ick the Preferences button or choose Preferences from the 1D 1 Click the Prefi b h Pref fi he ID Image Analysis Tools FMBIO Gel menu then click the Band Information tab Preferences FMBIO Image Analysis Tools Variable Contents mm Migration distance between the top migration line and band peak line OD Optical density of the peak fluorescent signal on the band peak line IOD Amount of fluorescent signal in a band Band volume IOD Band volume as a percentage of the total volume of all bands in the lane
96. ed for Volume Calculation i N Band Area Per Area Parameter Detected Area Background Area 1 The area directly adjacent to either side of the band of interest that is used in the background calculation Background Area 2 The area between bands of interest that is used in the background calculation 5 43 Image Analysis Tools FMBIO r T Background Area 2 f N Wi DEAE N nd i tLe c Detected Area x TN Detected Area Backer und Area 1 Background Specifies the correction value for the calculated back ground value This Background value should range from 1 to 100 Multi Band Color Separation Window To use the multi band color separation window you must select a lane containing a minimum of one medium intensity band of each color 1 Find a portion of a lane containing a minimum of one non overlapping band of each color Note Two or more bands of each color is recommended 2 Click the Lane Selection Tool button t or choose 1D Gel gt Tool gt Lane Selection Drag down and then across that portion of the lane A Ml 3 Click the Automatic Band Detection button al or choose 1D Gel Function Auto Band Note Auto Band detects bands in all channels To view the detected bands in a channel select the channel in the Image Setting dialog box See Displaying Detected Bands on page 5 12 5 44 FMBIO Image Analysis Tools 4 After Image Analysis has
97. efined uw jen mm Cancel 5 Choose either Automatic Lane Detection and Fitting or Manual Input If you choose Automatic Lane Detection you can also choose to check the uniformity of the lanes If you choose Manual Input enter the number of lanes in the sequence then choose to either automatically determine the lane width or specify a width Lane width may range from 1 0 to 100 00 mm in 0 1 mm 6 Click OK By default Image Analysis displays a central line down each defined lane with additional lines making the lane width When the lane is selected square edit handles are displayed on the lane lines 7 If any of the lanes need adjustment click the lane to highlight it and move it to the correct position The Lane Style button toggles among different display options center line with lane borders center lines only 6 11 1D Gel Analysis FMBIO lane boundaries only and lane display off You can also choose lane styles from the 1D Gel menu 8 Enter the number of lanes and verify that Auto Lane Width is selected Click OK Image Analysis creates the lanes automatically 9 If any of the lanes need adjustment click the lane to highlight it and move it to the correct position The Lane Style button l toggles among different display options center line with lane borders center lines only lane boundaries only and lane display off You can also choose lane styles from the 1D Gel menu Set Migration L
98. ent Color Separation v Show Image Setting Dialog Get Information Save Image as Transparent Background Line Selection Area Selection Show spectrum Gray Level Adjustment Color Separation Damla Image Information Show Image Setting Dialog Command Description Gray Level Adjustment See Gray Level Adjustment on page 5 13 Color Separation See Color Separation on page 5 29 Show Image See Image Setting Dialog Box on page 5 Setting Dialog 10 Image Information View sample image and scanning infor mation from the FMBIO Read Image file Save Image As Save an image or selected area as a TIFF or BMP file See Saving an Image on page 5 7 5 5 Image Analysis Tools FMBIO Command Description Transparent Background See Transparent Background on page 5 35 Line Selection Draw a line to select a location on the image Area Selection Draw a rectangle to select an area on the image Show Spectrum See Spectrum on page 5 25 In addition you can use the Zoom tools to magnify or reduce the image Zoom Out Zooming Tool Zoom In Fit Window Moving Tool QQ o s m Magnification Input List Box Tool Description Zoom Out Each click reduces the image Zoom In Each click to enlarges the image Magnification Displays current magnification Enter the Input List Box desired magnification or select a magnifi cation from the drop down list FMBIO
99. eps used in the analysis of a single color image While this summary utilizes diagrams from a single color mul tiplex the process is also applicable to other types of single color images including gels stained with ethidium bromide Create a New Project page 5 3 1 In the File menu click the New Project button n or choose New Project A new Project window is displayed Projecti 2 x E3 2 Inthe Project menu choose Add Image then select a file that contains a digital image of a ID gel Image Analysis adds an icon representing the image file to the project tree 6 8 FMBIO 1D Gel Analysis Samplelmagel Iof x E Samplelmage1 The Image menu appears in the menu bar and its corresponding tool buttons become active Create 1D Gel analysis page 5 13 e Choose New 1D Gel Analysis from the Project menu Project Image Aray Mindo New 1D Gel Experiment New Array Experiment The 1D Gel Document window Image Setting dialog box 1D Gel menu and its corresponding tool bar appear Adjust Gray Level page 5 13 1 Click the Gray Level Adjustment button AJ or choose Gray Level Adjustment from the Image menu The Gray Level Adjustment window is displayed 6 9 1D Gel Analysis FMBIO EE e Equal Range e ind 7352 f 864 Channel Mapping Type Background High Signal 167 f 4256 i Template Histogram Show Selected Range
100. er version 10 22 10 23 message ID 10 22 message type 10 22 type 10 22 version 10 23 CODIS 10 10 10 12 preparing data for 10 21 specimen information 10 26 codisdbf xls 10 9 color separation excluding a channel 5 34 individual band 5 31 modifying results 5 34 Color Separation window 5 31 Command menu 4 10 to 4 12 4 21 comments 4 18 comparing analyses 10 17 10 20 creating grid 7 6 Medium Grid document 7 3 oval spot 7 5 rectangle spot 7 5 Index 2 creation date time 10 22 cutoff threshold 4 11 5 13 5 78 fields 5 16 5 23 histogram 5 17 D Display Mode 5 11 displaying spreadsheet 7 18 standard curve 7 15 dpi 4 8 Draw menu 8 2 8 2 objects 8 14 8 14 text attributes 8 13 tools 5 89 8 2 8 7 A 13 Draw 1 tools 8 2 8 7 Draw 2 tools 8 2 8 8 Draw 3 tools 8 2 8 10 Duration 5 62 dyes and stains table 1 8 E edge filter long pass 4 25 Edit menu 4 20 tools A 2 editing lane boundaries 5 53 eject filter 4 22 electrophoretic mobility 6 6 emission filter 4 25 error codes 4 32 detected 3 3 light 9 3 evaluating alleles 10 9 Excel 10 9 excluding lane 10 24 exporting image 5 7 spreadsheet 7 20 10 8 to CODIS 10 21 F File menu ReadImage 4 20 tools A 2 filter 605 nm 4 13 eject 4 22 emission 4 25 long pass 4 25 orange glass 4 25 red glass 4 25 table of dyes and stains 1 8 FMBIO II location requirements 2 2 menu 4 13 4 22 parameters 4 4 printer requirements 2 5 shipment package 2 1 shutdown 9
101. ere is a rectangle inside the total scan area it represents a pre defined scan area that is smaller than the total scan area Pre defined Scan Area a Total Scan Area Lo p180 T 20 B 200 L Scan Area Coordinates When you choose PreRead Read Image produces a low resolution ras terized image of the scan area in the PreRead Display You can then modify the scan area to include only essential information The total scan area is 200 mm x 430 mm The minimum scan area is 20 mm x 20 mm 4 17 Read Image Software FMBIO Note To reduce the file size of a scanned image avoid including the glass plate spacers and areas outside the sample in the scan area During a PreRead scan the optical unit moves under the scanning bed and a rasterized image begins to appear on the monitor To preview the entire scan area e Ifthe PreRead display is empty the All Area button is not active Click the PreRead button to preview the entire scan area e Ifthe PreRead display contains a smaller pre defined scan area the All Area button is active Click All Area to preview the entire scan area To preview a pre defined scan area Click the PreRead button to preview the pre defined scan area 2 To redefine the scan area place the cursor in the scan area and drag it to the area of interest You can change the size of the scan area by dragging the sides of the rectangle Adding Comments You may enter memo infor
102. es If you are using an internal standard such as Promega s CXR Fluorescent Ladder choose Layer and select the corresponding channel After the bands have been detected and edited the length of the fragments in each band can be calculated Mamea s Registering Markers To register markers in your gel l Click the Marker Setting button MI or choose 1D Gel gt Marker A Marker Setting dialog box is displayed The Marker Setting dialog box lists the defined lanes In brackets adjacent to each lane is the 5 68 FMBIO Image Analysis Tools number of defined bands Marker Setting Note This is a good time to again check the number of expected bands in each marker lane Compare the number to the actual number of defined bands in the lane 2 Select a channel and a marker mode from the drop down list boxes There are three marker modes Separate markers are selected for each channel The results based on markers for other channels are ignored Mix selected markers are used to calculate the results for all channels Layer markers are selected for each channel The calculated results for the channels are layered 3 Click on the lane numbers in the list that contain the same marker To select more than one lane hold down the Ctrl key then click each lane Image Analysis Tools FMBIO 4 Select the marker template from the Marker list 5 Click the Set Marker to gt gt button 6 Continue this proc
103. ese parameters may subject the pur chaser to waive any liabilities by Hitachi Software Engineering Co Ltd In addition any claims deemed to arise due to failure to thoroughly read this manual may result in the waiver of any liabilities by Hitachi Software Engi neering Co Ltd The following symbols are related to safety concerns Please ensure that you understand the meaning and implications of each symbol before continuing to read this manual WARNING This symbol indicates the possibility of loss of life or serious injury if you accidentally operate the equipment in ignorance of the information highlighted by this symbol CAUTION This symbol indicates the potential for personal injury or damage to equipment if the equipment is operated in ignorance of the information highlighted by this symbol Examples of Schematic Symbols Each symbol indicates the type of warning illustrated Cautions the user of a particular hazard in this case of potential electrical shock Q This symbol represents prohibited actions FMBIO WARNING Proper Handling Do Not Disassemble or Repair Please do not disassemble or attempt repairs Tampering with the S internal components risks exposure to such hazards as electrical shock and laser radiation Shield the Scanning Unit from Water Shield the Scanning Unit from water Never place objects con Q taining water or other fluids on the Scanning Unit A spill can lead to short circuits or e
104. eset to the original magnification Capture Image Copy the graph to the clipboard Pickup Spots in the Range Display the spot data in the Selected Spots spreadsheet 2 Click the Show Spot Label button m A label with the grid coor dinate appears on each spot FMBIO Array Analysis 3 Double click on a spot 120 000 000 eMf_ 115 000 000 T O KEUES 110 000 000 E Volumel a EN The Overlay View filecard is displayed and the spot is selected on the grid 4 To change the size of the spots click the down arrow next to the Spot Size button and choose the spot radius from the drop down menu of xd Data Sheet Overlay View Grid Comparison Scatter Plot Selected Spots m amp aaa 1 pixel Volume2 Array Analysis FMBIO 5 To magnify an area of the plot drag the cursor to form a rectangle enclosing the area of interest 6 Release the mouse button The plot is magnified so that the area occupies the entire graph 7 Click the Pickup Spots in the Range button The comparison data FMBIO Array Analysis for the spots is displayed in the Selected Spots spreadsheet olume1 olume2 Comment Comment2 98841774 71220894 Spot Spot 82048830 69511883 Spott3 Spott3 78543650 66485095 Spot25 Spot25 103445736 58398659 j Spot37 Spot37 104332741 68847142 Spotg Spot9 107071161 20745438 SpotB1 SpotB1 80043806
105. ess for each marker or marker group Note To deselect a marker lane remove the checkmark adjacent to its lane number Marker Setting fre reset 7 Click OK to complete the marker registration Importing and Exporting Markers You can import a set of markers including markers exported from the Macintosh FMBIO Analysis software or export a set of markers To import a marker set 1 Click the Import button The Open File dialog box is displayed 5 70 FMBIO Image Analysis Tools Open L2 x1 2 Select a marker set file and click the Open button The size markers are added to the BP Values list To export a set of markers 1 Click the Export button The Save As dialog box is displayed 2 Enter a name for the marker set and click Save Image Analysis Tools FMBIO Working with Marker Templates You can edit an existing marker add new markers duplicate rename or delete markers Adding Marker Templates To add new marker templates 1 Click the Add button The New Marker dialog box is displayed New Marker oR cance 2 Enter the name of the marker template then click OK 5 72 FMBIO Image Analysis Tools 3 Select the new marker then enter the length of the marker band in the BP field 4 Click Add The new band length appears in the BP list Delete 5 Repeat this process for each band in the new marker Base Pair Standard Curve Image An
106. ff peak and range bands displayed and only peak bands dis played 6 5 1D Gel Analysis F MBIO Analyzing 1D Gels With the tools and commands in the 1D Gel menu you can identify lanes and bands of interest Image Analysis then calculates the following band parameters mm migration distance of the band from the top limit line in millimeters OD optical density of the peak fluorescent signal relative to a standard marker IOD average band volume based on the fluorescent signal intensity IOD band volume as a percentage of the total volume of all bands in the lane Rf relative electrophoretic mobility bp estimated size of the fragment in basepairs DNA only concentration Summary of Analysis Steps Image Analysis of a 1D gel consists of these steps SOP oor ON Uv de ORS GENS m 10 11 6 6 Open project Create 1D Gel analysis Adjust gray level Set lanes Set migration lines Perform color separation if required Review image Autoband Set markers Calculate base pairs View results FMBIO 1D Gel Analysis Single Color Experiment Flowchart Open Project y Create 1D Gel Analysis N Adjust Gray Level H y Set Migration Lines v Autoband i Calculate Base Pairs H Export Spreadsheet to STaR Call 6 7 1D Gel Analysis F MBIO Single Color Experiment Procedure The following section is a summary of st
107. ge Analysis Tools amp 4 Whole Image window Zoom cursor amp Zoomed Area window Selection cursor Click the Set button to make the color of the selected band represent the fluorophore Image Analysis calculates the percent overlap between selected fluorophore and each of the other channels The percentages are displayed in the Emission Overlap fields e You can click the Preview button at any time to test the color separation process e Click the Original button to revert to the original color settings Use one of the other channels in the Selected Fluorophore list Repeat this process for each of the remaining channels If the Color Separation parameters are to be used again in the future click the Template button Click Add and assign the template a unique name When you are satisfied with the color settings click OK If you want to remove the color separation uncheck the Show Color Separated Images checkbox in the Image Setting box You can now repeat the color separation process 5 33 Image Analysis Tools FMBIO Using a Color Separation Template To perform this function a previously created Color Separation template must have been saved 1 Click the Color Separation button 1 or choose Color Separation from the Image menu 2 Once the Color Separation window has opened click the Template button The Template dialog box is displayed 3 Select a templ
108. gnostic routine has been completed without detecting a problem You may now use the scanning unit Error Detected When the self diagnostic routine detects an error the Ready light shuts off and the Error light begins to glow Turn off the Power switch for at least five seconds then turn on the Power switch The FMBIO repeats the self diagnostic routine If the routine detects an error again contact your nearest authorized Hitachi Software Engineering service representative 3 3 Operating the Scanning Unit FMBIO CAUTION Spills on the scanning unit may damage the electronics or laser inside the FMBIO Heavy objects on top of the unit may throw the scanning surfaces out of alignment Avoid placing objects on top of the scanning unit WARNING Normal FMBIOe operation uses a 50 mW micro laser beam Exposure to laser radiation can seri ously damage eyes and vision A set of built in safety switches places a shutter in front of the laser beam for your protection e Do not override the laser safety interlock system e Do not look directly into the open optical filter door e Do not remove the external panels and covers of the FMBIO at any time WARNING v Unauthorized repair attempts may expose you and your coworkers to extremely high voltage and hazardous laser light and will also void your warranty or service contract Always call Hitachi Software Engineering for repairs and service Autofocus Calibration After a powe
109. ground and fluorescein Percent entry fields Preview the color separation If you are satisfied with the results click OK on the Color Separation box When the message Create the Separated Images this time appears click OK again If you want to remove the color separation uncheck the Show Color Separated Images checkbox in the Image Setting dialog box You can now repeat the color separation process Transparent Background You can choose to make the background of an image transparent and position it over another image to compare bands EE 0725011owmassladderethFMBIOIl 1D Gel Experi GE 072501 ethlle 1D Gel Experiment To make the background of an image transparent select Transparent Back ground from the Image menu Image Analysis Tools FMBIO Transparent Background TTG a EIEEGTDITIT 5 36 FMBIO Image Analysis Tools 1D Gel Menu and Tools 1 Click the New Project button or choose New Project from the File menu 2 Choose New 1D Gel Experiment from the Project menu The 1D Gel Experiment window Image Setting dialog box 1D Gel menu and its corresponding tool bar appear Ioram S EHI Image Analysis Tools FMBIO Overlay Trace Set Marker Volume Calculation Open Spreadsheet aly Multi Band Color Separation Preferences Quantification Setting Open BP Curve Tool Description Overlay Trace Displays new overlay trace of selected lanes Set Marker Displ
110. gs until you are satisfied with the signal and background levels Repeat the procedure for each channel 10 When you are finished click OK Image Analysis recalculates the gray levels for the entire image using these values as high signal and background limits Note The raw image data is not affected by the gray level settings 5 22 FMBIO Image Analysis Tools Using the percent and number fields You can also adjust the high signal and background cutoff thresholds by entering values in the percent and number fields Select a channel and mapping type Enter numbers in either the percent or absolute value fields and then click OK 2 Click the Try button to apply the modified gray levels to the actual image To return to the original settings click Reset 3 Continue to try different gray level settings until you are satisfied with the signal and background levels 4 Repeat the procedure for each channel 5 When you are finished click OK Image Analysis recalculates the gray levels for the entire image using these values as high signal and background limits Creating a template If you have a series of gels that have been run under the same conditions and produce similar images you might want to save your gray level adjustment values as a template for future use l Click the Template button 2 Click the Create New button The Template dialog box is displayed 3 Typeaunique name for the template then cli
111. h signal and background limits Note The raw image data is not affected by the gray level settings Visually defining signal and background pixels You can define the signal and background intensities using the Zoom and Selection cursors 1 2 Whole Image Zoomed Area JAINA t rx um VEHI t meene FORERTTIRET FAILURE ELULUUUL Zoom Cursor Selection Cursor Select a channel and mapping type In the Whole Image window drag the Zoom Cursor to a region con 5 21 Image Analysis Tools FMBIO 9 taining average intensity bands background If necessary adjust the size of the Zoom Cursor to include the desired area The area appears in the Zoomed Area In the Zoom window surround a band with the Selection Cursor Whole Image Zoomed Area i LN JAINA VORERITIRET FAILURE ELUUUUE Click the High Signal button The image displays appear with the new high signal cutoff threshold and the values are displayed in the High Signal Percent and High Signal Number fields Move the Selection Cursor to a background area away from the band Click the Background button The selected background cutoff threshold is applied to the Whole Image and Zoomed Area windows and the values appear in the Background Percent and Background Number fields Click the Try button to apply the modified gray levels to the actual image To return to the original settings click Reset Continue to try different gray level settin
112. h the sample and the scanning unit Orientation of glass plates Scanning begins in the upper left corner of the scanner bed near the sample alignment surface See Figure 3 5 The longer of the two glass plates should rest on the sample supports Removing spacers It is not necessary to remove the spacers on the glass plates Important For optimal results and to minimize image file size do not include the spacers in the defined scan area Note Begin the scan as soon as possible after electrophoresis to ensure a clear scan image 1 Set the Power switch to the On position and open the cover of the scan ning unit 3 10 FMBIO Operating the Scanning Unit Clean and thoroughly dry the surfaces of the glass plates You may want to mark the front of the plate so that it can be distinguished on the scan Before putting the glass plate on the scanner bed adjust the sliding sample support so that it is ready to accommodate the length of the plate Carefully and firmly place the end of the longer glass plate up against the sample alignment surface See Figure 3 5 on page 3 10 As you carefully begin to lower the rear end of the glass plate to lay flat on the scanner bed finely adjust the position of the sliding sample plate so that it offers full support When both ends of the glass plate are firmly supported on the scanner bed gently close the scanning unit cover to avoid vibrations that might unsettle the glass plate You can
113. he Evaluate STR Data function are copied to this column and used as the basis for calculating ranges Stutter band reporting To evaluate stutter STaR Call calculates the OD or IOD ratios of adjacent bands If a band is in the stutter position n 4 and the related ratio falls within a user defined cutoff range that ratio is reported in the Percentages column Information on this band is not exported to CODIS see page 10 21 and does not appear in the Merged Landscape worksheet see page 10 19 10 12 FMBIO Allele Calling with STaR Call e To include a stutter band in either CODIS or the Merged Landscape worksheet delete the value in the Percentages column e To change the cutoff range choose Edit STR Cutoff Values from the STaR Call menu Creating editing or deleting a STR Lookup table STaR Call STR Lookup Table 2 x r Create New Lookup Table Sheet Name 010 L0 l jJ New r Edit or Delete Lookup Table PowerPlex 2 1 FL PowerPlex 2 1 TMR PowerPlex CTTv PowerPlex CTTv Am PowerPlex CTTv 9 3 PowerPlex CTTv Am 9 3 Delete GammaSTR DDDD new D7 zi e Inthe STaR Call menu choose STR Lookup Table The STR Lookup Table dialog box is displayed To create a STR Lookup table 1 Enter a valid sheet name in the Create New Lookup Table field 2 Click the New button To edit a STR Lookup table 1 In the STaR Call menu choose STR Lookup Table The STR Lookup Table di
114. he Software is subject to restrictions and controls imposed by the United States Export Administration Act the Act and the regulations thereunder You agree and certify that neither the Software nor any direct product thereof is being or will be acquired shipped transferred or reexported directly or indirectly into any country prohibited by the Act and the regulations thereunder or will be used for any purpose prohibited by the same GENERAL This agreement will be governed by the laws of the State of California except for that body of law dealing with conflicts of law Future updates of the Software will be available for purchase by licensees for a fee provided a registration card has been received by MIRAIBIO Registered Licensees will be notified of such updates as they become available Should you have any questions concerning this Agreement you may contact MIRAIBIO by writing to MiraiBio Inc 1201 Harbor Bay Parkway Suite 150 Alameda CA 94502 You acknowledge that you have read this Agreement understand it and agree to be bound by its terms and conditions You further agree that it the complete and exclusive statement of the agreement between us which supersedes any proposal or prior agreement oral or written and any other communications between us in relation to the subject matter of this Agreement Addendum Image Analysis v 3 0 This document contains information regarding the latest changes made for the Image Anal
115. he desired template in the list and click Apply Background Calculation You can specify the background value the method for averaging a back ground marker and the side of a rectangular spot to use as the background 1 Click the Preferences button KI or choose Preferences from the Array menu 2 In the Preferences dialog box click the Volume Calculation tab Preferences 7 11 Array Analysis FMBIO Background Option Description Gray Level Adjustment Local Spot Average User Defined Background Average Method Local and Spot Average Effective side s of Average Local Background Uses gray level background value for the image Uses average pixel value of spot grid cell boundary Does not use the background marker Uses average pixel value of background spot marker Uses value entered in the field Mean total volume number of pixels Median middle value e g 1 2 5 15 16 20 300 2000 5000 Mode most frequent value Side of cell used to calculate local background All Left Top Right Bottom For example if there is significant horizontal overlap of signal between spots you might want to select the top of each cell Registering the Background Value 1 Create a rectangle or oval spot surrounding the area that represents the background 2 Click the Set Selected Spots to Background button B or choose Set Selected to Background from the Array menu
116. he print job in several ways To customize a print job e Choose Print Setup from the File menu The Page Setup window is displayed Page Setup a wee do 5 94 FMBIO Image Analysis Tools Feature Description Header Footer Align Left Align Center Align Right Margins Scaling Fine Adjustment n project name d current date t current time p page number P total number of pages Top Left Right Bottom Header Footer Adjust to Enter scaling whole numbers Fit to a page Scaling determined by paper size e g if the figure is reduced if it is larger than the page size Horizontal and vertical 50 150 real numbers Print Setup To set the print options click the Options button Print Preview To preview your print job click Print Preview Printing To begin printing click the Print button g or choose Print from the File menu 5 95 Image Analysis Tools FMBIO Batch Analysis A set of templates can be saved and loaded for use in a different analysis There are four sections e Gray Level Adjustment e Band Settings Lane Template e Color Separation 1D Batch Control Dialog Procedure 1 Choose Batch Control from the 1D Gel menu The 1D Batch Control dialog box is displayed 5 96 FMBIO Image Analysis Tools Item Description Gray Level Select a template from the drop down menu Adjustment Band Setting Lane Template C
117. hear a clicking sound when the cover shuts WARNING Laser radiation can leak from the scanning unit when the cover is not closed tightly Stray radia tion can damage vision and interfere with accu rate image scanning Carefully check that the cover is closed tightly before beginning a scan 3 11 Operating the Scanning Unit FMBIO Scanning the Image The sensitivity of the scanning unit is dependent on a number of factors including the dyes or stains used the quality of the reagents gel density and the diffusion rate of the sample in the gel protocols and technique You can fine tune the image scan by adjusting the scanning parameters in the Read Image dialog box Before beginning a scan read Chapter 4 Scanner Software to become familiar with the features of this software Beginning the Scan Once you are satisfied with the Scanner software scanning parameters click the Read button on the Read Image dialog box to begin the scan Auto Focus After the Power switch is first turned on the FMBIO Read Image software automatically performs an autofocus before it begins the first scan During autofocusing the rotating mirror and other signal tracking and processing elements are adjusted to optimize optical alignment The entire process requires about thirty seconds to complete The 605 nm optical filter should be in Channel 1 during autofocusing After the initial autofocus you can turn autofocusing off and on with Read Imag
118. his range Enter a number in the Repeat field Focusing Point When you modify the focusing point value Read Image software adjusts the height of the focusing point at the scanning bed to read thick samples based on 5 0 mm glass plates For most acrylamide gels 0 35 0 4 mm thick and membranes the focusing point value should be set to zero 0 For a 5 0 mm agarose gel you may raise the focusing point to 1 2 mm WARNING The FMBIO scanning unit can accommodate a focusing point range from 3 0 to 2 0 mm Do not enter a focusing point value outside of this range or severe damage may result 4 9 Read Image Software FMBIO Settings in the Command Menu Command FMBIO II Help Read PreRead Set Parameter Set GrayLevel amp djustment mage UE y Orientation The Command menu contains two kinds of commands e Commands that correspond to buttons on the Scan Control window e Settings that affect the format and appearance of the scanned image These are displayed in the Scan Control window Preparing to Scan Before beginning a scan check the following settings displayed in the Scan Control window e Gray Level Adjustment including the Adjustment Type and the High and Low Cutoff Thresholds mage File Type Orientation Setting Gray Level Adjustment Proper setting of the Gray Level Adjustment parameters enhances signal and suppresses noise resulting in clean readable images Improper setting
119. ical range is normalized it is compressed to fit to the range of available pixels in an 8 bit file from 0 to 255 The low cutoff threshold becomes 0 the high cutoff threshold becomes 255 When Image Analysis encounters signal above the high cutoff threshold it extrapolates a value for these spots that may actually be greater than 255 The upper end of the vertical range reflects the value assigned to these high signals Straight Line Method 1 Click the Area Selection button or choose Line Selection from the Image menu 2 Drag the cursor in a straight line over the location you want to analyze Release the mouse button A selection line is displayed If necessary adjust the line by dragging the selection handles 5 27 Image Analysis Tools FMBIO 4 Click the Spectrum View button ata or choose Show Spectrum from the Image menu 5 28 FMBIO Image Analysis Tools Color Separation In multi color fluorescence imaging up to four fluorescent dyes fluoro phores can be detected in a single image Although each fluorophore exhibits its own characteristic emission spectrum with a distinct maximum at specific wavelength there may be some overlap bleed through between the spectra of the different fluorophores The purpose of color separation is to identify these areas of emission spectra overlap by quantifying the proportion of each fluorophore that is known to overlap with the other fluorophores Image Analysis a
120. ification Setting Displays Quantification Setting window Preferences Displays Preferences window Multi Band Displays Multi Band Color Separation Color Separation window 6 3 1D Gel Analysis FMBIO Select Tool Lane Selection Tool Multiple Lane Selection Tool Lane Alignment i Edit x e mx Delete Band Automatic Band Detection Lane Template Automatic Lane Fitting Tool Description Select Tool Select objects Lane Selection Tool Define a single lane Multiple Lane Selection Tool Define multiple adjacent lanes Lane Alignment Aligns lanes with migration lines Automatic Lane Fitting Automatically adjusts lane boundaries Lane Template Saves all lanes on images Automatic Band Detection Autoband selected lanes Delete Band Delete all bands in the selected lanes Band Edit Add edit or delete bands 6 4 FMBIO 1D Gel Analysis Show Hide Lanes Show Hide Band Information Show Hide Comment spe Band Style Lane Style Tool Description Show Hide Lanes Show Hide Band Information Show Hide Comments Lane Style Band Style Toggle display of lane boundary lines Toggle display of band information Toggle display of label information entered in spreadsheet Toggle among different display options center line with lane borders center lines only lane boundaries only and lane dis play off Toggle among the different band styles band display o
121. ile for export to other programs such as Microsoft Excel and Microsoft Word To export a spreadsheet 1 Click the Open Spreadsheet button E or choose Open Spreadsheet from the 1D Gel menu 2 Choose Export from the SpreadSheet menu SpreadSheet V Export 3 Inthe Save As dialog box select a location for the file and then enter a unique name 4 Click Save FMBIO Image Analysis Tools Overlay Trace In an Overlay Trace window you can compare the spectrums of different lanes This window also allows you to edit a band while viewing the graphic trace of its signal To use Trace Overlay you must first use 1D Gel analysis to define one or more lanes in an 1D Gel Experiment window See Analyzing 1D Gels on page 6 6 Note Your images must have defined lanes but creating an overlay trace does not require previously defined bands Overlay Trace Window 1 Open one or more 1D Gel Experiment windows that have defined lanes 2 Select the lane or lanes of interest by clicking the Arrow cursor on the desired lane Shift click to select more than one lane 3 Amaximum of 16 lanes may be converted into Lane Trace format at one time 4 Click the New Overlay Trace button or choose New Overlay Trace from the 1D Gel menu The Overlay Trace window is displayed and the Trace menu appears in the menu bar Image Analysis Tools FMBIO Trace Window Help Zoom In Vertically Zoom Out Vertically
122. ines page 5 50 e Adjust the top and bottom migration lines according to the user s laboratory protocol Autobanding page 5 60 With the lanes selected and the migration lines adjusted autoband determi nation can be performed 1 Select Normalized Volume IOD and choose Type 4 as the calculation type in the 1D Gel Preferences dialog box Preferences 6 12 FMBIO 1D Gel Analysis 2 Click the Automatic Band Detection tab to edit the autobanding parameters 3 Try these settings and adjust the settings as needed for different gels Gradient Start 2 5 End 2 5 Duration 0 2 Noise Level 20 Preferences 4 Choose Edit gt Select All to select all the lanes 5 Click the Automatic Band Detection button l or choose 1D Gel gt Function Auto Band You might need to adjust the Image Analysis automatic band calls The Band Style button kE toggles among the different band styles band display off peak and range bands displayed and only peak bands displayed You can also choose a band style from the 1D Gel menu 1D Gel Analysis FMBIO 6 Click the Band Edit button or choose 1D Gel gt Tool gt Band Edit then use the cursor to place bands Set Marker page 5 68 Click the Quantification Setting button or choose Quantification Setting from the 1D Gel Menu Select the standard curve fit from the drop down list box Logarithmic is the correct standard curve fit for most applications
123. ing the Software on behalf of any unit or agency of the United States Government the following provisions apply The Government acknowledges MIRAIBIO s representation that the Software and its documentation were developed at private expense and no part of them is in the public domain The Government acknowledges MIRAIBIO s representation that the Software is Restricted Computer Software as that term is defined in Clause 52 227 19 of the Federal Acquisition Regulations FAR and is commercial Computer Software as that term is defined in Subpart 227 41 of the Department of Defense Federal Acquisition Regulations supplement DFARS The Government agrees that D if the Software is supplied to the Department of Defense DoD the Software is classified as Commercial Computer Software and the Government is acquiring only restricted rights in the Software and its documentation will be as defined in Clause 52 277 19 c 2 of the FAR ID if the Software is supplied to any unit or agency of the United States Government other than DoD the Government s rights in Software and its documentation RESTRICTED RIGHTS LEGEND Use duplication or disclosure by the Government is subject to restrictions as set forth in subparagraph c 1 11 of the rights in Technical Data and computer software clause of DFARS 52 227 7013 MiraiBio Inc 1201 Harbor Bay Parkway Suite 150 Alameda CA 94502 EXPORT LAW ASSURANCES You acknowledge and agree that t
124. iography FMBIO images are processed without removing glass plates drying gels or developing film saving more time Using multi wave length scanning samples labeled by two or more different fluorophores can be read in one scan FMBIO Greater Accuracy Accuracy sensitivity and flexibility of experimental operations are improved with the FMBIO system 16 bit imaging allows detection of signal intensity over a much greater linear dynamic range than isotope based systems This enables both dark and light bands to be read in the same exper iment Many of the errors that stem from sample handling are virtually eliminated because glass plates are not removed gels are not dried and read ability of results does not depend on exposure time Because scanning does not require removing the gel from the glass plate gels can be run then scanned then run and scanned again increasing the number of readable bands per gel Lower Costs When the FMBIO system is in place the expense associated with X ray film storage phosphor screens shields badges Geiger counters low tem perature freezers and disposal services is eliminated Simplicity The FMBIO consists of a scanning unit and two software applications that run on Microsoft Windows 95 or 98 The Read Image scanning software controls the operation of the scanner and the Image Analysis software carries out the analysis of the scans The two software applications may be run on separate com
125. it 9 2 Shipping 9 2 Shutdown 9 3 Troubleshooting the Scanning Unit 9 3 Red Error Light Flashing 9 3 Responding to Problems 9 3 Water Spills Inside the Scanning Unit 9 3 Foreign Objects In the Scanning Unit 9 4 Equipment or Power Cord is Damaged 9 4 Unknown Cause of Damage 9 4 Allele Calling with STaR Call 10 1 Installing the STaR Call Software 10 2 STaR Call Installation Windows Version 10 2 Requirements 10 2 Installing STaR Call 10 2 Starting STaR Call 10 3 Removing STaR Call 10 3 STaR Call Installation Macintosh Version 10 3 Requirements 10 3 Installing STaR Call 10 4 Starting STaR Call 10 5 Removing STaR Call 10 5 Short Tandem Repeats 10 6 Preparing for Allele Evaluation 10 7 Exporting 1D Gel Analysis Results 10 8 Using STaR Call to Evaluate Alleles 10 9 Options 10 11 STR Lookup Table 10 11 Importing STR files 10 14 vii Adding an STR Recalculating Results Comparing Analyses Printing and Saving Results Exporting Data to CODIS Appendix A Software Reference Keyboard Shortcuts Icons Image Analysis Tools File and Edit Tools Image Tools 1D Gel Tools Multi Band Color Separation Tools Trace Overlay Tools Array Tools Drawing Tools Tools Index viii FMBIO For Safe Operations Precautions Preventing Mechanical Failure Electric Shock Fire and Exposure to Hazardous Laser Radiation For your safety operate this equipment only as prescribed by the instructions in this manual Operation outside th
126. lane or on an entire gel Read Image collects emission data from all active channels during a scan Active channels are preceded by a checkmark in the FMBIO menu and are listed in the Scan Control window Channel 1 is the default active channel Note Channel 1 does not have to be active if it is only in place for an autofocus 1 Before beginning a scan place the appropriate filters in the filter holders 2 Choose the corresponding channels in the FMBIO menu to make them active Read Image collects emission data from each active channel during the scan 3 Check the reading sensitivity setting for each active channel in the FMBIO Parameter window See page 4 8 Important Each active channel must have an associated filter in the filter holder If you make a channel active in Read Image the corresponding channel on the FMBIO scanning unit must contain a filter During a scan if an active channel does not contain a filter or if the filter holder is not in place the resulting scanned image is white If an active channel contains an opaque plastic barrier the resulting scanned image is black 4 14 FMBIO Read Image Software See Inserting Optical Filters on page 3 5 for directions on changing optical filters See page 4 22 for information about the other commands in the FMBIO menu Scan Control Window Feature Name Description Total scan area Displays available scan area and a pre defined scan area Scan area
127. le Spot tool in Medium Grid Toolbar Oval Tool on Draw Toolbar and Create Oval Angle Spot in Medium Grid Toolbar Text Tool on Draw Toolbar Select Tool on Draw or 1D Tool Toolbar or Medium Grid Toolbar Lane Selection Tool on 1D Tool Toolbar Multiple Lane Selection Tool on 1D Tool Toolbar 3 MAGNIFICATION Magnification Range has been extended 1D Gel Experiment up to 3200 This new feature allows ge 1D Gel Window Help end user to magnify the image that is y scanned with high resolution of 25 um ani In addition images with 72 Dpi Screen Resolution can be loaded without resulting in an error 4 OD CALCULATION Preferences In 1D Gel gt Preference dialog box there Band Information Multi Band Color Separation Parameters is an extra box where end users can Automatic Band Detection Calculation select either Overall Maximum Background Value Intensity or Average Pixel Intensity Channel x algorithm to calculate OD values The Background of Gray Level Adjustment 767 default setting is Average Pixel User Defined Intensity Use Least Square Method for bp calculation zx eii Normalize Volume IOD In general Average Pixel Intensity can Beis give better result for most images Average Pixel Intensity Overall Maximum Intensity Type C Typel Type2 Type3 Typed 5 COLOR SEPARATION Users must set background noise value for each channel before clicking on Set button to calculate Channel Parame
128. lease contact your FMBIO Sales Representative 10 1 Allele Calling with STaR Call FMBIO Installing the STaR Call Software The STaR Call software is available for both the PC and Macintosh Both versions require that Excel be previously installed The Macintosh version will run with either Excel 5 or Excel 98 The PC version will run under either Windows 95 or Windows 98 STaR Call Installation Windows Version Requirements e Atleast 2 MB of free memory to load the STaR Call macro and the data file e Microsoft Windows 95 or Windows 98 with Excel 95 or Excel 97 Consult the documentation that came with the Microsoft Excel software for Microsoft Excel s memory requirements Installing STaR Call Note To install and use the STaR Call software you must have Excel loaded on your computer If Excel is not already installed on your hard drive install it before attempting to install STaR Call 1 If you have just installed Microsoft Office or Excel run Excel once to allow it to complete some automatic configuration steps 2 Make sure that Excel is closed 3 Insert the STaR Call disk into the drive 4 Selectthe directory corresponding to the version of Excel that is installed either Excel 95 or Excel 97 5 According to which version of Excel you have e Ifyou have Excel 95 installed double click the INSTALL xla file e Ifyou have Excel 97 installed double click the install xla file 6 Ifa dialog box appears asking
129. lect Mono as the display mode Image Setting Blend td mz g Mono El roae rode When a black and white image is displayed the user is ready to adjust the grayscale 2 Click the Gray Level Adjustment button E or choose Gray Level Adjustment from the Image menu The Gray Level Adjustment window is displayed 6 20 FMBIO 1D Gel Analysis In the Full Image window drag the Zoom Cursor to a region containing average intensity bands background If necessary adjust the size of the Zoom Cursor to include the desired area The area appears in the Zoom window In the Zoom window surround a band with the Selection Cursor then click the High Signal button The new High Signal cutoff threshold is applied to the Full Image and Zoom windows and the values appear in the High Signal Percent and Background Number boxes Whole Image Zoomed Area B d B B B Ee 8 arum tt tut Move the Selection Cursor to a background area away from the band and then click the Background button The new Background cutoff threshold is applied to the Full Image and Zoom windows and the values appear in the Background Percent and Background Number boxes e All regions of the image with gray level values below these Back ground values will appear white e All regions of the image with gray level values above the High Sig nal values will appear black e All other elements of the image that fall within the range between High
130. lectric shock If there is a spill immediately turn the power switch off disconnect the power cord and contact your nearest authorized Hitachi Software Engineering Service representative No Foreign Objects Keep foreign objects out of the Scanning Unit Do not allow paper clips hairpins metallic objects paper or other combustible S material to fall in or otherwise enter the Scanning Unit Foreign objects in the Scanning Unit can lead to short circuit fire or electric shock If this should happen turn the power switch OFF disconnect the power cord and contact your nearest authorized Hitachi Software Engineering service representative FMBIO WARNING Handling the Electric Power Unit S A Use Only the Specified Power Cable Ground the Scanning Unit with the proper power cord with the three prong plug provided with the Scanning Unit Before turning on the Scanning Unit check that the power cord is NOT damaged Using the damaged power cord can lead to short cir cuits fire or electric shock If the power cord is damaged turn the power switch OFF disconnect the power cord and contact your nearest authorized Hitachi Software Engineering Service representative for a replacement Use only a properly grounded three prong power cord as a temporary replacement Do Not Use a Non standard Power Supply Use the specified power supply AC100 V and 50 60 Hz Use only with the proper voltage and frequency indicated on the specification
131. lines in the histogram e Visually defining signal and background pixels Entering numbers for the high signal and background values Using the histogram 1 Selecta channel and mapping type 2 To adjust the high signal cutoff threshold value place the pointer over the blue line The pointer transforms into a horizontal double headed arrow 3 Drag the line along the gray level axis until the desired value is dis played The new high signal cutoff threshold value is applied to the Whole Image and Zoomed Area windows and a new High Signal Percent value is calculated 4 To adjust the background cutoff threshold value place the pointer over 5 20 8 9 FMBIO Image Analysis Tools the red line The pointer transforms into a horizontal double headed arrow and the value appears in the upper left corner of the window Drag the line along the gray level axis until the desired value appears The new background cutoff threshold value is applied to the Whole Image and Zoomed Area windows and a new High Signal Percent value is calculated Click the Try button to apply the modified gray levels to the actual image To return to the original settings click Reset Continue to try different gray level settings until you are satisfied with the signal and background levels Repeat the procedure for each channel When you are finished click OK Image Analysis recalculates the gray levels for the channels using these values as hig
132. ll continue to be subject to the terms and conditions of this Agreement d Transfer the Software and all rights under this Agreement to another party together with a copy of this Agreement if the other party agrees to accept the terms and conditions of this Agreement If you transfer the Software you must at the same time either transfer all copies whether in printed or machine readable form including all modifications and portions of the Software contained in or merged into the other programs to the same party or destroy and copies not transferred RESTRICTIONS You may not use copy modify or transfer the Software or any copy modification or merged portion in whole or in part except as expressly provided for in this Agreement Any attempt to transfer any of the rights duties or obligations hereunder except as expressly provided for in this Agreement is void YOU MAY NOT RENT LEASE LOAN RESELL FOR PROFIT OR DISTRIBUTE TERM This Agreement is effective until terminated You may terminate it as any time by destroying the Software together with all copies modifications and merged portions in any form This Agreement will immediately and automatically terminate without notice if you fail to comply with any term or condition of this Agreement You agree upon termination to promptly destroy the Software together with all copies modifications and merged portions in any form LIMITED WARRANTY MIRAIBIO warrants for the period of ninet
133. ll menu STaR Call copies the STR Lookup tables evaluates the STR files and creates the Merged and Merged Landscape worksheets Comparing Analyses To compare two analyses 1 Choose Compare Analyses from the STaR Call menu The Compare dialog box is displayed 10 20 FMBIO Allele Calling with STaR Call STaR Call Compare 2 x File 1 Sampleab xls Browse eo TE Erose Compare lane information 9 ce we 2 Selectthe two analysis workbooks in the drop down list boxes Click the Browse button to open a workbook Select the Compare lane information option if required 4 Click OK Printing and Saving Results Your evaluation is now complete You may print and save the STaR Call table of genotypes and stutter percentages Save the file either as a tab delimited text file or an Excel workbook Exporting Data to CODIS You can add CODIS information to the table of STaR Call results and prepare a file to upload your allele calls to the CODIS database To export to CODIS 1 In the STaR Call menu choose CODIS STR Export The CODIS STR Header dialog box appears 10 21 Allele Calling with STaR Call FMBIO STaR Call CODIS STR Header Information Imag ORI Lab ORI 06 Jun 1999 00 00 00 02 Jun 1999 Title Description CMF Header Version CMF Message Type CMF Message ID Imaging System Organization Company Imaging System Utilized Imaging S
134. llected light signal is considered as viable data The higher the voltage the greater the sensitivity and the more background signal is included in the data Material with inherent high back ground signal such as membranes require lower reading sensitivity When you use more than one filter to produce an image one or more fluo rescent signals may be noticeably stronger than the others You can adjust the sensitivity of each channel to equalize the intensity of the signals Important You must use the FMBIO menu to activate all the channels used in the scan See page 4 13 e Enter a value between 0 100 for each channel Read Image automatically assigns a value of 0 to sensitivity fields that are left blank Setting Repeats During a scan the laser scans across each line repeatedly before moving on to the next Read Image software retains median signal values for each line and throws out high and low values This process of throwing out extreme values reduces anomalies due to temperature shifts in equipment and sample during processing 4 8 FMBIO Read Image Software The ideal number of repeats for any sample is difficult to predict and is dependent on a number of factors including gel density and sample concen tration Increasing the number of scan repeats per line increases the ratio of signal to noise and also increases the scan time You may begin with 150 to 256 repeats per line but do not hesitate to use values outside t
135. llow at least 10 cm of clearance next to the rear and left panels Do not place heavy objects on top of the scanning unit Keep the scanning unit away from flammable or corrosive gases Important The lasers on the FMBIO requires a constant power source If the power specifications on page 1 5 are not met a power shortage could result and cause instrument failure 2 2 FMBIO Installation Installing the FMBIO Software FMBIO system requirements IBM or 100 IBM compatible personal computer e Windows 2000 e Pentium II 300 MHz or greater is recommended e 128 MB RAM minimum 256 MB or more recommended e 10 MB free minimum of hard disk storage space is required for program loading Image and spreadsheet files could require substantially more storage space e CD ROM disc drive is required for installation e Resolution 1024 x 768 or higher 16 bit color display or higher Mouse or other pointing device e Keyboard The FMBIO software can be obtained on a CD To install the FMBIO software contained on a CD 1 Turn off the screensaver e In the Windows Taskbar click on the Start button Choose Settings Control Panel e Double click the Display icon e Click the Screensaver tab then select None Disable all virus protection programs On the CD open the ReadMe file to find out about changes and updates Click the Start button then choose Run phage Murs Enter the pathname for the setup exe file on
136. llows two methods for color separation e Color separation by individual band selection e Multi band color separation Multi band color separation is a feature of the 1D Gel analysis tools See page 5 41 Color Separation Theory The following is a theoretical treatment of color separation for an image with two channels Below are shown the spectra for an image from a lane with two channels In this example the pixels in region B of Channel 2 bleed through into area A of Channel 1 so the following type of color separation is performed Channel 1 Basic Channel Channel 2 Image pixel value Electrophoresis Electrophoresis distance mm distance mm Image Analysis Tools FMBIO Remove the background portion N4 N gt from each channel Image pixel value Area A Integral value Sa Integral value Sg Area B e Electrophoresis Electrophoresis distance mm distance mm 1 Calculate the bleed through rate Bleed through rate RAg S A Sp 2 The bleed through rate Rap is calculated for each pixel value in Channel 2 and the corresponding pixel value in Channel 1 This rate is multiplied by the image pixel values in Channel 2 and the result is sub tracted Then the spectra in Channel 2 is as follows For any point on the image P x y the image pixel value in Channel is I1 and the image pixel value in Channel 2 is I2 After color separation the pixel value in Channel 1 is I52L
137. lly selected 2 Click on the band line to select it The band line changes from yellow to violet 3 Drag the band line to the new position Deleting bands There are two methods to delete bands Area Selection method 1 Draw a rectangle around the bands you want to delete Image Analysis Tools FMBIO The edit handles for the bands are displayed 2 Press the Delete key Note If you want to delete bands in multiple areas hold down the Shift key while selecting the additional areas Band Edit Method 1 Double click on the lane you want to edit The lane is selected and the Band Edit button is automatically selected 2 Click on any band line to select it The band line changes from yellow to violet 5 66 FMBIO Image Analysis Tools 3 Press the Delete key Hiding bands The Show Hide Band Information button and the 1D Gel View Band Information menu item control the display of the band migration values and lane numbers Displaying band lines The Band Style button l toggles among the different band styles band display off peak and range bands displayed and only peak bands displayed You can also choose a band style from the 1D Gel menu Image Analysis Tools FMBIO Standard Markers Standard markers serve as reference points for base pair length calculations You may load more than one marker in the same lane You can load markers in sample lanes and intersperse marker lanes with sample lan
138. log Box Display Mode Displaying a Channel Changing Channel Color Changing Intensity Level Displaying Detected Bands Gray Level Adjustment FMBIO 4 14 4 15 4 16 4 17 4 18 4 18 4 20 4 20 4 21 4 22 4 23 4 25 4 25 4 27 4 32 5 1 5 3 5 3 5 3 5 3 5 4 5 7 5 9 5 10 5 11 5 11 5 11 5 12 5 12 5 13 iii FMBIO Gray Scale Adjustment Options Gray Level Adjustment Window Procedure Image Diagnostic Guide Spectrum Area Selection Mehod Straight Line Method Color Separation Color Separation Theory Individual Band Color Separation Using a Color Separation Template Modifying Color Separation Results Transparent Background 1D Gel Menu and Tools Multi Band Color Separation Band Selection Parameters Multi Band Color Separation Window Indicating Lanes Defining Lanes Displaying Lane Lines Editing a Lane Boundary Copying and Deleting Lanes Selecting Multiple Lanes Lane Templates Defining the Migration Area Defining Bands Automatic Band Detection Manual Band Input Standard Markers Registering Markers 5 15 5 16 5 20 5 24 5 25 5 25 5 27 5 29 5 29 5 31 5 34 5 34 5 35 5 37 5 41 5 41 5 44 5 50 5 51 5 53 5 53 5 54 5 54 5 55 5 58 5 60 5 60 5 64 5 68 5 68 FMBIO Importing and Exporting Markers 5 70 Working with Marker Templates 5 72 Adding Marker Templates 5 72 Base Pair Standard Curve 5 73 Calculating Volumes and Peak Heights 5 77 Background Value 5 78 Calculating Normalized Volume IOD 5 78
139. lso select channels in the Image Setting dialog box Lane Place a checkmark in the box next to the lane you want to display 5 90 FMBIO Image Analysis Tools Item Description View Mode Separated On Lane The spectra for each lane is shown One spectral graph is shown for each lane and spectra from all channels are shown Separated On Channel One spectral graph shows one channel and all lanes are shown over lapped Overlapped The spectra of all channels are shown overlapped Lane Image Show Select the show lane image hide image check box Width Enter the width of the lane image Spectrum Enter the width of the spectral graph Show Hide Y Rf axis Show Hide X Value axis Adjustment Margin Enter the spacing for lanes and spectra V Increment Enter the units for magnification reduction for the overlay trace To increase magnifi cation enter a higher value Exporting Lane Trace Data In some cases it may be desirable to export the Lane Trace data from Image Analysis into an Excel spreadsheet The data will be displayed in the spread sheet as migration distance mm and intensity OD Define the desired lanes in the images 2 View the lane traces if desired The lane trace data can be exported to a folder as a set of Microsoft Excel spreadsheets by choosing Export Lane Trace from the 1D Gel 5 91 Image Analysis Tools FMBIO menu The Select Folder dialog box is dis
140. ly turn the power switch Off disconnect the power cord If the Scanning Unit emits an unusual smell smoke noise or excessive heat e Immediately turn the power switch to OFF e Disconnect the power cord e Contact your nearest authorized Hitachi Software Engineering Service representative If you continue to use the scanning unit under these circumstances fire or electric shock may result Water Spills Inside the Scanning Unit If there is a spill e Immediately turn the power switch to OFF 9 3 Maintenance FMBIO e Disconnect the power cord e And contact your nearest authorized Hitachi Software Engineering Service representative If you continue to use the scanning unit under these circumstances fire or electric shock may result Foreign Objects In the Scanning Unit If foreign objects are discovered or may have dropped inside the equipment e Immediately turn the power switch to OFF e Disconnect the power cord e And contact your nearest authorized Hitachi Software Engineering Service representative If you continue to use the scanning unit under these circumstances fire or electric shock may result Equipment or Power Cord is Damaged If the Scanning Unit or the Power Cord is damaged e Immediately turn the power switch to OFF e Disconnect the power cord e Contact your nearest authorized Hitachi Software Engineering Service representative If you continue to use the scanning unit under these circum
141. lysis Noise Level 20 Preferences 4 Choose Edit gt Select All to select all the lanes 5 Click the Automatic Band Detection button l or choose 1D Gel gt Function gt Auto Band You might need to adjust the Image Analysis automatic band calls The Band Style button toggles among the different band styles band display off peak and range bands displayed and only peak bands displayed You can also choose a band style from the 1D Gel menu 6 Click the Band Edit button ki or choose 1 D Gel gt Tool gt Band Edit then use the cursor to place bands Set Marker page 5 68 Click the Quantification Setting button E or choose Quantification Setting from the 1D Gel Menu I1D Gel Analysis FMBIO Select the standard curve fit from the drop down list box Logarithmic is the correct standard curve fit for most applications 2 Click the Marker Setting button MI or choose Set Marker from the 1D Gel menu 3 Highlight the lanes containing the marker by holding down the Shift key while clicking on them with the mouse Select the correct marker from the Template list then click Adopt Marker The selected marker appears next to each lane in the list Marker Setting crete Remove Erter Dupicate Rename Dete Calculate Base Pairs page 5 77 1 Click the Volume Calculation button El or choose Volume Calculation from the 1D Gel menu to calculate base pair sizes in 6 3
142. m H 550 x 400 mm Up to five orders of magnitude 25 um maximum spatial resolution 16 bit 65536 gray scale levels 10 50 mW solid state YAG laser 50 60 Hz 100 W 100 110 115 120 220 240 V Ensure that the voltage and frequency of the power source correspond to FMBIO power settings Check the power settings on the specification panel on the rear of the machine Verify that the building power supply has a func tional ground Software Features The FMBIO software consists of two modules FMBIO Read Image and Image Analysis These applications function independently and need not be installed on the same computer FMBIO FMBIO Read Image This application controls the FMBIO scanning hardware synchronizes the fluorescent input signal with the scanning mirror and optical unit and converts this data into a bitstream Read Image is used to easily set the scan area scan resolution image orientation and photomul tiplier sensitivity The user can also choose filters add comments to be saved with the scanned image and perform hardware checks The experimental data collected are converted by Read Image into a 16 bit digital TIFF file for analysis with the Image Analysis software The scanning software provides a 16 bit gray scale image of fluorescence intensity with a broader linear dynamic range than possible with conventional autoradiography Image Analysis This application offers a user friendly interface for viewing and an
143. mation about the scan such as date researcher s name or particular experiment features in the Comment field in the Scan Control window The comment field holds up to 255 characters Comments are saved in a small text file associated with the image file You can also use Image Analysis to add comments to the image file Read When you have defined the scan area and all the scan settings you can begin scanning the sample e To begin a scan click the Read button Before the scan begins a Save As dialog box appears Use this window to name the image and select its folder location When you click the Read button Read Image estimates the size of the image file that will be created and compares that size to available disk space If the amount of available free disk space is inadequate an alert box appears to warn you of insufficient space and Read Image cancels the scan If there is adequate disk space the scan continues 4 18 FMBIO Read Image Software Depending on the size of the image and the stringency of the scan param eters scanning may take between five and ninety minutes As the scan progresses a low resolution raster image appears in the PreRead Display window When scanning is complete the data is assembled into the digital image file This last step takes a short time 4 19 Read Image Software FMBIO Additional Features The menu bar in Read Image software contains many standard commands found in IBM compati
144. n you can also choose to check the uniformity of the lanes If you choose Manual Input enter the number of lanes in the sequence then choose to either automatically determine the lane width or specify a width Lane width may range from 1 0 to 100 00 mm in 0 1 mm 6 Click OK By default Image Analysis displays a central line down each defined lane with additional lines marking the lane width When the lane is selected square edit handles are displayed on the lane lines 7 If any ofthe lanes need adjustment click on the lane to highlight it and move it to the correct position The Lane Style button l toggles 6 27 1D Gel Analysis F MBIO among different display options center line with lane boundary center line only lane boundary only and lane display off You can also choose a lane style from the 1D Gel menu Set Migration Lines page 5 50 Adjust the top and bottom migration lines according to the user s laboratory protocol Autoband page 5 60 With the lanes selected and the migration lines adjusted autoband determi nation can be performed 1 Select Normalized Volume IOD and choose Type 4 as the calculation type in the 1D Gel Preferences dialog box Preferences m mw 2 Click the Automatic Band Detection tab to edit the autobanding parameters 3 Try these settings and adjust the settings as needed for different gels Gradient Start 2 5 End 2 5 Duration 0 2 6 28 FMBIO 1D Gel Ana
145. nalysis for step by step procedures 5 2 FMBIO Image Analysis Tools Image Analysis Projects Double click the Image Analysis shortcut icon to start the Image Analysis program When the Image Analysis window opens a new project is automat ically created for you and a new Project window is displayed E Projecti ioj x B You can use this new project create another new project or open an existing project Creating a New Project e To create a new project click the New Project button or choose New Project from the File menu Opening a Project e To open an existing project click the Open Project button z Or choose Open Project from the File menu Saving a Project e To save a project click the Save Project button fel or choose Save Project from the File menu Give the project a name and click Save 5 3 Image Analysis Tools FMBIO Image Menu and Tools 1 Inthe Project menu choose New 1D Gel Experiment or New Array Experiment Project Window Help New 1D Gel Experiment New Array Experiment 2 Select an image file from the directory window The image icon is added to the project tree PPlex11 OE x Ff PPlext1 3 Double click the image icon to display the selected image Once an image is displayed the Image menu appears in the menu bar and the Image tool buttons become active 5 4 FMBIO Image Analysis Tools Image 1D Gel Window Help Gray Level Adjustm
146. nalysis allows you to save lane templates for future use When preparing a lane template it is recommended that the total length of each lane extends beyond the lowest band in the gel to allow for increased migra tion distance in future gels A lane template may be applied to any 1D Gel experiment Note When defining lanes for a template it is suggested that the total length of each lane extend beyond the lowest band in the gel to allow for increased migration distance in future gels To create a lane template l Click the Lane Template button Is or choose Lane Template from the 1D Gel menu The Template dialog box is displayed Template Delete Duplicate Rename Close 7Z5nly 2 Click the Add button In the Lane Template Saving dialog box deselect any lanes you do not want to save Image Analysis Tools FMBIO Lane Template Exporting 3 Click Save 4 Assign a unique name when prompted then click OK Lane Template Name Input Dial E3 5 The template name is listed with the number of saved lanes in brackets 5 56 FMBIO Image Analysis Tools To apply a lane template 1 Click the Lane Template button El or choose Lane Template from the 1D Gel menu then select the template from the list 2 Click Apply Image Analysis Tools FMBIO Defining the Migration Area You can adjust the top and bottom migration lines according to your labora tory s protocol Some la
147. ne Style Toggles among different display options center line with lane borders center lines only lane boundaries only and lane dis play off Band Style Toggles among the different band styles band display off peak and range bands displayed and only peak bands dis played 5 40 FMBIO Image Analysis Tools Multi Band Color Separation In a 1D Gel Analysis Image Analysis lets you perform separation all colors simultaneously using multiple bands within a lane The colors assigned to the bands collectively constitute a Multi Band Separation Parameter Set which is used to calculate the spectral overlap between the individual fluorophores Band Selection Parameters Before performing multi band color separation can be performed you assign values for the Band Selection Parameters which define the criteria for calcu lating the background band area and overlap between bands in a lane 1 Choose New 1D Gel Experiment from the Project menu Project Image Array windo New 1D Gel Experiment New Array Experiment The 1D Gel Experiment window Image Setting dialog box 1D Gel menu and its corresponding tool bar appear 2 Click the Preferences button q or choose Preferences from the 1D Gel menu 3 Click the Multi Band Color Separation tab This filecard displays the default Band Selection Parameter set 4 Try these settings and if necessary adjust them for different gels e Overlap 0 0 mm e Band A
148. nearest to the service door Close the optical filter service door 3 7 Operating the Scanning Unit FMBIO Preparing for Scanning You can use the FMBIO system to scan and generate an image of e Gels 1D and 2D e Membranes Southern Northern Western dot slot and TLC e Low and medium density arrays Opening the Cover Below is a figure that shows the location of the button which when pressed causes the top cover to open Press button to open cover Figure 3 4 Front view of FMBIO scanning unit When the cover is open you can access the glass plates and the scanner sample stage 3 8 FMBIO Operating the Scanning Unit Important When the cover is open the scanning unit cannot scan and the computer cannot start a scanning operation When closing the cover be sure to firmly press it down so that it is securely closed and the scanning unit can operate Glass Plates The scanning unit accommodates a glass plate that is 400 mm wide x 600 mm long and 5 0 mm thick 0 1 mm For best results only use low fluorescence borosilicate glass of Tempax or similar grade The use of other grades or sizes of glass may result in poor scanning quality The sample stage supports the sample plates on two sides only to allow the maximum area to be scanned and when the glass plate is properly seated on the scanner bed the scannable area is 400 mm width x 550 mm length Be careful when loading the sample stage
149. ned images in which very small or subtle differences in the elements of the image may be discerned The resolution or density of pixels dots in the digital image may be 75 150 or 300 dots per inch dpi in each direction Dot density of the image is determined in Read Image before scanning Due to two limitations inherent in video display monitors the image as it appears on the monitor is a rough representation of the actual image data present in the digital file First video monitors use 8 bit processing which means only 28 or 256 signal intensity gray scale levels are displayed per pixel Thus file signals that are close but not identical to one another in gray scale value appear to have the same intensity on the monitor Signals that are slightly above the low cutoff threshold may also be submerged in back ground on the monitor Second most video monitors have a resolution of 72 dpi Thus an image scanned at 150 x 300 dpi would contain 45 000 pixels in in the digital file while the monitor is capable of displaying only about 72 x 72 or 5184 pixels in File pixels are averaged together for display purposes with a con sequent loss of resolution in this case at a resolution loss ratio of nearly 9 1 The combined effect of these two limitations is that display images are apt to appear coarser grainier or blurrier than they actually are You can compensate for these limitations in a number of ways e Adjust gray scale
150. ng the STaR Call macro e Click Cancel to cancel the installation 8 Atthe end of the installation the following message box will inform you of a successful installation Click OK 10 4 FMBIO Allele Calling with STaR Call 9 Restart Excel STaR Call should automatically load when Excel is started It will appear as a menu item as shown below and the STaR Call spreadsheet will open Starting STaR Call STaR Call will automatically start whenever you load Excel If you want use Excel for other than STaR Call you may unload it by selecting Unload from the STaR Call menu To use STaR Call again you must restart Excel If you do not want STaR Call to start each time you run Excel you must remove it as described below Removing STaR Call 1 Make sure that Excel is not running 2 Locate the Excel startup folder The Excel startup folder location depends on which version of Office you are using Note If you are having trouble finding the Excel startup folder use the Find application Enter Command F from the desktop select Find items on local disks and name and contains Then type fmbiostr xla in the contains box and click the Find button 3 Remove the files fmbiostr xla and codisdbf xls from the startup folder Allele Calling with STaR Call FMBIO Short Tandem Repeats Short tandem repeats STRs are short repetitive DNA sequences two to seven base pairs long with alleles differentiated
151. not close the codisdbf xls table while using STaR Call because STaR Call uses it for allele calling If you do not see the STaR Call menu on the Excel menu bar choose New from the File menu to make it appear STaR Call Import STR Recalculate Create Merged Landscape Edit STR Cutoff CODIS STR Export Compare Analyses STR Lookup Table Options Help About Allele Calling with STaR Call FMBIO Command Description Import STR Imports up to 6 Image Analysis generated DAT files You can choose Plain with OD or with lOD Recalculate Recalculates all the DAT files in the selected analysis workbook and assigns alleles to each base pair Recalculate the DAT files after changing a STR Lookup table Edit STR Cutoff Allows you to edit the cutoff percentage values for assigning stutter bands CODIS STR Export Displays two options e Export v2 0 exports a STaR Call analysis to a CODIS import file Update CODIS values updates the valid CODIS values using an export file generated from the CODIS system Compare Analyses Compares the Merged sheets of two analyses workbooks STR Lookup Table Allows you to create edit and delete STR Lookup tables Options Allows you to choose the order of merged allele sorting and whether the phenotype or genotype is listed in the merged landscape worksheet Help Displays the help text About Displays information about the STaR Call p
152. oefficient of the spectrum curve It recognizes a signal peak as a band if the linear differentiation of the spec trum curve is greater than the value set for the gradient start and continues to exceed that value across a specified duration The gradient and duration values must also exceed a baseline or noise level You can adjust these four parameters in the Preferences dialog box Values you enter in the Automatic Band Detection filecard depend on the quality of your gel Heavily loaded and smeared lanes require more gradual gradients longer gradient durations and higher background settings Gels with faint or partially hidden bands require more stringent settings for these parameters Note For optimum band identification adjust the gray level to improve band appearance in the 1D Gel Experiment window See Procedure on page 5 20 5 60 FMBIO Image Analysis Tools Automatic Band Detection Preferences To set the Automatic Band Detection preferences e Click the Preferences button K or choose Preferences from the 1D Gel menu and then click the Automatic Band Detection tab Preferences Image Analysis Tools FMBIO Duration Noise Level Gradient End Title Description Gradient Start Gradient End Duration Noise Level Defines the minimum change in signal intensity necessary to signal the leading edge of the band This parameter combined with the duration parameter defines the slope of
153. olor Sepa ration Note Special care should be given to match the template with the correct channel Select the templates for the Band Selection Parameters and the Band Information from the drop down menus Select the calculation type from the drop down menu Select the Lane Template from the drop down menu If you want to use auto lane detection click the On radio button to select it Choose the Conventional or Multi Band separation method If you choose the Conventional method select a tem plate from the drop down menu If you choose the Color Separation method select a Color Separation template and a Parameters template from the drop down menus Choose the templates and enter the appropriate parameters in the fields If you want to use this set of Batch Control settings again click on the Save Load Setting button The Template dialog box appears Click the Add button and enter a name for the Batch Control template Alternatively if you want to Load a previous Batch Control template select the template by clicking on it s filename in the list box Then click the Apply button When you have finished entering the parameters click OK in the Batch Control dialog box Image Analysis processes the 1D Gel experiment 5 97 6 1D Gel Analysis One dimensional 1D analysis entails studying the migration patterns of molecules that have been separated and spread out in one linear dimension by the passage of a
154. or Codes In the process of using Read Image software you may occasionally see an error code number appear on the screen Error code 161 162 163 164 165 167 or 169 Perform the following steps 1 Press the Power switch to the Off position and wait at least 5 seconds 2 Press the Power switch to the On position The FMBIO performs a self diagnostic routine e If the error code appears again repeat steps 1 and 2 one more time 3 If the error code persists call Hitachi Software Engineering service personnel Error code 166 Insert a filter holder in either Channel 1 or Channel 2 and then restart the scan Error code 168 Firmly close the scanning unit cover Error code 192 There is an internal memory shortage Error code 193 There is an external memory shortage Error code 198 The optical unit did not return to the home position 4 32 5 Image Analysis Tools With the FMBIO scanning unit and Read Image software a single scan can create a multi color image of a gel that contains one two or more fluoro phores in one scan In Image Analysis you can view and analyze the scanned image You can run fluorophores of two or more different emission spectra in the same lane or gel for comparative allele analysis Highly accurate size deter minations can be achieved when an internal lane standard is run in the same lane with a sample If a sample contains two or more fluorophores and you designate two or more
155. plate on the rear of the equipment Use of other than the proper power specifications may lead to accidents and equipment malfunction xi 1 Overview Advantages of the FMBIO System Thank you for purchasing Hitachi Software Engineering Company Limited FMBIO scanning unit a device for reading the electrophoresis patterns of fluorescent dye marked samples FMBIO represents a maturing of fluo rescent technology that has been applied to life sciences labs since the 1960 s Some of the major benefits associated with the system are improved safety speed accuracy and lower cost Read this manual carefully before attempting to operate the system and care fully heed all safety warnings Improved Safety The FMBIO system eliminates the risks to lab personnel the need for radio active shielding and the storage and procurement problems associated with handling I C S and P Faster Results With the FMBIO speed of experimental throughput is substantially greater because scanning and analysis are independent of electrophoresis Many more gels can be run and scanned with the FMBIO than with a dedicated electrophoretic sequencer The FMBIO reads gels membranes and plates without the need for x ray film or costly phosphor screens Over and under exposure problems are eliminated Scan times for the FMBIO are typically five to ninety minutes considerably less than the eight hours development time required for conven tional rad
156. played Choose folder x ase sie gal en fes m 4 Specify the folder to store the files and click Select Note The window will automatically choose the Image Analysis program folder as the default folder and create filenames using previously entered information i e Lane Project Name ch csv 5 Open the desired file in Microsoft Excel C dickson logger data Folder 6 12 01 4 21 PM C3 Intergen Folder 1 18 01 10 41 AM mavis noab Folder 4 19 01 1 39 PM Folder 6 9 00 8 13 AM 3KB Microsoft Excel 1 18 01 11 27 AM 3KB Microsoft Excel 1 18 01 11 27 AM Image1 60sloc txt 19KB Text Document 12 4 00 2 19 PM 6 The spreadsheet displays the migration distance mm and the intensity 5 92 FMBIO Image Analysis Tools OD of the trace Vol 13485968 12342197 12438081 13639419 12599474 13372057 13017182 114023307 13347073 113321282 12214062 13477255 12813153 13619474 Spons Spott6 Spot 17 Spot8 SpotiS Spot20 Spot21 Sen Spot23 Spo24 Spot25 Spot26 011699 Spot27 13739011 Spot28 13692676 Spot23 13063096 Spot30 13720078 Spot31 13164886 Spot32 IE 5 93 Image Analysis Tools FMBIO Printing Image Analysis allows you to print an image experiment standard curve spreadsheet or overlay trace You can also customize t
157. puters if desired FMBIO FMBIO System Scanning Unit Features The FMBIO scanning unit detects laser induced fluorescent signals on gels blots and thin layer chromatograms It accommodates plate sizes up to 600 x 400 mm with a maximum reading size of 550 x 400 mm The excitation sources are two or three solid state laser The laser s sample unit moves across the optical unit Y direction as its laser beam is directed onto the sample X direction via a polygon mirror rotating at high speed The resulting fluorescent light signals emitted from the excited fluorophores are then collected by two optical fiber arrays The instrument also features two photomultiplier tubes a large scan area of 55x40 cm and a linear dynamic range of four orders of magnitude The FMBIO produces extremely high resolution images capable of resolving even single base microvariants Fluorescent probes that are responsive to the frequency of the laser beam emit fluorescent signals upon being excited The emitted light is collected by a lens and directed into a fiber optic array which passes it to an interference filter The targeted fluorescence wavelength passes through the interference filter and then to a photomultiplier where the light signal is converted to a digital signal Conversion of the digital signal to experimental data takes place in the data acquisition circuits where fluorescence detected at the pho tomultiplier is synchronized with the angle of
158. r to the software license and warranty agreement No part of this document may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying and recording without the express written permission of Hitachi Software Engineering Co Ltd Hitachi Software Engineering Co Ltd reserves the right to make changes and improvements to the FMBIO product at any time and without prior notice Trademarks and Patents The FMBIO fluorescent scanning system is protected by U S Patents Nos 5 121 320 5 069 769 5 242 567 5 246 866 and 5 190 632 The Apple logo and Macintosh are registered trademarks of Apple Computer Inc IBM is a registered trademark of International Business Machines Inc FMBIO and DNASIS are registered trademarks of Hitachi Software Engineering Co Ltd Other brands and product names are registered trademarks trademarks or tradenames of their respective owners Polymerase Chain Reaction PCR is protected by U S Patent No 4 683 202 Devices and accessories used in conjunction with FMBIO may be covered by brand and product names trademarks or patents not specifically referred to herein Copyright November 1999 T152 7 003 E All Rights Reserved Image Analysis for Windows r v3 0 Software License Agreement BEFORE INSTALLING THIS SOFTWARE YOU SHOULD CAREFULLY READ THE FOLLOWING TERMS AND CONDITIONS BY OPENING THIS PACKAGE YOU AGREE TO BECOME BOUND BY THE TERMS AND CON
159. r up the FMBIO automatically performs an autofocus before beginning the first scan Channel 1 does not have to be active during an autofocus but it must contain the 605 nm filter After the initial autofocus you may remove the 605 nm filter and replace it with a filter that accommodates a detection range compatible with the stain or dye you are using on your sample 3 4 FMBIO Operating the Scanning Unit Inserting Optical Filters CAUTION Scratched or dirty filters interfere with precise laser detection Avoid directly touching the surface of the optical filter Use a lens blower to remove dust on the filter For more stubborn dirt use lens cleaning paper Prepare for a scan by placing the appropriate filters in each channel Only active channels collect image data during the scan See Selecting Active Channels on page 4 14 for information about active channels The FMBIO scanning unit can accommodate two filter holders Each filter holder can hold two filters You must place the appropriate filters in the scanning unit before beginning a scan Once the scan begins the optical unit moves out of home position and the filters are no longer accessible Note If this is the first scan after turning on power to the FMBIO unit insert the 605 nm filter in the Channel 1 position If the automatic initial autofocus has been completed turn off Auto Focus in the FMBIO menu and replace the 605 nm filter with a filter appropriate to your sc
160. re 2 3 Understanding Personal Computers 2 4 Setting Color Display 2 4 Printer 2 5 Peripheral Storage Devices 2 6 Operating the Scanning Unit 3 1 Starting the FMBIO Scanner 3 2 Self diagnosis Normal 3 3 FMBIO Error Detected Autofocus Calibration Inserting Optical Filters Preparing for Scanning Opening the Cover Glass Plates Scanner Sample Stage Loading the Sample Scanning the Image Beginning the Scan Stopping a Scan Scanning Guidelines Read Image Software Opening Read Image Read Image Files Setting the Parameters Additional Scanning Parameters Features of the Parameters Window Defining the Parameter Name Selecting SCSI ID Setting the Reading Resolution Reading Sensitivity Setting Repeats Focusing Point Settings in the Command Menu Preparing to Scan Setting Gray Level Adjustment Defining Image Orientation Settings in the FMBIO Menu Performing an Autofocus 3 3 3 4 3 5 3 8 3 9 3 9 3 10 3 12 3 12 3 12 3 13 4 1 4 10 4 10 4 10 4 12 4 13 4 13 Selecting Active Channels Scan Control Window Using the Scan Control Window Defining Scan Area Adding Comments Read Additional Features File Menu Command Menu FMBIO Menu Imaging Basics Multi Color Images Long Pass Filters Example Scanning Process Read Image Error Codes Image Analysis Tools Image Analysis Projects Creating a New Project Opening a Project Saving a Project Image Menu and Tools Saving an Image Viewing a Multi Color Image Image Setting Dia
161. re activated 2 Inthe View menu choose to display the Draw 1 Draw 2 Draw 3 and Text Attribute toolbars 8 2 FMBIO Drawing Tools Right click Menu You can also choose the line and paint attributes by placing the cursor over the window and right clicking the mouse Line Width Setting the Margins To create a margin for annotations around the image 1 Click the Paper Position button F The Comments Area Settings dialog box is diplayed 2 Type in the desired top bottom left and right margins in pixels and 8 3 Drawing Tools FMBIO click OK The margins are added to the image Da I VTITT INI TTI Rotating Text To rotate text boxes 1 Select the text box you want to rotate GammaSTR Loci D16 8 4 FMBIO Drawing Tools 2 Click the Rotate text button The Rotation Setting dialog box is displayed Rotation Setting 3 Choose to enter a free rotation angle or select a fixed rotation from the drop down list box and specify the angle Rotation Setting The text box is rotated clockwise through the angle Drawing Tools FMBIO 4 Position the text box as desired 0 o a e e G E E 0 e 1 ut 645 58 8 6 Draw Tools FMBIO Drawing Tools Tool Draw 1 Function Select Tool Line Tool Rectangle Tool Ellipse Tool Paper Position x s n o e e A c Select Tool Line Tool Rectangle Tool Ellips
162. rea 0 1 mm e Background Area 1 0 5 mm e Background Area 2 0 5 mm e Background 100 Note If you plan to use these parameter settings in the future it is nec essary to save a template with a unique name 5 41 Image Analysis Tools FMBIO Preferences 1 Overlap Specifies the width of the band used in the area calculation when checking for band overlap e fthis Overlap value is 0 0 mm the overlap will be the width detected by the Auto Band tool e If there is no overlap the band area is the total that is calcu lated by the Auto Band tool e When the Overlap value X is greater than 0 mm the width used to calculate the area will X to X from the peak maxi mum e Overlap values may range from 0 99 9 mm with a minimum increment of 0 1 mm 5 42 FMBIO Image Analysis Tools a Peak Maximum N N N N Manually Set Overlap Width a Peak Maximum Detected Area m Overlap Per Overlap Parameter e A Manually Set Overlap Width Detected Area Band Area Specifies the area that is used to calculate the volume for color separation e Ifthis Band Area value is 0 mm the band area will be the area detected by the Auto Band tool e Ifthe Band Area value X is greater than 0 mm the band area will be X to X from the peak maximum e Band Area values may range from 0 to 99 9 mm with a mini mum increment of 0 1 mm Peak Maximum an Area Us
163. rid document 7 3 menu 7 10 tools 7 2 A 11 Medium Grid menu 7 1 membrane 4 7 merged landscape 10 19 worksheet 10 18 migration distance 6 6 modifying grid 7 9 spot boundary 7 5 mol calculation 7 18 Mono display mode 5 11 Multi Band Color Separation tools A 9 window 5 44 multi band color separation 5 41 multi color project assigning color 5 11 multiple lanes defining 5 51 N n 4 stutter percentage 10 15 net migration area 5 58 new project 5 3 No of Reading s 10 23 noise level 5 62 Normalized Volume IOD 5 78 O opening ReadImage settings 4 20 operating scanner 3 1 fo 3 13 optical density 6 6 orange glass filter 4 25 orientation of glass plate 3 10 of scanned image 4 12 Over display mode 5 11 Overlap 5 42 Overlay Trace Setting window 5 90 Overlay Trace window 5 87 vertical zoom tools 5 89 P Parameter Name default settings 4 7 FMBIO Index Parameters window features 4 6 to 4 9 partial quantity 7 16 Paste Values 10 12 pause scan 3 5 peak area background subtraction 5 78 photomultiplier tube 4 8 9 2 pixels 4 10 5 13 population group 10 25 Power switch 3 2 preparing allele evaluation 10 7 data for CODIS 10 21 PreRead 3 13 4 18 prescan image 4 17 print preview 5 95 setup 5 95 printing STaR Call 10 21 Project menu 5 4 5 37 6 2 window 5 3 project creating 5 3 opening 5 3 saving 5 3 Project menu 7 3 Q quantification marker 5 79 quitting ReadImage 4 20 Index 5 Index FMB
164. rogram 10 10 FMBIO Allele Calling with STaR Call Options To open the options dialog box choose Options from the STaR Call menu STaR Call Options You can choose the sorting order for the alleles in the merged worksheet The worksheets for the individual DAT files will sort alleles in ascending order You can also select whether single values representing phenotypes or duplicate values representing genotypes are displayed in the merged landscape worksheet Note If you are exporting data to CODIS choose Phenotype STR Lookup Table A STR Lookup table is used when evaluating an STR table The lookup tables are saved in a file called codisdbf xls 10 11 Allele Calling with STaR Call FMBIO J Ele Edt view Insert Format Tools Data Window Help STR Cal BEE TAPETE amp 4 mes as S S 2 promot T At E PowerPlex CTTv with Am 9 3 A B c ESTEE G 1 Sa a M 1 PowerPlex CTTv with Am 9 3 STR OD IOD Cutoff Table 2 3 Ranges Cutoff 4 From To Genotype Paste Values Expected Locus From To 5 32494 32694 CSFIPO 15 100 100 325 94 327 00 ICSFIPO 15 B 32114 32314 CsFIPO 14 100 1 00 322 M 323 00 TPOX 0 151 ER 31722 319 22 CSF1PO 13 100 1 00 318 22 319 00 THO T 0 151 B 313 29 315 29 csF1PO 12 10 10 31429 31500 Mwa i 15 9 30936 3113
165. rotation of the scanner mirror and with the horizontal motion of the sample unit to produce positional values in the sample See Figure 1 1 FMBIO Lens assembly Polygon mirror Laser source Photomultipliers Figure 1 1 Interior view of FMBIO scanning unit Multicolor imaging is achieved using band pass detection filters to discrim inate light from fluorescent dyes emitting between 300 and 700 nm Up to four filters can be stored in the instrument and accessed through software Two filters can be used simultaneously to detect emissions from two dif ferent dyes In multicolor analyses the gel is scanned after electrophoresis using optimal wavelength band pass filter to detect various flurorescent dye labeled products These images can be overlaid into a multicolor image or viewed separately The maximum number of overlay is 4 color 1 4 FMBIO Specifications Weight Outside dimensions Operating temperature Operating humidity Laser excitation wavelength Emission wavelength Detection sensitivity with polyacrylamide gel Plate size max Scanning area max Dynamic range signal readability Resolution Laser power Power frequency Power consumption Power supply AC FMBIO 80 kg 87 cm W x 65 cm D x 35 cm H 15 30 C 20 80 relative humidity 635nm 532 nm 488nm optional Selectable 300 700 nm Rhodamine 0 1 fmol band Fluoroscein 0 2 fmol band 600 mm W x 400 mm D x 5 m
166. rray Analysis Set Selected Spots to Background Set Selected Spot s to Marker Calculation Open Standard Curve Ungroup Open Spreadsheet Amela Grid Comparison Set Grid Marker Group Array Template Preferences Creating an Array Analysis To create an array analysis experiment 1 Create a new project 2 Choose New Array Analysis in the Project menu Project Image Array Windo New 1D Gel Experiment New Array Experiment 3 Select a file that contains a digital image A new Array experiment is added to the project tree and the Array menu appears in the menu bar 7 3 Array Analysis FMBIO gridtest Inverted oject gridtest Inverted J Medium Grid Document 4 Double click the Array Experiment icon An Array Experiment window containing the image is displayed ERE ER MA B Document 5 To deselect an item remove the checkmark adjacent to it 6 Click OK 7 4 FMBIO Array Analysis Analyzing Spots in an Image To analyze spots in an image you must first create a boundary surrounding each spot of interest You can either create a single boundary or create an evenly spaced grid over a region with many spots A single spot can be a rectangle or an oval In addition you can specify a region to analyze within the spot area Creating a rectangle or oval spot boundary l Click the Create Rectangle Spot or Create Oval Spot i5 hie 2 Drag the cursor to draw a rect
167. sis 3 Use the Automatic Band Detection tool to define bands see page 6 11 Check bands and edit as necessary Register marker lanes using the Logarithms curve fit see page 6 14 5 Inthe 1D Gel menu choose Preferences In the Calculation filecard choose Normalized Volume and Type 4 see page 5 78 Note Excel spreadsheets accommodate up to 256 data columns If the gel image contains many lanes you may need to limit the number of result types on the table to assure all data is imported into Excel You can safely eliminate mm Rf and IOD 6 Click the Volume Calculation F3 button to generate results see page 5 85 10 7 Allele Calling with STaR Call FMBIO Exporting 1D Gel Analysis Results 10 8 Click the Open Spreadsheet button ai or choose Open Spreadsheet from the 1D Gel menu or to display the table of analysis results If the image is a multi color project Image Analysis displays results for each channel In the SpreadSheet menu choose Export Indicate where you want to save the tab delimited text file name the file then click Save Save any changes to the project FMBIO Allele Calling with STaR Call Using STaR Call to Evaluate Alleles After you analyze a 1D gel and export the results you are ready to use STaR Call Genotyping Software for allele calling 1 Open Excel STaR Call appears as a menu on Excel s menu bar and a table named codisdbf xls appears Important Do
168. sition the tool at a corner of the lane above the top migration line Drag the cursor down to the opposite corner below the bottom migration line until the bands are contained in the boundary Click the Automatic Band Detection button l or choose 1D Gel gt Function gt Auto Band After Image Analysis has autobanded the lane click the Multi Band Color Separation button or choose Multi Band Color Separation from the 1D Gel menu Using the Band Merging parameters Image Analysis assigns a color to each band and the Multi Band Color Separation window is displayed The color of the Detected Band number indicates the color assigned by Image Analysis The numbers of the detected bands appear in the Status 6 23 1D Gel Analysis F MBIO 10 11 Edit Sheet Lane am araneieneer Template Lane Selector II No Status NON ON Lane Window L j Magnification fio gt Copy gt gt Merge Setting Selector ce If you are not satisfied with the separation of the colors adjust the Multi Band Color Separation preferences Click the Set Multi Band Parameters button and change the parameter values see Band Selection Parameters on page 5 41 When you click OK the colors are automatically separated according to the new values After the Multi Band Color Separation window is displayed click the Copy gt gt button or load a previously generated Multi Band Separation
169. spots and grids to change their parameters simultaneously To Group spots grids 1 Hold down the Shift key and then click the single spots or grids you want to group Any type of spot or grid may be selected 7 9 Array Analysis FMBIO o e 2 Choose Group from the Array menu se 3 Change the spot parameters Your changes will affect all spots in the group To Ungroup spots grids e Click the group to select it and then choose Ungroup from the Array menu Note In the Array menu if Group is grayed out and Ungroup is active then the spots grids are already grouped If Group is active and Ungroup is grayed out then the spots grids are not grouped Creating a Template Image Analysis allows you to save spots or grids as templates for future use You can also duplicate and rename saved templates To create an Array template l Click on the Array Template button Bl Array Template dialog box is displayed 2 Click Add The Select Spots and Grids dialog box lists the spot and grid elements in the image Select the spots and grids you want in the tem FMBIO Array Analysis plate by clicking the checkboxes or using the Select All and Clear buttons 3 When you are finished click the Save button The template appears in the list preceded by the number of elements contained in it To apply a template 1 Click on the Array Template button The Array Template dialog box is displayed 2 Click on t
170. stances fire or electric shock may result Never under any circumstances repair the equipment by yourself Unknown Cause of Damage If the cause of damage is unknown and the prompting image fails to appear then refrain from using the Scanning Unit e Immediately turn the power switch to OFF e Disconnect the power cord e Contact your nearest authorized Hitachi Software Engineering Service representative 10 Allele Calling with STaR Call STaR Call Genotyping Software converts base pair values generated by Image Analysis to allele names STaR Call takes advantage of the quanti tation features in Image Analysis to perform automated genotype calling and analysis of artifact stutter bands First the calculated band sizes are imported into STaR Call STaR Call then compares these values with a selected STR Lookup table that contains size ranges for each allele Based on comparison of band sizes with allele ranges bands are assigned a locus name and repeat number Weak bands occurring in the stutter position are iden tified as artifacts and are excluded from the final output Genotype data generated by STaR Call are easily converted to common message format CMF files for export to the Combined DNA Index System CODIS forensic database This chapter discusses how to apply STaR Call to evaluate alleles and stutter band artifacts If you are interested in evaluating STaR Call Genotyping Software or obtaining a quotation p
171. stogram to show the range between the high signal and background cutoff thresholds Mapping Type Selector The mapping type defines the method used to translate 16 bit gradation 0 to 65535 into 8 bit gradation 0 to 255 The gray level gradations between the high signal cutoff threshold and the background cutoff threshold are divided into 255 equal parts To view the mapping curve select the Show Mapping Curve option FMBIO Image Analysis Tools Mode Description Equal Range Equal Area Range of the histogram between the high signal cutoff threshold and background cutoff threshold divided into 256 equal parts 10000 Note Use the Equal Range mode to display the relative concentration of the bands Area of the histogram between the high signal cutoff threshold and background cutoff threshold divided into 256 equal parts 10000 Note Use the Equal Area mode to enhance detection of faint bands this mode is NOT recommended for determining the relative concentration of bands 5 19 Image Analysis Tools FMBIO Mode Description User Defined e Drag any of the four rectangular points to define the mapping curve 0 10000 Note Use the User Defined mode to customize your mapping curve Procedure The gray level for each channel is adjusted by setting the background and high signal cutoff thresholds You can set these thresholds in three different ways e Drag threshold boundary
172. t Merged Landscape Shows a summary of allele calls It does not display the following e Lanes labeled as allelic ladders e Lanes marked Exclude in the CODIS portion of STaR Call e Bands with values reported in the Percentages column stutter bands e Bands marked Not in range 10 19 Allele Calling with STaR Call FMBIO Ir A B DIEFIGI H t J K E M N O P G zi 1 Population CSFIPO TPOX Amelogenin THO1 vWA D16S539 D7S820 2 Lane No Specimen No Group A22 see en he I ee Ss Ree 39 Lane2 10 8 11 X 5 193 9i113 912 4 8i j L8 Lane3 9 7j10 X 519 8i12 8 tl B 7 1 11 i E Lanes 9 i to x Y 5 93 8 2 38 im 8 811 i 9 Lane6 10 12 7 9 i93 17 8 10 719 10 T i n i i l i z M4 WZFFFL TMR ABBNew3CH PowerPlex 2 1 TMR Merged Me 4 4 Carefully evaluate bands that STaR Call has not assigned to a genotype Some Not in range bands may be only slightly outside the allele range You can correct these calls as needed Adding an STR 1 Select the analysis workbook 2 Choose Import STR from the STaR Call menu 3 Click Yes when asked if you want to add a file to the current analysis 4 In the Import STR dialog box click any available Browse button to select a file 5 Selecta STR Lookup Table in the drop down list box Recalculating Results To recalculate results e Choose Recalculate from the STaR Ca
173. t Print Windows Help Paste Copy Name Usage New Project Open Project Save Project Cut Copy Paste Print Windows Help Create a new project Open an existing project Save a project Remove selected object and store in the clipboard Copy selected object and store in the clipboard Place contents of the clipboard at the cursor Print the contents of the active window Place the What s This cursor on a Windows element and click to see Help topic Appendix A Image Tools Zoom Out Zooming Tool Zoom In Fit Window Moving Tool Magnification Input List Box Tool Description Zoom Out Each click reduces the image Zoom In Each click enlarges the image Magnification Displays current magnification Enter the Input List Box desired magnification or select a magnifi cation from the drop down list Zooming Tool 1 Click the button The cursor transforms into a magnifying glass with a sign 2 Drag the magnifying glass cursor to create a rectangle surrounding a region of the image The image is magnified to show the region Fit Window Click to fill the entire window with the image Moving Tool Click the button and then drag the image Appendix A Gray Level Adjustment Color Separation Show Image Setting Dialog Show Spectrum Area Selection LIne Selection Image Information Tool Description Gray Level Adjustment
174. t in range it is not included in the stutter calculations After the evaluation is complete a workbook is created containing work sheets containing data from the individual DAT files the corresponding allele evaluations with the selected lookup tables and merged worksheets containing results from all the DAT files To display a worksheet click on its tab at the bottom of the window If you cannot view the entire worksheet click on the arrows in the scroll bar at the bottom right of the window If you cannot see the tab for a worksheet click on the arrows at the bottom left of the window The following types of worksheets are created Summary Worksheet Provides a summary of the analysis STR import and any comparison performed using the Compare Analysis function E Ele Edt view Insert Format Tools Data Window Help STaR Call 2181 x OSM S S me o wr mn lt s BLU EES A gt m Al ao E Summary E A B m D E F G H TT F 1 Summa 2 3 4 Sheet Name Lookup Table Comments Total Lanes BP Found OD Found IOD Found x 5 DAT File 1 CTIvVA amp BNew3CH PowerPlex CTTv Am 9 3 T YES YES 6 DAT File 2 IDDDDAS BNew4CH GammaSTR DDDD new D7 i YES YES 7 DAT File 3 FL A amp BNew4CH PowerPlex 2 1 FL y 4 YES YES 8 DAT File 4 TMR A amp BNew3CH PowerPlex 2 1 TMR T YES YES 9 DAT File 5 D DAT File 6 Additional Information 13 DAT Conversion Type 00 15
175. t on page 5 13 pou eE 5 pixel r Channel Setting Target Channe Ch 1 s Color Red b Gray Bottom 1 096 78402218 Gray Top 1 0 127930156 01 Gray Setting Note You can also use the Gray Settings dialog box to adjust these threshold values 4 Click the Gray Scale button The Gray Scale Setting dialog box is 7 26 FMBIO Array Analysis displayed Gray Scale Setting 5 Choose to use percentage of total pixels or raw values as the gray scale unit for the thresholds Type values for the top and bottom gray scale thresholds in the fields If desired you can choose to use the optimal gray mapping mode This mode is equivalent to the Equal Area mode See Mapping Type Selector on page 5 18 8 Click the Screen Setting button The Screen Setting dialog box is displayed 9 Specify the Screening and Viewing options Note You can choose to view a simple subtraction of the Channel 2 value from the Channel 1 value for each spot or increase the gray scale value of weak spots The latter can be used for detection but is not recommended for quantification by visual inspection Screening Setting 78 402 218 00 Array Analysis FMBIO 10 Apply the screening settings to the grid overlay by clicking the On Off Pickup Mode button Q 11 If desired you can copy the image to the clipboard by clicking the Capture Image button S 12 Click the Pickup Screened Spots bu
176. ter Focusing Repeat Parameters for the scan Horizontal and vertical scan resolution SCSI ID assigned to the FMBIO scanning unit User defined label for experiment User defined label for channel Percentage of the collected laser signal is amplified by the PMT at each channel Filter used for the channel Focal length range 2 to 3 mm Number of laser passes per scan line FMBIO Read Image Software The most appropriate parameter values for your experiment depend on image quality signal to noise ratio sample volume heavy bands or faint bands and fluorophore chemistry Improper scan parameters may lead to images saturated with signal or images so faint that missing important elements such as weak bands are missing If in doubt it may be helpful to scan a portion of the image a number of times using different parameters then choosing one set of parameters for the entire image 1 In the Parameter Name pull down list choose the material type you want to scan Each parameter name is associated with default settings for the scanning parameters 2 Examine the parameters in the window and modify them as needed to accommodate your particular experimental conditions 3 Click OK to apply the scanning parameters to your scan See page 4 15 for information about defining scan area Defining the Parameter Name The Parameter Name list contains various types of scannable material Five parameter names are available
177. ters for color separation Color Separation Select Fluoro ed phore ac EN Background Noise 767 Set Emission Overlap Ch 1 Ch 2 Ch 3 Ch 4 v v v v Mode To select a background for a particular channel locate a band that is known to be in that channel Then select an area that is blank next to that band and then click Set button User must do this for each and every channel before Le 102DAWNF 1 mend mi 102DAWNF z 0 m 102DAWNF BE 102DAWNF 4 o o Set Jesi Preview Template setting each color separation parameters Also while in Image gt Color Separation dialog box users CANNOT assign or change any channel s color The color assignment has to be changed in Image Settings dialog box before bringing up Color Separation dialog 6 FOUR CHANNEL SUPPORT To have four channels support to function properly the image must be in OVER mode or MONO mode Contents For Safe Operations Precautions ix Overview 1 1 Advantages of the FMBIO System Improved Safety Faster Results Greater Accuracy Lower Costs Simplicity FMBIO System Scanning Unit Features FMBIO Specifications Software Features Fluorescent Labels Installation 2 1 ee ae a ee a a a ch o ima Onnwo dNNNM aA a oa Preparing for Installation 2 1 Contents of FMBIO Shipment 2 1 Installing the Scanning Unit 2 2 Installing the FMBIO Softwa
178. the function Defines the minimum change in signal intensity necessary to signal the trailing edge of the band This parameter combined with the duration parameter defines the slope of the function Defines the gradient length in the leading and trailing edges of the band Defines the baseline signal intensity Gradient and duration must exceed noise level if the signal is to be recognized as a band Choose a value between 0 and 255 Choose higher values for gels with dirty or noisy back ground Choose lower values for clean or quiet gels Evaluate the noise level of your gel by trying different Gray Scale Adjustment values see Procedure on page 5 20 5 62 FMBIO Image Analysis Tools Using the Auto Band Tool To use the Automatic Band Detection tool 1 Choose Select All from the Edit menu to select all lanes for automatic detection or hold down the Shift key and click the lanes Click the Automatic Band Detection button gt or choose 1D Gel gt Function gt Auto Band b X DUDUULUE For each detected band Auto Band creates a Band Start line Band Peak line and Band End line The migration distance is also calculated Band Start line amp Band Peak line DET Band End line Check the accuracy of the automatic detection Switch from one to the other channel by selecting the desired channel on the Image Window Setting dialog box See Image Setting Dialog
179. ther saturated or faint images The Gray Level Adjustment parameters modify the signal intensity of each dot or pixel in the scanned image A pixel is the smallest unit of a scanned image and each channel in an FMBIO II file contains 16 bits per pixel or 2 65 535 grayscale levels starting at 0 Most computer monitors are 8 bit and can therefore only display 256 shades of gray Image Analysis transforms the 16 bit image for display on an 8 bit monitor averaging the additional 16 bit values within each of the 256 8 bit grayscale levels to give an 8 bit value The Gray Level Adjustment tool tells your computer monitor how best to display your image It allows you to optimize your image by assigning these 256 shades of gray to only the region containing sample signal Note The raw image data is not affected by the gray level settings Cutoff Thresholds Image Analysis assigns a background cutoff threshold and a high signal cutoff threshold for each image Cutoff thresholds are applied to the range of 65 536 shades of gray generated during a scan Back ground or noise lies at the low end of this range signal lies near the high end of this range Signals below the background cutoff threshold appear white in the image signals above the high signal cutoff threshold appear as black The range between the background and high signal cutoff thresholds signals is trans formed into 256 gray levels Image Analysis Tools FMBIO imag
180. ting Select the target channel and color Histogram Slider adjusts the top and bottom gray scale signal threshold Gray Setting Displays the Gray Setting dialog box 7 24 Screening Setting On Off Pickup Mode Capture Image Pickup Screened Spots amp 9 amp E FMBIO Array Analysis Item Description Screening Setting On Off Pickup Mode Capture Image Pickup Screened Spots Displays the Screening Setting window Applies the screening settings to the overlay to detect spots Copies overlay image to the clipboard Displays the comparison data in the Selected Spots spreadsheet 1 Spot Radius 5 pixel Channel Setting Target Channe Ch 1 Color a Gray Bottom 1 0 78402218 Gray Top 1 0 127930156 01 na Adjust the spot radius by using the sliding control bar 7 25 Array Analysis FMBIO The spot size changes dynamically in the overlay image Data Sheet Overlay sev A v Ex 2 Choose the target channel and its color from the drop down list boxes 3 The histogram displays the pixels vs raw gray scale value Using the blue slider on the histogram to adjust the top gray scale threshold and red line slider to adjust the bottom gray scale threshold for the target channel The threshold values and percentages change dynamically as you move the sliders For an explanation of the histogram and thresholds see Gray Level Adjustmen
181. tion Show Hide Comment alel 0M Band Style Tool Description Show Hide Lanes Show Hide Band Information Show Hide Comments Lane Style Band Style Toggle display of lane boundary lines Toggle display of band information Toggle display of label information entered in spreadsheet Toggle among different display options center line with lane borders center lines only lane boundaries only and lane dis play off Toggle among the different band styles band display off peak and range bands displayed and only peak bands dis played Appendix A Overylay Trace Set Marker Volume Concentration Open Spreadsheet Multi Band Color Separation Preferences Quantification Setting Open Base Pair Standard Curve Tool Description Overlay Trace Set Marker Volume Calculation Open Spreadsheet Open Base Pair Standard Curve Quantification Setting Preferences Multi Band Color Separation Displays new overlay trace of selected lanes Displays Marker Setting window Performs a volume calculation Displays spreadsheet Displays Base Pair Standard Curve win dow Displays Quantification Setting window Displays Preferences dialog box Displays Multi Band Color Separation window Appendix A Multi Band Color Separation Tools Not Band Skip mE mum ee es s Overlap Band Colors None Tool Description Band Colors Select a band
182. tion rectangle is displayed If nec essary adjust the rectangle by dragging the selection handles Image Analysis Tools FMBIO 4 Click the Spectrum View Button ata or choose Show Spectrum from the Image menu The Spectrum window displays an overlay trace of the signals with the areas of signal intensity represented by peaks sm 10 Normalize Baseline Display Channel Ej Switch Axis 5 26 FMBIO Image Analysis Tools Tool Description Noramlize Baseline Normalizes vertical range Switch Axis Switches between the horizontal and vertical spectrum views Display Channel Toggles display of spectrum for individual channels 5 To change the dimensions of the graph resize the window Normalize Baseline tool Click the Normalize Baseline tool to normalize the vertical range by converting the low end of the range to 0 zero and the upper end of the range to 255 Use this feature to compare lane traces from different image files or from files created by different filters Before the vertical range is normalized it represents the range between the high and low Cutoff Thresholds on the Gray Level correction The low and high ends of the vertical range before normalization lie somewhere within the range of available pixels in a 16 bit file from 0 to 65 535 On this range spots on the image that appear black or saturated may actually have a signal above the high cutoff threshold However when the vert
183. to be repeated unless the scanning unit is moved or bumped The FMBIO always performs an autofocus at the beginning of the first scan after the unit is turned on If you do not check the Auto Focus command an autofocus occurs only once at the beginning of the first scan after you turn on the power switch which is on the front of the FMBIO scanning unit When a checkmark appears before the Auto Focus command an autofocus occurs before each scan The 605 nm filter should be in Channel 1 during the autofocus Performing an autofocus as part of a routine scan Choose the parameter settings and load a sample for a routine scan When FMBIO completes the 4 13 Read Image Software FMBIO autofocus it begins the scan If Channel is active Read Image creates an image file for the emission wavelength that passes through the 605 nm filter 1 Place the 605 nm filter in Channel 1 See page 3 6 for directions 2 Select Auto Focus in the FMBIO menu 3 Select the appropriate Read Image settings and load the sample 4 Click the Read button on the Scan Control window Before the FMBIO scanning unit begins the scan it performs the autofocus Note You can stop the scan after the autofocus See page 3 12 Selecting Active Channels The FMBIO scanning unit has two optical filter holders and each filter holder can hold two filters As a result the FMBIO scanning unit can simul taneously read four different emission wavelengths in the same
184. tton The Selected Spots spreadsheet is displayed containing the comparison data for the screened spots Data Sheet Overlay View Grid Comparison Scatter Plot Selected Spots Gria METTE Volume2 Commenti Comment2 98841774 71220894 Spott Bt 82048830 69511883 Spot3 78543650 66485095 Spot25 Di 103445738 58398659 Spot37 Et 104322741 58847142 Spot49 107071161 20745438 Spot61 80043606 62517539 Spot73 e Ht 106379898 70319071 Spot amp 5 a amp 118364588 57411895 Spot2 112332471 55668639 Spot14 c2 84851472 65221922 Spot26 111256720 64628690 Spot38 02 ot E2 110558639 55677439 Spot5 rg F2 113266345 24836703 Spot62 G2 110948473 63499039 Spot74 A2 1076785860 58737835 Spot86 A3 118110654 49871391 Spot3 B3 120329464 28526550 Spot15 c3 90461732 65839102 Spot27 03 95101378 70305471 Spot38 114542829 56815107 Spot51 Grid Comparison Filecard In the Grid Comparison filecard you can compare histograms of the expression and volume ratios in the grids 1 Click on the Grid Comparison tab The expression and volume ratios for the channels is displayed in histograms The color of the channel with the greater expression and volume is dis played The width represents its relative expression and the height rep resents its relative volume 7 28 FMBIO Array Analysis Grid Comparison Buttons The Grid Comparison bu
185. ttons allow you to view the expression and volume ratios separately On Off Graduation Show both expression ratio and volume ratio Show expression ratio Show volume ratio 1 Click on the Show expression ratio button E The expression ratio histograms and numerical values for the two channels are displayed The color of the channel with the greater expression is displayed The width represents its relative expression 2 Clickon the Show volume ratio button a The volume ratio histo Array Analysis FMBIO grams and numerical values for the two channels are displayed The color of the channel with the greater volume is displayed The height represents its relative volume Scatter Plot Filecard You can view a scatter plot of the data from the two grids and automatically track spots of interest to the grid in the Overlay View An area of the plot can be magnified to view selected spots and numerical spot data viewed in a spreadsheet FMBIO Array Analysis 1 Channel 2 mE Gridi Channel 3 Grid1 Scatter Plot Buttons Show Spot Label Plot Size Reset Magnification Pickup Spots in the range Capture Image 7 31 Array Analysis FMBIO Item Description Show Spot Label Display grid coordinates on each spot Plot Size Adjust the spot radius Reset Magnification R
186. u want to exclude lane s from being exported to CODIS Displays a list of lane names where any lanes highlighted will be excluded from export You can enter up to 8 alphanumeric characters 10 24 FMBIO Allele Calling with STaR Call Title Description Specimen Category Tissue Type Tissue Form Population Group You must enter a valid CODIS value up to 40 alphanumeric characters long You must enter a valid CODIS value up to 15 alphanumeric characters long You must enter a valid CODIS value up to 10 alphanumeric characters long You must enter a valid CODIS value up to 15 alphanumeric characters long values Enter the required information Click the button to add or delete list To copy the corresponding control value from the currently selected lane to the remaining lanes click the gt gt button e To copy the Sample ID Specimen Category Tissue Type Tissue Form and the Population Group values for the currently selected lane to the remaining lanes click the All gt gt button Click Export to convert data to a CODIS compatible data file 10 25 Allele Calling with STaR Call FMBIO Save As Import dat All Files 6 Enter a unique name and location 7 Click Save A CODIS worksheet is added containing the header information 306 07 CSFIPO 281 54 TPOX 243 46 TPOX 196 58 Amelogenin 158 58 THO1 185 81 THO1 276 83 D153539 9 3
187. uonnq HO AWD 5 15 JeuUeYD uee 10 eJnpe2oJd siu eedey vong rufis ufi ws ND eeun pawooz ay jo esse pa isep etf oj 10 SIN uop3ejes ear BAO amna urdd w ppa apop Budde peupjaq iasn 6uisn ore nok Ji Kex 18103 ay ssaid pue xoq ppa eur or Ajoa np 10 anjea jeubjs yry 16103 ebeur eui jo eere persep 84 01JOSIn u1007 AOW uong pundiBype g ws ND Aysueyut pessep eu 01 MOpUIA we BOISI 9t ur 89i jeuiS ur aaow 9Bewi pewooz ay jo ese peursep eui oj J0 SiN t 01 39 9S Bary ACW i Aysuajur pests ap BY oj MOpUiA Uueibojst oyj ur diy p noifi33e g GAIN Kay sez sseid pue xoq upa ayy oj j2eup anjen punosfixaeg 19103 ef eu jo ease peitsep y 0 JOSin U 007 IAOW epo Buiddew peres jeuueu peles Image Analysis Tools FMBIO Gray Level Adjustment Window rae Click the Gray Level Adjustment button AJ or choose Gray Level Adjustment from the Image menu The Gray Level Adjustment window is displayed Gray Level Adjustment Whole Image Zoomed Area Mapping Type Equal Range Background 73 52 9 864 High Signal 1 67 96 4256 Template een Histogram I Show Selected Range Iv Show Mapping Curve Channel Selector The gray level must be adjusted for each individual channel Select the channel from the Channel drop down list box The Gray Level Adjustment window displays the image data for the selected channel
188. use both hands lower the sample plate gently onto the stage supports and close the scanning cover gently Sample plates break easily and broken glass inside the instrument exposes the operator to cuts and the equipment to mechanical damage Fingerprints reagents and other foreign objects on the glass plate surface can cause noise in the image and may even show up as a signal e Keep the surfaces of the plates clean Use soap and water plain water or 7096 methanol MeOH to clean plates Avoid using ammonia based glass cleaners Wipe plates until dry to avoid streaks Scanner Sample Stage Dust and particles on the sample stage reflect stray light which can increase background signal or cause loss of signal e Keep the sample stage clean particularly the autofocus strip and adja cent areas e Regularly use the cleaning mode to clean the mirror and lens that passes below the sample Wipe the stage and interior areas with a damp low lint cloth e Immediately wipe off any gel or samples that fall onto the sample sup ports or into the unit itself 3 9 Operating the Scanning Unit FMBIO Loading the Sample E zd Sliding sample support Figure 3 5 Loading the glass plates on to the scanner bed To maximize the scannable area on the glass plates the scanner supports the sample plate only on two ends Take great care when placing the glass plates on the scanner bed to avoid breaking the glass plate and damaging bot
189. ustrate the application of various FMBIO functions Turn on power to the computer Turn on power to the FMBIO The switch is behind the pull down panel on the front left After electrophoresis has been performed keep the polyacrylamide gel plate assembly together Carefully clean and dry the outside of the plates The gel plate assembly does not need to be taken apart unless post staining is necessary for your application Note Maintaining the integrity of the gel plate assembly can allow for 4 better separation of larger STR loci because the gel can be returned to the electrophoretic apparatus after scanning for additional electrophoresis If you have an agarose gel polyacrylamide mini gel or membrane place them on a 5 mm thick low fluorescent glass plate Avoid dripping by removing excess buffer on stain Smooth out bubbles under gels as they can interfere with the scan Membranes should be mounted wet face down between two pieces of glass Polystyrene and other clear plastics may also be acceptable for holding sample s but may result in high levels of background Place the gel plate assembly in the scanner a Press the button on the top right of the front of the instrument and open the sample door b Slide the right side stage bar to the proper distance to support the plate without obscuring the area to be scanned c Place the plate assembly long plate face down on the stage resting the plate assembl
190. voltage through a gel Image Analysis provides a versa tile assortment of display options and tools for 1D gel analysis Chapter 5 Image Analysis Tools describes how to use Image Analysis tools to create single and multi color image project files This chapter describes how to use the 1D Gel tools to analyze these single and multi color images The following summaries are provided e Single color analysis example See page 6 8 e Multi color analysis example See page 6 16 You can also use the Drawing tools to enhance the results of 1D analysis See Chapter 8 Drawing Tools Note The procedures and parameters settings contained in this chapter are recommendations that can be modified to achieve the best results for your images 6 1 I1D Gel Analysis FMBIO 1D Gel Menu and Tools 1 Choose New 1D Gel Analysis from the Project menu The 1D Gel Document window Image Setting dialog box 1D Gel menu and its corresponding tool bar appear DentniCaton Setting 6 2 FMBIO 1D Gel Analysis Overlay Trace Set Marker Volume Calculation Open Spreadsheet Multi Band Color Separation Preferences Quantification Setting Open Standard Curve Tool Description Overlay Trace Displays new overlay trace of selected lanes Set Marker Displays Marker Setting window Volume Calculation Performs a volume calculation Open Spreadsheet Displays spreadsheet Open Standard Curve Displays standard curve Quant
191. y 90 days from the date of delivery of the Software to you as evidenced by a copy of your receipt or until the Software is modified by you whichever period is shorter that 1 The Software unless modified by you will perform the function described in the documentation provided by MIRAIBIO Your sole remedy under the warranty is that MIRAIBIO will undertake to correct within a reasonable period of time any marked Software Error failure of the Software to perform the functions described in the documentation MIRAIBIO does not warrant that the Software will meet your requirements that operation of the Software will be uninterrupted or error free or that all Software Errors will be corrected 2 The media on which the Software is furnished will be free from defects in materials and workmanship under normal use MIRAIBIO will at its option replace or refund the purchase price of the media at no charge to you provided you return the faulty media with proof of purchase to MIRAIBIO or an authorized dealer MIRAIBIO will have no responsibility to replace or refund the purchase price of the media damaged by accident abuse or misapplication THE ABOVE WARRANTIES ARE EXCLUSIVE AND IN LIEU OR ALL OTHER WARRANTIES WHETHER EXPRESS OR IMPLIED INCLUDING THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE NO ORAL OR WRITTEN INFORMATION OR ADVICE GIVEN BY MIRAIBIO ITS EMPLOYEES DISTRIBUTORS DEALERS OR AGENTS SHALL INCREAS
192. y on the black bars at each end and between both of the stabilizing screws on the bars Make sure the plate is level 4 27 Read Image Software FMBIO Note and secure For polyacrylamide gels place long plate face down Start the FMBIO Read Image program Click the Setting button The Read Image Parameter dialog box is displayed Set the Scanning parameters The example show described below is for illustration only and must not be interpreted as a suitable procedure for any particular case a Choose the Parameter Name In this example Acrylamide is entered b Choose the resolution 150 dpi is an example for the initial setting c Enter the sensitivity In this example 80 is entered for Channels 1 and 3 and 10046 for Channels 2 and 4 One of the factors influencing this choice is the filter types used d Enter the focusing 0 is shown in this example It may need to be lower for the thinner plates used in pre cast gels Note that the range of focusing is ONLY 2 mm to 3 mm Use of values outside of this range can seriously damage the instrument e Choose the number of repeats An initial setting example is 256 4 28 FMBIO Read Image Software SettingDialog Acrylamide Gel fso sj fiso gl EN Not Found str allele C EE a Click OK to return to the Scan Control window 10 11 Note In the FMBIO menu set the active channels A separate image is collected for each
193. you if you want to enable or disable macros click the Enable Macros button The STaR Call Installer dialog box is displayed Click the Install button 10 2 FMBIO Allele Calling with STaR Call Starting STaR Call STaR Call will automatically start whenever you load Excel and a STaR Call spreadsheet will open If you want use Excel for other than STaR Call you may unload it by selecting Unload from the STaR Call menu To use STaR Call again you must restart Excel If you do not want STaR Call to start each time you run Excel you may remove it as described below Removing STaR Call 1 Make sure that Excel is not running 2 Locate the Excel startup folder The Excel startup folder location depends on which version of Office you are using If you are having trouble finding the Excel startup folder use the Find application Click the Start button select Find gt Files or Folders Then type fmbiostr xla in the Named field and click the Find Now button 3 Remove the files fmbiostr xla and codisdbf xls from the startup folder STaR Call Installation Macintosh Version Requirements e Atleast 2 MB of free memory to load the STaR Call macro and the data file e Microsoft Excel 5 or Excel 98 Consult the documentation that came with the Microsoft Excel software for Microsoft Excel s memory requirements If you have less memory than is required by Excel it will be necessary to have the virtual memory turned on If you h
194. ysis v 3 0 1 LANE BENDING When a handle point is clicked and dragged on a lane outline the outline will swing in a curved path making it easier for users to manually fit them on an irregularly shaped lane If the end points of the outline i e top or bottom handle points are clicked and dragged the corresponding portion i e top or bottom portion of the lane will bend like a fishing pole being pulled If any of the mid handle points are pulled then the lane will bend from that handle point resulting in a small arc When a control key is pressed while dragging handle points on a lane then only a particular segment i e between two handle points will be moved in a straight line and not in a curved path 2 POWER SCROLLING For images bigger than the size of the window on the screen any selection tool or rectangle shaped tool will cause the image to scroll to the end when the mouse cursor is moved beyond the window border Suppose an image being analyzed is too big to fit in an application window and the user has about 25 lanes for automatic lane detection With this Power Scrolling feature selection tools and rectangle shaped tools will cause the image to scroll as the mouse cursor crosses the window border until the user stops moving the mouse or when the image cannot be scrolled any further As of now the following tools now have this new feature Ellipse tool on Draw Toolbar Rect Tool on Draw Toolbar and Create Rect Ang
195. ystem ORI Laboratory ORI Creation Date Time CMF Type This is the Common Message Format header version This is the message type Accepts numeric values only You can enter up to 64 alphanumeric characters You can enter up to 64 alphanumeric characters You can enter up to 10 alphanumeric characters You can enter up to 10 alphanumeric characters Use the format DD MMM YYYY HH MM SS This is the results type 10 22 FMBIO Allele Calling with STaR Call Title Description CMF Version This is 1 0 Technology This is the technique used Reading by You can enter up to 20 alphanumeric characters Reading Date Use the format DD MMM YYYY Reading date must be in 4 digit format Reading Time Use the format HH MM SS No of Reading s This accepts numeric values only 2 Enter the required information Click the button to add or delete list values 3 Click Next The CODIS STR Specimen Information Dialog Box is displayed 10 23 Allele Calling with STaR Call FMBIO STaR Call CODIS STR Specimen Information Title Description Lane Marker Name Specimen Number Use marker name as specimen number Exclude Lane Included Excluded Lane List Sample ID Default is lane number You can enter up to 24 alphanumeric characters Check this box if you want to use the lane marker name as the specimen number Copies row 1 to row 2 Check this box if yo

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