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SlideBook 4.2 Ratio and FRET Manual

1. Rmin Rmax rn t Concentration Error Conditions The following conditions in addition to those that prevent r n t from being computed will prevent c n t from being computed r n t lt Rmin r n t gt Rmax SlideBook Ratio FRET Manual Appendix E Direct FRET Calculations Terms AD AA AF Fa Da Fa Aa Iip t x y L a t X y L t X y ROI n ROI Background FRET Donor emission wavelength FRET Acceptor emission wavelength FRET Transfer emission wavelength Donor emission bleedthrough Direct excitation of acceptor Intensity of image measured through FRET Donor filter set of time point t at position x y Intensity of image measured through FRET Acceptor filter set of time point t at position x y Intensity of image measured through FRET Acceptor filter set of time point t at position x y Region of interest n a subset of all possible x y positions The number of positions in the region of interest n The background is computed for each time point for FRET donor acceptor and transfer images by averaging intensities over the background region of interest ROI b p t 1 ROIL Zrom Lp t x y b a t 1 ROL Erom halt x y b r t 1 ROIb Erom L r t x y Three Channel Corrected FRET FRET Computation FRET a t is computed for ROI n t by first computing average intensities for the donor acceptor and transfer channels i n t p n 1 ROL p t Zro
2. Camera Any Remove Associated Channel Type Ratio 2 Denominator Ale RAGE Color FRET Donor dul Default Color Display None Red Green Blue Pseudocolor Color Pseudocolor Intensity RGB Settings Monochrome Filter Configuration Parameters l x Filter Configuration Ee Add Parameters Name FRET Light Source Transmitted Mode Unknown O Prompt if mode ts not available for current objective f Fluorescence Emission 0 527 wavelength pm Requires UY objective Filter set Fixed Excitation wheel position Internal turret position Emission wheel position LCD BF filter position Unmounted Camera Any Remove Associated Channel Type RGE Color FRET Donor FRET Acceptor Cancel OF ql Default Color Display None Red Green Blue Pseudocolor Color Pzeudocolor Intensity RGB Settings Monochrome x 2 4 SlideBook Ratio FRET Manual 3 Close and restart SlideBook to update your preferences 2 2 1 2 Two Channel Corrected FRET For Two Channel Corrected FRET define a ratio channel with the FRET channel set as the numerator and the acceptor or donor channel set as the denominator see page 2 1 2 2 2 Acceptor Photobleaching FRET In order to perform acceptor photobleaching FRET you must define five channels donor prebleach acceptor prebleach photobleach donor postbleach a
3. 3 Either type in 26 into the Timepoint edit field for Rmax or if your main view is on timepoint 26 simply press Set to current selection at current timepoint If your main view is not currently displaying timepoint 26 you may visit that timepoint by pressing Visit after entering the timepoint in the edit field 5 9 Chapter 5 Ratio Calibration Intracellular Ratio Calibration e_N 4 Now repeat steps 2 and 8 for Rmin In this example Rmin 1s located between timepoints 50 and 65 After timepoint 65 the sample begins to photobleach SlideBook Ratio FRET Manual Ald ax Set to current selection at current timepolnt Timepoint 26 ROI f i 340 668 38 background 81 93 390 701 14 background 223 44 Aman 1 2277 Rin Set to current selection at current timepolnt Timepoint feo ROI f uff 340 261 81 background 45 31 390 1359 19 background 223 81 Rmin 0 1907 Beta 2 3768 Ed jo Refresh Cancel 5 Enter the dissociation constant Kd for your fluorophore This value is specific for your fluorophore and biological system A Kd calculator for Fura 2 is available at the Molecular Probes website http probes invitrogen com resources calc kd html Please enter the Kd value in units of nM concentration In this example we will use 225 nm 5 11 Chapter 5 Ratio Calibration Intracellular Ratio Calibration gt E Y 6 Click OK to complete the calibration Sc
4. Open alternate Source Next Capture 00 00 00 Time Remaining 00 00 10 Elapsed Time 18 32 09 Capturing channel Fura 380 timepoint 9 of 20 Graph Channels Regions _ f Individual Showy Fura 340 Fal By Type Select All Set Background Cancel Stop Continue The window has the following features e Start Stop begins ends the semi live camera readout e Open Fluor Close Fluor opens and closes the fluorescent shutter e Open Bright Close Bright opens and closes the transmitted light shutter e Filter moves motorized filters into position when you select Go e Z Stage allows you to refocus your sample using precise movements To use this window press Start open the desired shutter select the appropriate filter and focus your sample When you are finished close the shutter press Stop and then Continue SlideBook Ratio FRET Manual 4 Post Acquisition Display and Settings Ratio data is generally best displayed using Pseudocolor settings This will be the default setting if you define filters used for ratio imaging as discussed in Chapter 2 on page 2 1 We will now explore display of ratio data using the example slide ratiosample sld This slide is included in the file RatioFRET zip If you are a new user please complete the Quick Tour tutorial in the SlideBook User s Manual before beginning this tutorial 4 1 Viewing Ratio Data in Pseudocolor To begin this demonstration 1 Open S
5. Select Capture Ion Saturated Image The region of interest that you selected in the background image collection will be used for these captures Continue collecting ion free images by selecting Capture Ion Saturated Image When you have finished collecting the image or images click on Compute Parameters The guide will automatically compute the Rmin Rmax and beta values see definitions on page 5 1 For the exact mathematical equations used to compute these parameters please see Appendix B Buffers For Ratio Calibration Enter the dissociation constant Kd for your fluorophore This value is specific for your fluorophore A Kd calculator for Fura 2 1s available at the Molecular Probes website http probes invitrogen com resources calc kd html Please enter the Kd value in units of nM concentration Enter the Riow and Rnigh values see definitions on page 2 8 In general you should set Riow and Rhign to be equal to Rmin and Rmax initially After running experiments you may wish to revise the Riow and Rnigh values to reflect the actual range observed You may access these values post capture by selecting Image gt Ratio Settings Click OK to exit the Ratio Calibration Guide Your ratio calibration data will be used to generate concentration data for ratio images 5 4 SlideBook Ratio FRET Manual 5 2 Intracellular Calibration As described above SlideBook provides an in vitro calibration tool for Ca imaging usin
6. Dy post 1 ROI rom I a x y Photobleach FRET pbFRET Computation pbFRET n is computed for ROI n by first computing average intensities for the prebleach and postbleach channels i n t pre n 1 ROL pre n t EROI pre n t bat X y i n t post n 1 ROL post n t Zror2 n t lpk X y and then computing the background corrected pbFRET pbFRET n G0 post i b pre im xpre n 3 b post t G post j by pre
7. 14 Elapsed Time 00 00 06 Capturing channel Fura 380 timepoint 5 of 20 Graph Channels Regions n f Individual Show Fura 340 IC By Type Select All Set Background Cancel Stop Continue 3 2 1 Selecting Regions of Interest You may select regions of interest for graphing either before capture has started or during capture ROI tools may be used on test images generated before capture or on the live image display during capture please see Creating ROIs and Graphs to Monitor Regions of Interest in Chapter 7 of the SlideBook M User s Manual If you choose a background region the average value of this region will be subtracted from the average values in the other regions of interest before ratio computation The background region will also be stored in the Ratio Settings dialog box see next Chapter 3 2 2 Graph Display To display the ratio graph during capture simply select the channel that you wish to graph from the drop down list then press the Show button in the Graph Channels section of the Capture Status dialog Repeat for any additional channels you wish to display or simply select the channels from the channel menus on the graph Note that you will only be able to select channels of the same type e g independent or ratio from the channel menus Thus you must have at least two graphs open to display 340 380 and a ratio 3 5 Chapter 3 Real time Ratio Display ES Sold Fura 2 340 C
8. Data Graphing and Export 4 Type in the desired name of the text file where your data will be written 5 Open the data file using Excel You may wish to repeat steps 2 and 3 for different fluorophores 6 2 Multiple Region Graphing and Export from ROI selections You may also export data from multiple regions that you selected during capture You may also add or delete regions post capture 230 63 32 063 Raitio 100 339 _Fura 2 380 SERE Bkend None ee eee ESE EES ES CCC ESSE SES C OLEOLE SES CCC C CCC CCE C CCC Sess C OLEOLE CC lisesi ssi esi it eli ssl ists C OLEOLE CCC CCC CC CCE TT TETTE RETTE TT TETTE TETTE TT CEC CCC TT TTT TET EE TT TE TETTE ATTTTTTTTTTTTTTARTTTTTTTTTTTTTTTTATTTT TTT A 6 2 1 Working with ROls You may display regions that you chose during capture add or delete selections and select a background region e Display ROIs select View gt Annotations gt Regions e Create new ROIs use the ROI tools to draw regions please see the SlideBook User s manual for a detailed description of ROI tools and usage e Delete ROIs click on the ROI identification number then right click and select Delete e Select Background Region click on the ROI to select it then right click and select Set as Background 6 2 SlideBook Ratio FRET Manual 6 2 2 Graphing and Exporting Data To graph your ROI data select View gt Graph and select the channel you wish to display You may explo
9. E data three channel FRET FRETc must be normalized for either donor or acceptor concentration using masks statistics see Obtaining FRETc Statistics on page 7 12 7 Click OK to close the dialog box 8 Repeat steps 1 7 for the Mutant image 7 3 Viewing FRET Data It is very easy to view your corrected FRET data with SlideBook s pseudocolored main views Further explanations and an example for viewing data in pseudocolor can be found above in Viewing Ratio Data in Pseudocolor on page 4 1 1 Open a Main View of Dual Label 2 Go to View gt Pseudocolor Two channel menus will be available The first channel will be displayed as a pseudocolored image ranging from saturated red to saturated blue known as Pseudocolor Color 3 Select lt FRETc D gt from the Hue top drop down channel menu The second channel menu will act to gate the pseudocolor image based on intensity This second channel is called Pseudocolor intensity Thus an area with a high pixel intensity for the first channel and a low pixel intensity on the second channel will appear as dim red An area with a low pixel intensity for the first channel and a high pixel intensity on the second channel will appear as bright blue This helps distinguish areas with high intensity from background 4 Select CFP from the second channel drop down menu Your image will appear similar to the following SlideBook Ratio FRET Manual Ta directFRETsam 5
10. Filter Configuration FITC post Add Parameters Name Aire post Associated Channel Type Virtual AGB Green Light Source Virtual RGB Blue FRET Donor Frebleach e Fluorescence FF E T anor Fostbleach Emission 0 535 wavelength um oth um Default Color Display Transmitted None Mode Unknown ki Prompt if mode is mot available for current objective Remove E ql Cancel Red Green oe e Blue Alternate Source Pseudocolor Color Position Ma Fseudocolor Intensity AGE Settings Monochrome Filter set Fixed Excitation wheel position Unmounted pect eeIINeE L Wavelength Internal turret position Unmounted Emission wheel position Urmounted Requires UV objective LCD EF filter position Unmounted Camera es 0a Any v Filter Configuration Parameters f 1 Filter Configuration Rena Add lx Parameters Name cra after Associated Channel Type Light Source Ratio 1 Numerator l Ratio 1 Denominator Transmitted Ratio 2 Numerator Mode Unknown O Prompt if mode ts mot avallable for current Mone objective peu Remove OK Fell Cancel Default Color Display f Fluorescence Green Emission 0 57 wavelength um Blue Pseudocolor Color I Requires UV objective Fseudocolor Intensity RGB Settings Filter set Fixed y Monochrome Excitation wheel position Unmounted Inte
11. NO F 1 PHOTOBLEACH FRET PBFRET COMPUTATION Sulle F 1 SlideBook Ratio FRET Manual Contacting Intelligent Imaging Innovations Inc If you have any questions or experience any problems with SlideBookTMIM please contact us by either phone or email Our phone number is 303 607 9429 and our email address is support intelligent imaging com SlideBook Ratio FRET Manual Manual Conventions Typographic Conventions Menu commands are written in bold and follow the order of menu navigation For instance choosing Open from the File menu is written as File gt Open Dialog fields and other interface items are written in bold as in Initial Offset References to other sections of the manual are underlined as in Image Capture New terms are italicized SlideBook Ratio FRET Manual 1 Introduction to Ratio and FRET Modules The Ratio and FRET Modules add functionality that simplifies a wide variety of quantitative techniques Ratio imaging 1s a live cell imaging technique that measures pairs of wavelengths Quantification of ratio pairs is relatively unaffected by issues such as photobleaching uneven dye loading and dye leakage Thus it 1s an excellent technique for performing quantitative measurements Commonly ratio imaging techniques employ ratiometric dyes that indicate ion concentration Examples of such dyes are Fura 2 for Ca2 and BCECF for pH Ratio imaging techniques may also be employed when performing l
12. box 4 5 Chapter 4 Post Acquisition Display and Settings Ratio Settings x Ratio 1 Fura 2 340 Fura 2 380 Image Mame T Cell Calcium Flux Background RUI Numerator Fura 2 540 Stark fp Denominator Fura 2 380 Exposure Factor 1 000000 Calibration Parameters Ed 0 0000 Height fo Import from Active View Amn 0 0000 Amas 0 0000 beta Sf Sb2 00000 Thresholds for Ratio Computation Make Default Mum Lowy eo Calibrate Mum High jo Ratio Display Range Den Low jo Low 0 2 Den High jo High f Import from Default Renom Redefine Ratios OF Cancel 4 2 1 Adding and Viewing Ratio Channel Information The numerator and denominator are displayed for a given ratio channel as well as the exposure factor which is the ratio of exposure times for the numerator and denominator wavelengths The channels used for calculating the ratio can be edited by selecting Redefine Ratios which brings up the Add Ratio Information dialog box 4dd Ratio Information x Ratio 1 Numerator Channel Denominator Channel Fura 2 340 Ratio 2 Numerator Channel Denominator Channel Caros SlideBook Ratio FRET Manual 4 2 2 Calibration Parameters The Ratio Calibration Guide may be used to calibrate or recalibrate an image by selecting Calibrate The Ratio Calibration Guide is discussed in Chapter 5 4 2 3 Ratio Display Range You may sele
13. courtesy of Dr Luis Marrero LSU Health Sciences Center 8 2 SlideBook Ratio FRET Manual 2 Use an ROI tool to mark a background area with no signal A region will appear 3 Select the region by clicking on it the number will turn white then right click on the region and select Set as background 4 Select Image gt FRET gt FRET Settings The following dialog box will appear FRET Settings Er x Fiter Bleedthrough Factors Donor Fd d jo Acceptor Fa Aal jo Use Defaults FRET Channel Display Signal Threshold jo Display Low jo Display High jo Reset to Display Range to Min M ax Display Normalization Image Name TNF TACE FRET CONTROL 1 Participating Channels Channel Type Name Background FRET Acceptor Cys after 0 0 Donor Prebleach FITC Before 105 8 Donor Postbleach FITC postbleach 100 0 Redefine Three ChannellEorrection FRE Tc Transfer Fd Dd Donor Fara Acceptor O Two Channel Correction f None FRE Tc Transfer Fd Dd Donor C Donor Acceptor Photobleaching pbFRET Postbleach Prebleach Postbleach 0 Acceptor OF Cancel 2 Select Acceptor Photobleaching and then OK 3 Return to the Main View of your image and select lt pbFRET gt from any of the channel menus to display and view your acceptor photobleaching FRET values 4 To alter the display values in your image alter the edit fields in the FRET Channel
14. follows Three Channel Corrected FRET For Three Channel Correct FRET three images are necessary 8 Control containing donor only imaged with donor and FRET filters 9 Control containing acceptor only imaged with acceptor and FRET filters 10 Experimental sample containing both donor and acceptor imaged with donor acceptor and FRET filters Appendix A FRET Introduction and Theory For demonstration purposes let us assume CFP is the donor and YFP is the acceptor When CFP excites a large portion will emit normally corresponding to the CFP emission spectrum If the CFP FRETs a radiationless energy transfer will occur that causes YFP to emit The CFP emission spectrum overlaps with the YFP emission spectrum therefore some of the normal CFP emission will be visible through the YFP emission filter This cross talk confounds the signal due to FRET and must be corrected The first image the CFP control is used to calculate the spectral bleedthrough of CFP emission visible through FRET filter set Additional corrections must be made for non FRET signal due to the overlap of CFP and YFP excitation spectra This overlap causes normal YFP fluorescence to be visible through the FRET filter set The second image the YFP control is used to calculate the spectral bleedthrough of normal YFP emission through the FRET filter set The third image is the raw FRET image Corrected FRET or FRET is calculated with consideration of all thr
15. masks Masks are flexible and can be copied to multiple images in a slide Please see Chapter 9 of the SlideBook User s Manual for a complete discussion of masks To perform multiple region export from a main view of your image 1 Create a mask by choosing Mask gt Create and draw your regions using mask drawing tools Please see Chapter 9 of the SlideBook User s Manual if you are unfamiliar with mask drawing tools 186 2 9 0 61 Ratio 100 187 _Fura 2 380 cli tel l mus 000 eee CC CCC CCE CC CC CCC CCC CE CELE COTE CCC iste sects lisesi CCCCCCCE CCC CCC CCC CC CCC CCC CCE C CCC C CCC CLE CCC C CCC CCE CCC CTE iii i i i E E E E i i E 2 Extend the selection to all planes by choosing Mask gt Copy This Plane To All 3 Divide the mask into objects so that SlideBook can individually find and number each region by choosing Mask gt Define Objects and selecting OK 4 To display a graph select Image gt Statistics gt Ratio Timelapse Data A graph similar to the following will appear 6 4 SlideBook Ratio FRET Manual Ratio Timelapse Data f Graph all regions Lea 0 31 Graph all channel i 0 75 i Regions E 0 69 0 63 0 56 F Se 0 50 ratio 0 4 0 033 0 31 pe o tl 0 25 Channels 0010 D 00 16 00 32 00 42 00 64 00 80 00 96 00 112 00 0 13 O Time Point Display type O Intensity f Ratio Della F F wi
16. the YFP and FRET channels 7 1 2 Experimental Sample Now bring your experimental sample into view and focus in the focus window Use the FRET filter when focusing your sample Then image your sample s capturing the CFP YFP and FRET channels Make sure that all exposure times are equal to each other If you are performing a live FRET experiment you may wish to take advantage of the live graphing features that are discussed above in Chapter 4 7 2 Post Capture Analysis Tutorial Next you will perform background subtraction calculate bleed through coefficients and generate the corrected FRET signal You may follow this protocol step by step using the example file directFRETsample4 1 sld 7 2 1 Performing Bleedthrough Calculations Bleed through is calculated using the two control images 1 Open the sample slide by selecting File gt Open Slide and navigating to directFRETsample4 1 sld 2 Open a main view of the CFP only control by double clicking on the thumbnail 3 Next go to Mask gt Segment Move the red bar on the histogram to the right to segment your image You should select a significant amount of sample to increase the accuracy of the bleed through calculation For this example a low value near 240 can be used 4 After segmentation is complete draw a region in an area with no signal using an ROI tool A region will appear 5 Select the region by clicking on it the number will turn white then right click on th
17. which a view displays the color corresponding to the minimum value Rhign the ratio value above which a view displays the color corresponding to the maximum value From these parameters calcium concentration can be calculated from the following equation Ca Kd R Rmin Rmax R Beta 5 1 Solution based in vitro Calibration This type of calibration requires you to capture images of buffer solutions When imaging buffer solutions you may wish to add a small amount of non fluorescent polystyrene beads to aid in obtaining focus Alternatively you may mark or scratch the surface of the coverslip or dish used for imaging Example buffer solutions for calibrating calcium concentrations when using Fura 2 may be found in Appendix B Buffers for Ratio Imaging NOTE Do not attempt calibration using an AM ester derivative of an intracellular probe AM ester derivatives have little to no response to ion concentration differences You must use a salt form of your fluorophore for calibration 5 1 1 Background Image The first ratio image that you must collect consisting of images of both the numerator and denominator channels is a background image This is usually an image or average of a set of images of the experimental setup with a buffer solution that has no fluoroprobe present Prior to starting the Ratio Calibration Guide you should place buffer solution on the stage 9 1 Chapter 5 Ratio Calibration and adjust
18. 8 12 38 300 246 754 920 529 1521 391 125 192 0 174486 308 529 Along with the ratio settings SlideBook writes out for each time point the following fields Plane plane number Time time of day that the plane was captured Elapsed s or ms elapsed time in seconds or milliseconds Numerator channel Fura 2 340 average numerator intensities for the region of interest at the given time point value is not background subtracted Denominator channel Fura 2 380 average denominator intensities for the region of interest at the given time point value is not background subtracted Num Bkgnd or Den Bkgd average numerator and denominator intensities for the background region at that time point Ratio computed background subtracted ratio for qualified pixels Qualified number of voxels within the region whose values satisfied all threshold conditions see Thresholds for Ratio Computation on p 4 7 to set conditions only qualified pixels are used for computing ratio values 6 3 Chapter 6 Ratio and Concentration Data Graphing and Export e Total total number of voxels within the region e Total concentration computed concentration if calibration data is present See Chapter 3 Note The exact mathematical equations used for computing Ratio data can be found in Appendix D Ratio Data Export Calculations 6 3 Multiple Region Export from a Mask You may also export data from multiple regions by creating
19. Book 4 1 as shown in the following protocol This example protocol uses the common FRET pair Cy3 and FITC For further explanation on the theory and calculations for Acceptor Photobleaching FRET please see Appendix A FRET Introduction and Theory on page A 1 This protocol includes procedures for Capturing Images Performing Post Capture Analysis Viewing Photobleaching FRET Data Obtaining PB FRET Statistics 8 1 Capturing Images In order to perform Acceptor Photobleaching FRET you must first configure your filter definitions as discussed in Defining FRET Channels for Acceptor Photobleaching FRET on page 2 5 You will need to determine an appropriate photobleaching time before capturing your desired sample To begin capture 1 Mount your sample on the stage and bring it into view and focus using the Focus Window See Chapter 5 of the SlideBook User s Manual for instructions on using the Focus Window 2 Use the field diaphragm to create a small area of illumination on the sample You will be using this small area to determine an appropriate photobleaching exposure time 3 Select Image gt Capture The Capture dialog box will appear 4 Now you must determine an appropriate exposure time for photobleaching Select the Photobleach channel and enter an approximate time for photobleaching 5 Select Test to generate a test exposure Increase the illumination area and observe the amount of photobleaching that has occurred via the o
20. Display section of the dialog box e Signal Threshold the intensity above which pbFRET values will be calculated This threshold is background subtracted Therefore if you 8 3 Chapter 8 Protocol for Acceptor Photobleaching FRET select a value of 50 any pixel that 1s above 50 after background subtraction will be used for calculating pbFRET e Display Low the pbFRET value that corresponds to black in RGB display or blue in pseudocolor display See next section for further explanation of pseudocolor display e Display High the pbFRET value that corresponds to saturated color in RGB display or red in pseudocolor display 8 3 Generating Photobleaching FRET Statistics To obtain Photobleaching FRET statistics 1 Create a mask using either segmentation or manual techniques Please see Chapter 10 of the SlideBook User s Manual for information on creating masks 2 Select Mask gt Statistics and select Photobleaching FRET from Cross Channel statistics 3 Select Display This will generate statistics on a region basis using the following formula PB FRET Donor Post Donor Pre Donor Post where the intensities in parentheses are first averaged for each region before performing division NOTE Confirm that the appropriate channels are being used for the calculation by checking the FRET settings dialog box Image gt FRET gt FRET Settings 4 To export the data select Export enter a location for t
21. ET calculation you wish to perform 1 Open a Main View of the Dual Label image 2 Select a background region as you did when calculating bleedthrough coefficients 3 Go to Image gt FRET gt FRET Settings The FRET Settings dialog box will appear 7 8 SlideBook Ratio FRET Manual FRET Settings 3 xj Filter Bleedthrough Factors Image Name Dual Label Participating Channels Donor Fd Ddl 0638765 Acceptor Far8al 0 0677131 Use Defaults FRET Channel Display Channel Type Background FRET Donor 217 8 FRET Acceptor 240 0 FRET Transfer 226 1 signal Threshold jo Display Low jo Display High 99 7143 Reset to Display Range to Min Max Display Normalization Three Channel Correction FRE Tc Transfer Fd Dd Donor Fara Acceptor Two Channel Correction Mone FRETC Transfer Fd Dd Donor 0 Donor Acceptor Photobleaching pbFRET Postbleach Prebleach Postbleach 0 Acceptor OF Cancel 4 Select the Three Channel Correction radio button for this example You will be asked whether or not you would like to import default bleedthrough settings 5 select Yes and the bleedthrough coefficients will be imported You may have one set of bleedthrough coefficients set as default at any time If you do not wish to use defaults simply type in the bleedthrough coefficients in the edit fields Once the bleedthrough coefficients are imported a new chan
22. INTELLIGENT IMAGING INNOVATIONS SlideBook 4 2 Ratio and FRET Modules for Windows 2000 XP User s Manual Latest Revision 1 15 07 SlideBook Ratio FRET Manual Table of Contents TABLE OF CONTENTS cia ni II CONTACTING INTELLIGENT IMAGING INNOVATIONS INC ccccsssssssssssssssssssssssssssssssssssssssssssssses IV MANUAL CONVENTIONS cali iii LL LIL DLL LILLO V TYPOGRAPHICCONVENTIONS ollare V 1 INTRODUCTION TO RATIO AND FRET MODULES ccccmmmmmms 1 1 1 1 RATIO AND FRET SYSTEM CONFIGURATION 1 1 1 2 RATIO LOOSE 1 2 T21 CADA GUAE oeie i r E dd E l 2 1 2 2 Real time ratio and concentration display ii 1 2 1 2 3 Post acquisition ratio and concentration display ii 1 2 1 2 4 Ratio and concentration data expott ii A A A ad AAA Tdi 1 2 1 3 FRET TOOL e dao 1 2 1 3 1 Channel bleed through calculation ooccccinnnnnnnnnnnnnnnnnnnnnnncnnnnnnnnnnnnn nono nnnnnnnnnrnnncnnnnnnn nn nn nn nnnnnnnn 1 2 1 3 2 Post acquisition calculation of Sensitized Emission Direct FRET MMM ii 1 2 1 3 3 Post acquisition calculation of Acceptor Photobleaching FRET ii 1 2 2 CONFIGURING YOUR SYSTEM FOR RATIO OR FRET IMAGING cocccccccoccccccccccccccccccccccccccccccccnnss 2 1 ZA DFHNINGRAFIOCHANNE Scritte 2 1 22 DPEFNNG FRE CHANNELS ici 2 2 2 2 1 Direet Sensitized Emission FRET sc illeso 2 2 2 2 2 Acceptor Photobleaching FRE 2 5 Y REAL FIMERA
23. NG AND EXPORT FROM ROI SELECTIONS cccsceccececcececcsceccececcscucescecesccess 6 2 6 2 1 Working with TOUS acces ll LEI 6 2 6 2 2 Graphing and Exporting Data iii 6 3 6 3 MUETIPEE REGION EXPORT FROM A MASK silla acireale ees 6 4 7 PROTOCOL FOR DIRECT FRET SENSITIZED EMISSION FRET cccsccsccccsccccccscccccccccees 7 1 7 1 CAPTURINGIMAGES real ieri rana OEE ei bada ll nie 7 1 PENA COMOROS diede 7 1 742 Experimental Sample iaia ei ira 7 2 7 2 POSTCAPTURE ANALYSIS TU TORA aan 7 2 7 2 1 Performing Bleedthrough Calculations MM iii 7 2 Vigil USING TRET SOUINGS A NA AS 7 8 7 3 WNIEWINCERETDATA enone tn nec a a ean eet et 7 10 7 4 OBTAINING FIRED C S TALIS TICS oo acacia 7 12 8 PROTOCOL FOR ACCEPTOR PHOTOBLEACHING FRET ccccsccccccccccccccccccccccccccccccees 8 1 8 1 CAPTURINGIMAGES ariete italia 8 1 8 2 PERFORMING POST CAPTURE ANALYSIS sccscscoscecoscscscecascscascscesesceccscscescesscssscscsescesescesescssescssescesass 8 2 8 3 GENERATING PHOTOBLEACHING FRET STATISTICS cccecceccececcececcscececcsceccscesescecescecescecscscesescesesces 8 4 9 SELECTED REFERENCES Glielo dille lbs bia 9 1 APPENDIX A FRET INTRODUCTION AND THEORY 22 ceccccccccccccccccccccccccccccccccccccccccccccccccees A 1 ACCEPTOR PHOTOBLEACHING RECOVERY A italia lele nt Loa er lana A 2 DIREGT FRET SENSITIZED EMISSION PIR De pillole cant
24. TODISPLA Yorri n E E I ARO IIicOEaS 3 1 3 1 SETTING CAPTURE PARAMETERS FOR TIME LAPSE RATIO IMAGING iii 3 1 3 2 IDEE 3 3 3 2 1 Selecting ResionsofInterest 1 mc aerea aaa 3 5 322 Graph Display tl 3 5 3 2 3 AROCUS ME D ring CADUre nunca 3 6 4 POST ACQUISITION DISPLAY AND SET TINGS cccccssssssssssssssssscsssssccsccccccssccscccscscccsscscsscsssssssees 4 1 4 1 VIEWING RATIO DATA IN PSEUDOCOLOR ES dasa T aA 4 1 4 2 RATIO SETIINGS ios E E A E a E O N 4 5 4 2 1 Adding and Viewing Ratio Channel Information iii 4 6 422 CAMDITAMNOTT GTA CLOTS oaea e E E E EAI T A eo AAA 4 7 423 Rato DISMAY RANGE nera a o oa 4 7 ADA BACON ROD zenen ao iaa 4 7 4 2 5 Thresholds for Ratio Computation ii 4 7 420 Una IOS CUNO ta a A A EEEE ai 4 7 S RATIO CATIBRATION cout 5 1 dal SOLUTION BASED IN VITRO CALIBRATION sica 5 1 ILI BGC GTOUNG Image 5 1 SZ LAS O aia ea 5 2 513 on Sat rated IMALE sleale erro 5 2 5 1 4 Generating Ratio Calibration Data ii 5 2 5 2 INTRACELLULAR CALIBRATION aaa 5 5 5 2 1 Selecting a background ROI and an ROI within a cell nac c nc cnn cnn cnn cnn nn nono nnnrnrcnnnnnns 5 5 322 A dk deal ana 5 7 SlideBook Ratio FRET Manual 6 RATIO AND CONCENTRATION DATA GRAPHING AND EXPORT 2 cccsccccccccccsccccccccccees 6 1 6 1 QUICK GRAPHING AND DATA EXPORT sorio eta 6 1 6 2 MULTIPLE REGION GRAPHI
25. add ratio information post capture as discussed in the section Adding and Viewing Ratio Channel Information on page 4 6 The following example shows how to set up a ratio channel for Fura 2 NOTE Ratio Channels may also used when performing Two Channel Corrected FRET See Appendix A for an introduction to Two Channel Corrected FRET 1 Select Edit gt Define Optics gt Filter Configurations The Filter Configurations Parameters dialog box will appear 2 Adda new channel by clicking Add and then entering all of the necessary parameters A detailed explanation of all the settings is found in the SlideBook User s Manual Chapter 4 Sample settings for Fura 2 are shown below ROSSI IS REGINE CSS RISO FIN _T___ _ _ _ t _ _ _ _ _ _ _ _ _ _ _ _t _ _t _ _ mt_ y _ 1 SEL LI e2 ria Filter Configuration Pa Filter Configuration Mew Fluoraprobe A dd Remove Name Fura 340 Associated Channel Type T Independent Light Source Ratio 1 Numerator Ratio 1 Denominator Cancel f Fluorescence Ratio 2 Numerator Emission fas wavelength um atin um Default Color Display Transmitted None Ix Parameters Mode Unknown o 1 i CG T Prompt if mode is not available far E curent objective Blue Alternate Source Pseudocolor Color Position Unmaunt Pseudocolor Intensity C
26. alog box 9 Opena main view of the YFP only sample and create a mask and select a background region as you did for the CFP only sample Create the mask using Mask gt Segment and choose the YFP channel as the basis for segmentation A low value of about 300 is appropriate 7 5 Chapter 7 Protocol for Direct FRET Sensitized Emission FRET Ne ee ee ee a ee ee ee o ple4 1 YFP Only 5 x 168 8 0 228 _ FRET 50 220 YFP Er Halaj a al Bkgnd None E Bkad 10 Select Image gt FRET gt Compute FRET Bleedthrough The dialog will appear as follows SlideBook Ratio FRET Manual TAN PP 0 698974 Make Defaut Comcel 11 Select Compute Bleedthrough to compute the direct excitation of acceptor Chapter 7 Protocol for Direct FRET Sensitized Emission FRET Image Name YFF Only Control Compute donor bleedthrough Fd Dd f Compute direct excitation of acceptor Fa Dal Channels FRET acceptor YFF FRET transfer FRET Redefine Compute Bleedthrough Filter Bleedthrough Factors Donor Fd Dd 0 698574 Acceptor Fatal 0 0648636 Make Default DE Lancel 12 Select Make Default to retain the bleedthrough values you have generated Now you will determine FRET settings and generate a corrected FRET or FRETc channel for viewing your experimental sample images 7 2 2 Using FRET Settings FRET settings may be used to select the type of FR
27. any microscope parameters in the Focus Window please see the SlideBookTM User s Manual Chapter 5 for instructions on using the Focus Window 5 1 2 lon Free image The second ratio image that you must collect is an ion free image This is usually an image or average of a set of images of the experimental setup with a buffer solution that has fluoroprobe present The concentration of fluoroprobe should be approximately equal to the concentration of fluoroprobe present in loaded cells 5 1 3 lon Saturated image The third and final ratio image that you must collect is an ion saturated image For this image you will need a ion saturated buffer solution The buffer should contain the same concentration of fluoroprobe as the buffer used to capture the ion free images 5 1 4 Generating Ratio Calibration Data To generate your calibration data 1 Place your background buffer solution in your experimental setup and bring your sample into view and focus Please see the SlideBook User s Manual Chapter 5 2 Bring up the ratio calibration guide by selecting Edit gt Setup Guides gt Ratio Guide Alternatively you may bring up the calibration guide after acquiring a ratio image by selecting Image gt Ratio Settings and selecting Calibrate Ratio Calibration Guide Ea Instructions Back di Humber of to A AI ER lle Sei For ratio calibration the guide needs three Numerator 340 Fura Images or averages of images Exposure Time
28. atio display This will yield a maximal Fura 2 ratio e Add solution of 1 uM ionomycin and 2 mM EGTA and wait for ratio to stabilize This will yield a minimal Fura 2 ratio This is a guideline you may need to experiment to find out what concentration of calcium and EGTA is appropriate for your cells The Rmax and Rmin from the intracellular calibration must be higher and lower than that which you observe during your experiment If this is not the case you must adjust the calibration solutions A tutorial for intracellular ratio calibration follows Please use IntracellularCal sld for the tutorial 5 2 1 Selecting a background ROI and an ROI within a cell 1 Open a Main View of sample 1 2 Draw a region in an area with no signal using the square ROI tool 3 Select the region by clicking on it then right click and select Set as Background 5 5 Chapter 5 Ratio Calibration IA Ea IntracellularCal Capture 2 208 244 0 100 562___380 ja si Bkend None It is a good idea to scroll through time and make sure that no debris or floating cells enter this region during some later time point as the background level is evaluated for each time point If you want to change the background ROI simply select the ROI and click and drag it to a different spot or delete it and redraw NOTE The size of an ROI for calibration purposes does not need to be large Even a small ROI such as the background ROI pictured ab
29. ation l eS hd 1000 ins Display F Renormalize to t 0 Interval simultaneous Multiple Y Location Capture Stereology Current Location Entire ListiMontage Filter Set Fixed Return to computed reflcurrent location Adjust Exposure ND Current s Once Find Best correct Dark Field Flat Field Offset Expose 100 ms Test Gain Current T Intensify Current bl Jil 2 Move Up Move Down _ Move Up Move Down 0 65535 i Image Information Optional o Comment Mptics Objective 10x Dry OK Cancel Mag Changer q 3 Select the Time Lapse check box and specify the time interval between collections as well as the total number of time points Once you select Time Lapse you will activate other options in the capture window Note that currently the time interval tells SlideBook how many milliseconds to walt after completing the capture of one time point before initiating capture of the next time point 4 Select the appropriate checkboxes for live graphing e Renormalize to first time point uses the minimum and maximum pixel intensity values for the first time point in a timelapse capture series to determine the renormalization values of all subsequent time points This 3 2 SlideBook Ratio FRET Manual option only affects the image display during capture and will not affect the ca
30. ct the range of ratio values that are displayed in any view of the ratio image Any ratios below the Low value are displayed as blue and any ratios above the High value are displayed as red This operation will not affect the underlying data 4 2 4 Background ROI You may select a background region that will be used to subtract background intensities from the numerator and denominator channel prior to calculating ratio and concentration values 4 2 5 Thresholds for Ratio Computation This feature 1s important when generating statistical data These thresholds allow you to compute ratios for relevant data This feature allows you to use simple masks to define ROPs but only compute ratio data for objects that are within the desired intensity range This allows you to qualify pixels that should be used for ratio computation Only pixels that fall into the range for both the numerator and denominator values will be considered when statistics are calculated 4 2 6 Using Ratio Settings We will now continue the Ratio Tutorial 1 Make sure a view of the T Cell image 1s active and then choose Image gt Ratio Settings The following dialog box will appear 4 7 Chapter 4 Post Acquisition Display and Settings Ratio Settings xj Ratio 1 Fura 2 340 Fura 2 380 Image Mame T Cell Calcium Flux Background ROI Numerator Fura 340 Denominator Fura 2 380 A Stark Y Start jo Width jo Height jo Im
31. culars Repeat the testing procedure on fresh areas of sample until an appropriate photobleaching exposure time is determined 6 Use the Focus Window to move to an area of interest and if desired open the field diaphragm so that entire sample area is illuminated 7 Inthe Capture dialog box determine exposure times for CY3 pre and FITC pre by generating test exposures See Chapter 6 of the SlideBook User s Manual 8 Select the following channels for capture e FITC pre using the exposure time determined above 8 1 Chapter 8 Protocol for Acceptor Photobleaching FRET 8 2 Cy3 pre using the exposure time determined above Photobleach using the exposure time determined above FITC post using the FITC pre exposure time Cy3 post using the Cy3 pre exposure time Set camera parameters and other settings and select OK See Chapter 6 of the SlideBook User s Manual for a complete description of options in the Capture Dialog box Five channels will be captured in succession It 1s important that the channels are captured in the order shown in the bulleted section above You may alter the channel order by selecting the Move Up or Move Down buttons located below the channel listbox Performing Post Capture Analysis 1 Open a Main View of the image you wish to analyze 33 18 0 129 _ Cy3 before 100 86 _FITC before None 290 se None ELE ELLE CEC EEC ECC Image
32. d should be set to the Ratio channel The second channel selects what determines the intensity or brightness of the displayed colors If it is set to either the numerator or denominator channel the pseudocolored display will be scaled by the selected fluorescence intensity such as in the example above Thus in the example above an area with a high pixel intensity for the ratio channel and a low pixel intensity on denominator channel will appear as dim red An area with a low pixel intensity for the ratio channel and a high pixel intensity on the denominator channel will appear as bright blue This helps distinguish areas with real data from background This is often desirable as computed ratio values for regions of the image that are just slightly above background can vary greatly Please see Chapter 4 Default Color Display in the SlideBook User s Manual for further discussion of the pseudocolor view 4 Set the second channel menu selection to None The image is now displayed as a range of blue low ratio to red high ratio The brightness dimness of the hue is not scaled 4 2 SlideBook Ratio FRET Manual 5 Note the large amount of noise in this image and return the second menu to Fura 2 380 6 Set the Bkgnd drop down menu to Open A DIC image is now displayed along with the ratio data For further instructions on displaying a background image please see Chapter 8 Displaying a DIC image as Background of an RGB pseudoco
33. dix C Ratio Calibration Calculations Terms A1 Excitation wavelength used in ratio numerators A2 Excitation wavelength used in ratio denominators B n x y Intensity of numerator background image n at position x y B 2 n x y Intensity of denominator background image n at position x y Inin x y Intensity of numerator ion free image n at position x y In2 n x y Intensity of denominator ion free image n at position x y Ibn x y Intensity of numerator ion saturated image n at position x y Ip 2 n x y Intensity of denominator ion saturated image n at position x y ROI Region of interest a subset of all possible x y positions ROI The number of positions in the region of interest y Correction factor exposure time at 41 exposure time at i2 Background The background is computed from averaging intensities of N images 1 n at both Aland 22 over the region of interest b 1 1 N En 1 ROI Ero1 B 1 n x y b 2 1 N En 1 ROI Ero1 B 2 n x y Rmin Rmin n is computed for each set of images 1 n and the minimum is taken for the calibration value Rmin ini 1 ROT Eror Ini n x y ine n 1 ROI ZrorIne n x y Rmin n Ge byt y Gr 2 m b 2 Rmin Mi a Rmin n R max Rmax n is computed for each set of images 1 n and the maximum is taken for the calibration value Rmax ip1 n 1 ROT Eror Ib 1 n3 x y ip 2 n 1 ROT Eror Ib 2 n x y Rmax n io 16m b 1 y i
34. e are obvious anomalies in the field the region of interest selection permits you to exclude them Ratio Calibration Guide 3 Instructions Background Image s Number of Images to Average f On the numerator view select a region in Numerator 340 Fura Denominator 380 Fura which to compute intensities Exposure Time milliseconds 1000 Exposure Time milliseconds 1000 Parameters Kd Rmin Amas beta 5f2 5b2 User defined defaults Min 1002 Max 3560 Min 1001 Max 2024 Recapture Background Image 1 Cancel OF i 7 Continue collecting background images by selecting Capture Background Image 5 3 Chapter 5 Ratio Calibration 10 11 12 13 14 15 16 17 18 19 When you have finished collecting the background image or images click on Proceed to Ion Free Image Place your ion free buffer solution with fluoroprobe in your experimental setup and bring your sample into view and focus using the focus window Select Capture Ion Free Image The region of interest that you selected in the background image collection will be used for these captures Continue collecting ion free images by selecting Capture Ion Free Image When you have finished collecting the ion free image or images click on Proceed to Ion Saturated Image Place your ion saturated buffer solution with fluoroprobe in your experimental setup and bring your sample into view and focus
35. e ratio of the raw FRET channel to the donor channel allows for comparison of two channel corrected FRET values in various regions of the experimental sample SlideBook Ratio FRET Manual Appendix B Buffers For Ratio Calibration You may wish to purchase a Fura 2 Calcium Imaging Calibration Kit from Molecular Probes Cat F 6774 Sample buffer recipes are shown below Rmin Rmax and beta values will vary depending on your particular setup but typical values for Fura 2 may be Rmin 0 25 Rmax 2 5 and beta 5 Background Buffer 10X stock solution Dissolve in 90 ml H20 8 95 g KCI 1 2 M 0 29 g NaCl 50 mM 0 1 g KH2PO4 7 4 mM 0 42 g NaCOs 50 mM 4 77 g HEPES 200 mM Do not adjust pH Bring volume to 100 ml with H20 For 1X solution dilute 1 9 in H20 pH to 7 4 lon Free Buffer Add 25 50 uM final concentration fluorophore not AM ester derivative must be salt form to the Background Buffer Note that this value may vary based on your fluorophore and loading conditions You will want to approximate the final concentration of fluorophore in your loaded samples lon Saturated Buffer 10X stock solution CaCla Dissolve in 90 ml H20 0 22 g CaClz 20mM Do not adjust pH Appendix B Buffers for Ratio Calibration Bring to 1X using 1 part CaCls 10X solution 1 part 10X background buffer solution 8 parts H20 pH to 7 4 and add 25 50 uM final concentration fluorophore SlideBook Ratio FRET Manual Appen
36. e region and select Set as background 7 2 SlideBook Ratio FRET Manual fn e e e e e e e e E PAPAN E i directFRETs sloj xi 542 706 0 310 FRET 50 _ None DEE 209 CFP Hlal a fa Bkend None 6 Select Image gt FRET gt Compute FRET Bleedthrough The following dialog will appear Chapter 7 Protocol for Direct FRET Sensitized Emission FRET Image Name CFP Only Control f Compute donor bleedthrough Fd Dd Compute direct excitation of acceptor Fa Da Channels FRET donor CFP FRET transfer FRET Redefine Compute Bleedthrough Filter Bleedthrough Factors Donor Fd Od fo Acceptor Fatal fo Make Default Cancel NOTE If the channel selection is not appropriate you may redefine the channels as necessary 7 Select Compute Bleedthrough to calculate the donor bleedthrough Your dialog will look similar to the one below 7 4 SlideBook Ratio FRET Manual FRET Bleedthrough Computation x Image Name CFF Only Control f Compute donor bleedthrough Fdd Compute direct excitation of acceptor Fa Da Channels FRET donor CFP FRET transfer FRET Redefine Compute Bleedthrough Filter Bleedthrough Factors Donor Fd Dd 0 698574 Acceptor Fatal jo Make Default Cancel 8 Select Make Default to use this bleedthrough coefficient for subsequent calculations and then click OK to close the di
37. ed cece A 3 EhreesChannebkCorrected FREGIO cette RL LIL La A 3 I wo sCGhannel Correcie amp FRET ill ADERIRE ALOLILA ie ek A 4 APPENDIX B BUFFERS FOR RATIO CALIBRA TION 12u ceccccccccccccccccccccccccccccccccccccccccccccccccecceecs B 1 ION PREEBbUonrERe bieco B 1 ION SATURATED BUFTERi alitalia ail ae ati B 1 APPENDIX C RATIO CALIBRATION CALCULA TIONS cccccccccccccccccccccccccccccccccccccccccccccccees C 1 APPENDIX D RATIO DATA EXPORT CALCULATIONS occoccccocococcnccccoconcnccnococcocnocncconononoconcnacaccncnccnncnes D 1 TERMS EEE A alinea O lai i lella in D 1 BACEGROUND lai MAT Ren ad e i nie id ala MOORES IVAN AT Rea D 1 ROISAND THRESHOLDNG alcalini iaia D 1 RATIOCOMPUTATION alal D 1 RATIO ERROR CONDITIONS isla D 2 CONCENTRACION COMPU A TN e N N D 2 CONCENTRATIONERRORCONDITIONS polli laicale aida D 2 APPENDIX E DIRECT FRET CALCULATIONS sesesecscscscsesesesesesesecesecscccseseseseseseceseseoscscsesesesesesesesesesecssseso E 1 TERN PRE A OR RARE OR SANI ERO SANI OT E 1 BACK ROUN DENA a o ora allo E 1 THREE CHANNEL CORRECTED FRET FRET COMPUTATION re E 1 TWO CHANNEL CORRECTED FRET FRETC COMPUTATIONS cccecesceccsceccececccscescsceccscecescsceececscscescecesesces E 1 APPENDIX F PHOTOBLEACHING FRET CALCULA TIONS cccccccccccccccccccccccccccccccccccccccece F 1 TERMS RN F 1 BACK ROED NL ALLORA iii e REP I OUT OPE ne TN
38. ee images The following equation is used to subtract the non FRET portions from the raw FRET signal FRET FRET aw D DalCFP D Da YFP 4 where FRETraw CFP and YFP are the signals visualized through the FRET CFP and YFP filter sets respectively The constants Df Da and D Da are the transmissivity or bleed through constants describing donor emission visible in FRET channel and direct excitation of acceptor respectively Sorkin et al These constants are calculated using images 1 and 2 Failure to account for these intensities in raw FRET will result in erroneous data Two Channel Corrected FRET For Two Channel Corrected FRET a single two channel image of the sample is required The two channels that must be captured are the donor or acceptor channel and the FRET channel Continuing with the CFP YFP example above if the concentration of CFP equals that of YFP equation 4 reduces to FRET FRET raw D Da D Da CFP 5 Dividing through by CFP yields FRET CFP FRET aw CFP D Da D Da 6 Thus the normalized uncorrected values will differ from the normalized corrected values by a constant offset Further for two channel FRET if the donor and acceptor bleedthrough factors D Da and D Da are unknown changes in normalized corrected FRET FRET x can still be measured AFRETcn A FRET CFP A tFRET aw CFP 7 SlideBook Ratio FRET Manual Therefore simply measuring th
39. efore if the donor cannot FRET to the acceptor then the acceptor emission 1s increased In practice there are three steps to calculating FRET using Acceptor Photobleaching Recovery These steps are 1 Capture an image of the donor 2 Bleach the acceptor 3 Capture an image of the donor After taking these two images FRET can be calculated by subtracting image one from image three Direct FRET Sensitized Emission FRET Direct FRET Sensitized Emission FRET is a faster and less intrusive method of FRET imaging than Acceptor Photobleaching FRET at the cost of computational accuracy As the name of the technique indicates FRET 1s measured directly by imaging the sample through donor excitation and acceptor emission filters or FRET filters In theory this would be the only image necessary if the donor excitation and acceptor emission spectra were perfectly separated In practice the spectra do overlap and corrections for spectral bleedthrough must be performed There are two techniques for calculating Direct FRET e Two Channel Corrected FRET applicable when donor and acceptor are present in a fixed stoichiometric ratio e g Cameleon imaging e Three Channel Corrected FRET separately corrects for donor bleedthrough and direct excitation of the acceptor appropriate when donor and acceptor are not present in a fixed ratio We will now discuss the details of Three Channel Corrected FRET A discussion of Two Channel Corrected FRET
40. el FRET denominator channel Mask Scope O Three Channel FRET intensities Display O Entire Mask Object Three Channel FRET Donor Norm i C Three Channel FRET Acceptor Morr Export Primary Mask Mask 7 Cancel Objects will be automatically defined Remove objects smaller than o Some advanced features require a secondary mask Secondary Mask None ht Object All Images in Slide with the Same Mask Name Export Only Time Point Ratio of numerator and Remove objects smaller than fo Select Basic Clear 4 Select the Three Channel FRET statistic that you wish to calculate and click Display to display the results You may eliminate objects below a certain size threshold by selecting the Gate Objects by Size checkbox e Two Channel FRET FRETC for two channel corrected direct FRET Three Channel FRET FRETc for three channel corrected direct FRET Three Channel FRET Donor Norm FRETc normalized by the donor channel for three channel corrected direct FRET e Three Channel FRET Acceptor Norm FRETc normalized by the acceptor channel for three channel corrected direct FRET 5 After viewing your data you may select Export Your data will be saved as a txt file You may then open this using spreadsheet programs such as Excel 7 13 SlideBook Ratio FRET Manual 8 Protocol for Acceptor Photobleaching FRET Acceptor Photobleaching FRET can be performed with Slide
41. enormalization settings that you have selected for the Fura 2 880 channel will be displayed Note that the Fura 2 340 settings will be set to the min and max of the channel as it is not a channel that is displayed in the view Select File gt Save Slide to save your new settings In the next chapter you will learn how these ratio settings affect ratio statistics 4 9 SlideBook Ratio FRET Manual 5 Ratio Calibration Ratio channels can be calibrated so that ratio values correspond to concentrations SlideBook supports two different methods of calibration The first is a solution based in vitro method that may be performed pre or post acquisition This calibration may be applied to multiple experiments The second is an intracellular calibration that is performed post acquistion for each experiment This method performs calibration within a single cell and then applies those values to the entire data set With either method a ratio calibration guide assists you in determining three important numbers for correctly calibrated concentration display Rnin the minimum ratio value e g Fura 2 with no free Ca ions present Rmax the maximum ratio value e g Fura 2 in an ion saturated solution beta the ratio of the denominators of the minimum and maximum conditions The ratio calibration guide is also where you specify three other parameters Ka the dissociation constant of the fluoroprobe Row the ratio value below
42. ependency also allows FRET to distinguish between two fluorophores that are less than 200 nanometers apart Acceptor Photobleaching Recovery The underlying principle of Acceptor Photobleaching Recovery takes advantage of statistical probabilities Whenever a molecule is excited by a photon there is a statistical probability of either of three things happening These three things are A The specimen under questions fluoresces and emits the exciter energy as the electrons drop back to ground state B The specimen becomes photobleached C The specimen FRETs or rather it loses in the case of the donor energy through a dipole transfer Knowing these three outcomes it 1s possible to calculate FRET through a subtractive process Prior to photobleaching the defining equation for the donor is as follows A B C 100 before photobleaching 1 Once the acceptor is photobleached FRET cannot occur and C is set to 0 The equation is then reduced to A B 100 after photobleaching 2 Subtracting equation 2 from equation 1 and assuming the probability of spontanteous photobleaching of the donor remains the same yields SlideBook Ratio FRET Manual ATO before photobleaching A after photobleaching 3 Thus after photobleaching the amount of A is increased by C the amount of FRET occurring prior to bleaching The process of photobleaching a destructive process eliminates the possibility of FRET occurring in the acceptor Ther
43. g fura 2 However often cellular physiology interacts with fura 2 such that the dye s behavior inside a cell does not match the dye s behavior in calibration solutions For more accurate measurement of absolute Ca it is best to measure the range of ratios observed In vivo or within the cells themselves The intracellular calibration guide in SlideBook 4 2 allows you to select representative regions in a time series and use them as the basis for determining the maximum and minimum ratios that are observed within cells Typically intracellular Ca calibration is done as an end step in a live cell experiment After observing meaningful Ca dynamics during an experiment a calcium ionophore such as ionomycin is introduced into a cellular buffer solution that has a high free Ca This raises the intracellular Catt dramatically and after the cytosol reaches equilibrium will allow us to measure a maximum intracellular ratio Rmax as the 340 nm excitation signal will be at its highest and the 380 nm excitation signal will be at its lowest After that a calcium chelator such as EGTA is added to drop the intracellular Cat Again after equilibrium we can measure a minimum ratio Rmin A typical experimental protocol is outlined below e Perform experiment e Once the desired effect is observed add solution so that final concentration in media is 1 uM ionomycin and 1 8 mM Ca and wait for ratio to stabilize using live r
44. ght Source Ratio 1 Numerator Ratio 1 Denominator Ratio 2 Numerator hd Cancel ql Transmitted Mode Unknown ki Prompt if mode isnot available for current None bjective objective Ped Default Color Display f Fluorescence Green Emission 0 57 Blue wavelength pm Pseudocolor Color Requires UV objective Pseudocolor Intensity RGB Settings Filter set Fixed Monochrome Excitation wheel position Unmounted Internal turret position E Emission wheel position Unmounted LCD EF filter position Unmounted Camera Ary Filter Configuration Parameters E Filter Configuration Photobleach Add lx Parameterz Mame Photobleach Associated Channel Type Light Source Ratio 1 Numerator Ratio 1 Denominator Ratio 2 Numerator Remove Ok ql Cancel Transmitted Mode Unknown Prompt if mode is not avallable for current None objective ped Default Color Display f Fluorescence G reen E mission 0 57 Blue wavelength um Pseudocolor Color Requires UY objective Pseudaocolor Intensity RGB settings Filter set Fixed y Monochrome Excitation wheel position Unmounted Internal turret position Ro H Emission wheel position Unmounted LCD BF filter position Unmounted Camera Any 2 7 Chapter 2 Configuring Your System for Ratio or FRET Imaging Filter Configuration Parameters ig f x
45. hannel menus __ gt Dashed Fura 2 380 Dotted None ER e A 704 00 i ASIA ord LE NET 448 DO Fi 384 00 320 00 256 00 192 00 o 000 200 400 600 8 00 10 00 1200 1400 16 00 18 00 20 00 2200 2400 UA Zoom in Zoom in an Timepoint ala ALI Reset intensity to full range Intensit Zoom out Zoom out Li Reset time to full range Scroll bar Use the Zoom In and Zoom Out tools to explore your data At any time you may reset the data to view full range using the Reset buttons Once your capture is finished your graph will remain open for viewing To recreate this graph post capture on an open view display the ROIs View gt Annotations gt ROIs and then select View gt Graph 3 2 3 Focusing During Capture You may adjust the focus of your image during capture To do so go to the Live tab The Live view works similarly to the Focus Window NOTE Before using the Live tab you must first select Pause Once you are finished focusing you must select Continue to resume capture 3 6 SlideBook Ratio FRET Manual Capture Controls x status FRAP Motes Live stages Z Stage ave Position dia 0 25 um Adjust the Z position using software or hardware When you resume capture the new position will be used Go to VE Update z Update Y Update All z 0 um Filter Fura 240 30 Captured Images Show Snap Open Fluor Open Bright
46. he data and select Save You may now open your data file in Excel SlideBook Ratio FRET Manual 9 Selected References Bolsover S R et al 1993 Ratio imaging measurement of intracellular calcium and pH In Electronic Light Microscopy New York Wiley Liss 182 210 Dunn K et al 1994 Applications of ratio fluorescence microscopy in the study of cell physiology FASEB Journal 8 573 582 Grynkiewicz G et al 1985 A new generation of Ca indicators with greatly improved fluorescence properties Journal of Biological Chemistry 260 3440 3450 Moore E D W et al 1990 Ca2 imaging in single living cells Theoretical and practical issues Cell Calcium 11 157 179 Sorkin A et al 2000 Interaction of EGF receptor and Grb2 in living cells visualized by fluorescence resonance energy transfer FRET microscopy Current Biology 10 1395 1398 SlideBook Ratio FRET Manual Appendix A FRET Introduction and Theory Flourescence Resonance Energy Transfer FRET is a distance dependent interaction between the electronic excited states of two dye molecules where the excitation is transferred from a donor molecule to an acceptor molecule without the emission of a photon More specifically FRET is the radiationless transmission of energy from its site of absorption to the site of its utilization by resonance between chromophores Figure 1 Figure 1 There are three types of FRET two of which SlideBook currently sup
47. heory on page A 1 This protocol includes procedures for Capturing Images Performing Post Capture Analysis Viewing Direct FRET Data Obtaining FRETc Statistics 7 1 Capturing Images In order to perform direct FRET you must be able to calculate bleedthrough coefficients or cross talk for your particular filter sets In order to calculate bleedthrough coefficients two control samples must be imaged one that contains donor only and one that contains acceptor only 7 1 1 Control Images 1 Mount the donor sample on the stage and open the focus window in SlideBook See Chapter 5 of the SlideBook User s Manual for instructions on using the Focus Window 2 Select the CFP filter set and bring the sample into focus 8 Close the fluorescent shutter and then close the focus window 4 Open the Capture Window See Chapter 6 of the SlideBook User s Manual for instructions on using the Capture Window 5 Set the CFP exposure time by selecting the CFP checkbox and then pressing Test and Once See Chapter 6 for further instructions on setting exposure times 6 Set the FRET exposure time by selecting the FRET checkbox and manually entering the same exposure time from step 5 It 1s important that the exposure times of both fluors are equal to each other Chapter 7 Protocol for Direct FRET Sensitized Emission FRET 7 Click OK to capture your image 8 Repeat steps 1 7 with a sample containing only YFP capturing with
48. intensity for ratio denominator Background The background is computed for each time point for both the numerator and denominator images by averaging intensities over the background region of interest ROI b 1 t 1 ROL Erop L 1 t x y b 2 t 1 ROL Erom La t x y ROIs and Thresholding Individual regions of interest for analysis are restricted by the minimum and maximum thresholds for each wavelength This means that a delineated region of interest ROI n t actually yields two regions ROL 1 n t and ROI 2 n t which may not necessarily be identical ROL t Uxy such that x y e ROIM t 0 1 lt Lilt x y lt o 1 ROL 2 n t Uxy such that x y e ROI t 0 2 lt Lalt x y lt 0 2 Ratio Computation The ratio r n t is computed for ROI n t by first computing average intensities for the numerator and denominator i n u 1 ROL t Eroni t ts x y i n t 2 n 1 ROL 2 m t Eronen t L alt x y D 1 Appendix D Ratio Data Export Calculations and then computing the background and exposure time corrected ratio r n t Gn t 1 n b 1 t yG a t 2 m by 2 t Ratio Error Conditions The following conditions will prevent r n t from being computed ROL n t 0 IROL n t 0 i n t 1 n lt b 1 t i n t 2 n lt b 2 t Concentration Computation The concentration c n t is computed using the calibration parameters c n t Ka B r n t
49. ive Fluorescence Resonance Energy Transfer FRET experiments e g Two Channel Corrected FRET Measurement of FRET allows you to determine whether or not two molecules are colocalized at a distance less than 10 angstroms enabling measurements below the resolution of a light microscope FRET is a distance dependent interaction between the electronic excited states of two dye molecules where the excitation is transferred from a donor molecule to an acceptor molecule without the emission of a photon FRET experiments may be performed on live or fixed cells SlideBook M supports two FRET methods Sensitized Emission Direct FRET both Two Channel Corrected and Three Channel Corrected and Acceptor Photobleaching FRET For more information on FRET methods please see Appendix A FRET Introduction and Theory on page A 1 The Ratio and FRET Modules add the following components to the SlideBook base package Ratio Module Tools Ratio system configuration Ratio indicator calibration guide Real time ratio and concentration display during capture Post acquisition ratio and concentration display Ratio and concentration data export FRET Module Tools e Channel bleed through calculation e Post acquisition calculation of Sensitized Emission Direct FRET Herman equation e Post acquisition calculation of Acceptor Photobleaching FRET 1 1 Ratio and FRET system configuration Expanded filter definitions allow you to define two independent ratio cha
50. lideBook and select File gt Open Slide 2 Navigate to ratiosample sld and select Open 3 Double click on the T Cell Calcium Flux image to generate a main view The following image will appear Please see Chapter 8 of the SlideBook User s Manual for further information on data views i ratiosample TT 2 DI x 229 9 6 0 33 Ratio 100 519 _Fura 2 380 Ja 32 9 aja mo I ET Steet estes elise iets iiss sists tsi ssi CCC ECC CC CCC C CCC OLLE ECC tittle EVE TA TETTE TEVERE TETTE EEE EEE a z E E E E E E E E E E E E E E E E E E E E E E E E E E z E E E E E E a E E i T cell image courtesy of Dr Jackson Egen University of California Berkeley 4 1 Chapter 4 Post Acquisition Display and Settings Note that the image is displayed in pseudocolor with the Ratio channel appearing in the first channel menu The ratio channel name will always be called Ratio or Ratio 1 and Ratio 2 if two ratios are defined and captured Also note that SlideBook stores both the numerator and denominator time lapse image The ratio values are computed when the image is opened and are not stored with the image NOTE If you have not defined your ratio channels as discussed in Chapter 2 you may view your data in Pseudocolor by selecting View gt Pseudocolor In the pseudocolor view mode there are two channel selections The first channel selects the hue of the display an
51. lor image in the SlideBook User s Manual 4 3 Chapter 4 Post Acquisition Display and Settings 7 Blend the DIC image with the fluorescence image by selecting View gt Blend Background 8 Return the background channel to None 9 Open the renormalization dialog box using the icon on the tool bar The following dialog box will appear 4 4 SlideBook Ratio FRET Manual Lul ol 1038 Channel Fura 2 340 High I 038 Intensity FC Percentage Reset all to Image Minh ax 10 Move the red bar back and forth Note that the display does not change Fura 2 340 is not a displayed channel in this image 11 Select Fura 2 380 from the channel drop down menu and move the red bar back and forth Note that the hue or color of the display does not change Note also that the intensity of the color becomes dim as you move the red bar to the right and becomes bright as you move the red bar to the left Note that the hues that are displayed cannot be changed using the renormalization dialog box only the intensity of color may be changed To change the hues that are displayed you must alter the Ratio Settings as discussed in the next section 4 2 Ratio Settings SlideBook lets you adjust if necessary many of the settings that go into both post acquisition display as well as analysis These settings are accessed in the Ratio Settings dialog box A tutorial follows the explanation of this dialog
52. ly and the CFP and YFP emitters are in filter wheel positions 1 and 2 respectively NOTE Make sure to select the appropriate Associated Channel Type FRET Donor for CFP FRET Acceptor for YFP and FRET Transfer for FRET Filter Configuration Parameters i X Filter Configuration cP SSS O O Add Parameters BE Name CFP Associated Channel Type Light Source Ratio 2 Numerator Ratio 2 Denominator RGB Color Transmitted Mode Unknown T O Prompt if mode is not avallable for current objective Default Color Display T Hone Red f Fluorescence Emission 0 477 wavelength um Green Blue Pseudocolor Color Requires UY objective Pseudocolor Intensity RGE Settings Monochrome oe 8 Filter set Fixed Excitation wheel position h N Internal turret position n w Emission wheel position h N LCDEF filter position Unmounted Camera lary 2 3 Chapter 2 Configuring Your System for Ratio or FRET Imaging Filter Configuration Parameters E Filter Configuration Add Parameters Mame YFP Light Source Transmitted Mode Unknown O Prompt if mode is not available for current objective f Fluorescence Emission 0 527 wavelength pm Requires UY objective Filter set Fine Excitation wheel position Internal turret position Emission wheel position LCD BF filter position Unmounted
53. millizeconds Denominator 380 Fura Exposure Time millizeconds background ion free indicator solution 1000 and ion saturated indicator solution 1000 Parameters Kd jo Amin Capture Background Image 1 Cancel 5 2 SlideBook Ratio FRET Manual 3 Enter the number of images that you wish to average in the data entry field In general 3 images are sufficient 4 Enter the exposure time in milliseconds for both the numerator and denominator in the data entry fields Note that you should aim for an exposure length that yields only moderate intensity values since there is no fluoroprobe present Otherwise the subsequent capture of ion free and ion saturated images will certainly exceed the dynamic range of the camera 5 Generate a test image by selecting Capture Background Image 1 and observe the resulting intensity values reported beneath the image You may recapture the image if the maximum intensity is too high by selecting Recapture Background Image 1 6 After capturing the first background ratio image select a region of interest by clicking and dragging on the numerator image view in order to determine the area over which the average background intensity as well as ion free and ion saturated intensities will be determined In general try to select the largest region possible as it means the ratio calibration values will be computed from a larger sample However if ther
54. nd acceptor postbleach The common FRET pair FITC donor and CY3 acceptor are used to demonstrate filter configurations A full protocol for performing acceptor photobleaching FRET can be found in Protocol for Acceptor Photobleaching FRET on page 8 1 1 Select Edit gt Define Optics gt Filter Configurations The Filter Configurations Parameters dialog box will appear 2 Add a new channel by clicking Add and then entering all of the necessary parameters A detailed explanation of all the settings in the dialog box 1s found in the SlideBook User s Manual Chapter 4 Sample dialog boxes for the five required filter definitions are shown on the following pages The following example shows typical settings when using filter cubes Note that the Photobleach channel is the same filter cube as the Acceptor CY3 channels 3 Close and restart SlideBook to update your preferences Chapter 2 Configuring Your System for Ratio or FRET Imaging Filter Configuration Parameters FITC pre kd FITC pre Virtual RGB Red Virtual RGB Green Virtual AGE Blue FRET Donor Prebleach hd 055 Unknown gt El Sfomipt ih modets nak awalane For cumenti objective Settings iwed 7 Ml Unmounted Unmounted 7 Unmounted Unmounted SlideBook Ratio FRET Manual Filter Configuration Parameters l x Filter Configuration Parameters Add Remove Name cra before Associated Channel Type k Li
55. nel called FRETc will be created The Display Range will update to list the low and high intensity values of the new channel If you wish to change the values enter the new values in the edit fields for Low and High The corrected FRET values for your sample are calculated using equation 4 described in Appendix A FRET Introduction and Theory on page A 1 6 If desired you may choose to normalize your corrected FRET channel with either the donor or acceptor intensity This will create a new channel called lt FRETc D gt or lt FRETC A gt For this exercise choose Donor and enter 50 0 and 0 2 in the Signal Threshold Display Low and Display High edit fields respectively 7 9 Chapter 7 Protocol for Direct FRET Sensitized Emission FRET e Signal Threshold the intensity above which FRETc and normalized FRETc values will be calculated This threshold is background subtracted Therefore if you select a value of 50 any pixel that is above 50 after background subtraction will be used for calculating FRETc e Display Low the value that corresponds to black in RGB display or blue in pseudocolor display See next section for further explanation of pseudocolor display e Display High the value that corresponds to saturated color in RGB display or red in pseudocolor display NOTE The new channels lt FRETc gt lt FRETc D gt or lt FRETC A gt are created for viewing purposes only To obtain meaningful transfer efficiency
56. nnels Ratio Module and FRET acceptor donor and transfer channels FRET Module Ratio channel definitions allow for real time calculation of ratio values and subsequent viewing and quantification of ratio data 1 1 Chapter 1 Introduction to Ratio FRET Module 1 2 Ratio Tools 1 2 1 Calibration Guide SlideBook provides an easy to follow guide for calibrating a fluoroprobe such as Fura 2 using the technique outlined in Grynkiewicz et al 1985 1 2 2 Real time ratio and concentration display During capture SlideBook permits you to delineate multiple regions of interest including a background region and graph ratio or concentration change over time Simultaneously SideBook provides a pseudocolored image of ratio information across the entire field 1 2 3 Post acquisition ratio and concentration display All interactive SlideBook views main view three view and tile view allow you to display ratio or concentration information as a pseducolored image Further each view can display the computed ratio or concentration at the current cursor location 1 2 4 Ratio and concentration data export SlideBook can produce a tab delimited text file that includes ratio and concentration data for multiple regions of interest across time 1 3 FRET Tools 1 3 1 Channel bleed through calculation SlideBook allows you to characterize the bleed through coefficients of your particular imaging system Characterization of c
57. np ni t Lp t x y i n t a n 1 ROL a t Zrone n t Lat x y i n t r n 1 ROLF t Zrone n t Det x y and then computing the background corrected FRETc FRETc n t GM t r byr t Fa Da Gt inln byo t Fa Aa i n t a n b a t Two Channel Corrected FRET FRETc Computations FRET a t is computed for ROI n t by first computing average intensities for the donor and transfer channels Appendix E Direct FRET Calculations i n t p n 1 ROL p amp t Zronp n t I pv t x y i n t r n 1 ROLsr a t Exonr a t Det x y and then computing the background corrected FRETc FRETc n t Gn t r n by r t Fa Da i n t p n b p t SlideBook Ratio FRET Manual Appendix F Photobleaching FRET Calculations Terms Apre Donor emission wavelength measured before photobleaching Apost Donor emission wavelength measured after photobleaching I pre t x y Intensity of image measured through FRET Donor filter set of time point t at position x y I post t x y Intensity of image measured through FRET Acceptor filter set of time point t at position x y ROI n Region of interest n a subset of all possible x y positions IROI The number of positions in the region of interest n Background The background is computed for prebleach and postbleach images by averaging intensities over the background region of interest ROI bapre 1 ROL Erom lnk y
58. o AGE Settings Filter set Fixed Monochrome Userdefined El f Wavelength Excitation wheel position h Internal turret position Unmounted o He e M Requires UY objective Emission wheel position Unmounted LCD BF filter position Unmounted Camera Any 2 1 Chapter 2 Configuring Your System for Ratio or FRET Imaging x Filter Configuration New Fluoroprobe A dd Remove Name Fura 390 Associated Channel Type F Independent Light Source Ratio 1 Numerator Ratio 1 Denominator Cancel f Fluorescence Ratio 2 Numerator E miszion 0 510 wavelength um oth um Default Color Display Transmilted None Parameters Mode Unknown Si 7 i CG O Prompt if mode is not available for curent objective Blue C Alternate Source Pseudocolor Color Position Unmount Pseudocolor Intensity O AGE Settings Filter set Fired Monochrome O Userdetined M O Wavelength Excitation wheel position E Internal turret position JUnmounted Emission wheel position Unmounted gt IM Requires UV objective LCD BF filter position JUnmounted Camera any NOTE Make sure that the numerator and denominator configurations for a particular indicator are associated with the same ratio channel 3 Close and restart SlideBook to update your preferences 2 2 Defining FRET Channels FRET channels will be defined u
59. ove contains over 100 pixels which is plenty to remove the effects of signal noise in determining a reliable background level 4 Draw an ROI within a representative cell It may be helpful to scroll through time and find a cell whose max ratio is among the highest in the field of cells 5 6 SlideBook Ratio FRET Manual 163 1 27 1 51 Ratio 100 246380 34 8 04 Bkgnd None RUULCCCC CCC CCC CCC CC CCC CCC CEC CCC CCC CCC CCC CCE C CCC ECC eee EEC TETTE TETTE VOTE TETTE Eee Eee CCC eee eee eee CCC CCC CCC CC eee eee eee eee eee eee CCC eee eee eee eee eee eee l 5 2 2 Finding Rmax and Rmin 1 Select Image gt Ratio gt Intracellular Calibration to open the intracellular calibration dialog box Chapter 5 Ratio Calibration Intracellular Ratio Calibration ol Initially the dialog box shows the computed Rmax and Rmin based on the first timepoint and the first ROI 2 Graph the ratio data of the ROI by selecting View gt Graph and setting the dialog as follows A graph similar to the following will appear SlideBook Ratio FRET Manual 10 x Dashed None Dotted Hone PA 1 38 1 25 1 13 1 00 0 88 fi 0 75 0 63 0 50 0 38 ba 0 2 1 0 00 ae h 42 00 64 00 S0 00 96 00 112 00 4 Timepoint n Simply click on the highest ratio value in the graph and the main view will update to timepoint 26 The green line indicates the timepoint showing in your main view
60. p 2 n2 b 2 Rmax MAXn Rmax n C 1 Appendix C Ratio Calibration Calculations Beta B is computed as the quotient of average denominator intensity in the ion free images over the average denominator intensity in the ion saturated images B 1 N pan 12 2 n b 2 1 N Xin ib 2 n b 2 C 2 SlideBook Ratio FRET Manual Appendix D Ratio Data Export Calculations Currently there are two ways of defining a ROI for post acquisition analysis 1 Using a selection cube for generating ratios and concentrations for a single rectangular region across all time points included in the selection 2 Using a mask divided into submasks for contiguous isolated user delineated regions across all time points included in the mask Each method uses the same ratio and concentration calculations Terms A1 Excitation wavelength used in ratio numerators A2 Excitation wavelength used in ratio denominators Lu t x y Intensity of numerator image of time point t at position x y La t x y Intensity of denominator image of time point t at position x y ROI n Region of interest n a subset of all possible x y positions ROI n The number of positions in the region of interest n y Correction factor exposure time at 41 exposure time at i2 0 1 Minimum threshold intensity for ratio numerator 0 2 Minimum threshold intensity for ratio denominator 0 1 Maximum threshold intensity for ratio numerator 0 2 Maximum threshold
61. port from Ackve View Exposure Factor 1 000000 Calibration Parameters Ed 0 0000 Amn 0 0000 Amas 0 0000 beta Sf2 5b2 00000 Thresholds for Ratio Computation Make Default Mum Low Calibrate Num High jo Ratio Display Range Eves Lilt jo Lowy oz Den High jo High I Import from Default Renorm Redefine Ratios OF Cancel 2 Now change the Ratio Display Range High edit field to 0 5 and click OK Note that the display hues have changed and more cells are red een i ratiosample Den 10 x 298 221 6 0 56 Ratio 100 207 _ Fura 2 380 EA smi one 3 Bring up the Ratio Settings dialog box again by selecting Image gt Ratio Settings 4 8 SlideBook Ratio FRET Manual 9 Change the Ratio Display Range High edit field to 2 0 and click OK Note that display hues are all blue green Open the Ratio Settings dialog box again and return the Ratio Display Range High edit field to 1 0 and click OK Activate the main view of the image and draw a box using the marquee tool in a region that has no signal Open the Ratio Settings dialog box and click on the Import from Active View button in the Background ROI section of the Ratio Settings dialog box The ROI dimensions and location will automatically be entered into the data entry fields Next select Import from Default Renorm in the Threshold for Ratio Computation section of the dialog box The r
62. ports The three types of FRET are e Acceptor Photobleaching Recovery e Direct FRET Sensitized Emission FRET e Fluorescence Lifetime Measuring SlideBook supports Direct FRET and Acceptor Photobleaching Recovery and will support Fluorescence Lifetime Measuring in the near future As stated above FRET involves the non radiative transfer of energy from a fluorophore in an excited state to a nearby acceptor fluorophore In order for this transfer to take place the donor and acceptor molecules must be within 10 50A F rster distance and the emission spectra of the donor fluorophore must overlap the excitation spectra of the acceptor fluorophore In principle this overlap will allow the exchange of energy through a non radiative dipole dipole transfer The energy transfer manifests itself by quenching the donor fluorescence in the presence of the acceptor and increased sensitized emission of the acceptor fluorescence The figure on the next page illustrates the spectral overlap of the corresponding fluorphores Appendix A FRET Introduction and Theory Quenched Donor Sensitized Emission Emission due to FRET vaio PI AT due to FRET Emission Acceptor Donor Absorbance Absorbance Normal Acceptor Emission WAVELENGTH Figure 2 The distance dependent characteristics of FRET make it an excellent tool for investigating a variety of biological phenomena that produce changes in molecular proximity Further this distance d
63. ptured image 5 Specifiy a ratio capture by clicking on the checkboxes for both the ratio numerator and denominator channels and testing the exposure length for each It is easy to collect other data such as a GFP or DIC image during ratio capture Simply select additional channels to capture as in normal time lapse imaging You may also capture a channel such as DIC at a slower rate than your ratio data by selecting the Periodic tab found in the Capture Preferences dialog box use the Advanced button in the Capture Preferences section to access this dialog box Please see Changing the Channel Capture Frequency in Chapter 7 of the SlideBook User s Manual 6 Once you have captured a test image you may use the ROI tools to draw regions to monitor during capture To learn more about ROI tools please see Creating ROIs and Graphs to Monitor Regions of Interest in Chapter 7 of the SlideBook User s Manual 7 If you would like to record events such as reagent addition set up Notes using the Advanced button as discussed in the SlideBook User s Manual Chapter 7 Creating Notes 8 Select OK when you are ready to begin capture NOTE It is often advantageous to use binning for time lapse ratio capture Not only will this permit better temporal resolution but it will also dramatically decrease the total size of the collected data 3 2 Live Display During capture SlideBook can display a pseduocolored image of ratio val
64. re the graph as described on page 3 5 To export the ratio concentration data select Image gt Export gt Ratio Timelapse Data The output should look similar to the following Ratio information present Numerator channel Fura 2 340 Exposure Length ms 25 Denominator channel Fura 2 380 Exposure Length ms 25 Exposure factor 1 000000 Plane Time Elapsed s Fura 2 340 Fura 2 380 open Num Bkgnd Den Bkgnd Ratio Qualified Total O 18 07 38 0 127 389 264 921 636 115 134 283 1 18 08 40 62 156 293 349 773 92 584 184 468 2 18 08 56 78 136 427 291 306 896 681 167 429 0 3125 3 529 3 18 09 12 94 145 836 355 204 1613 015 149 349 0 181287 124 529 4 18 09 28 110 191 009 589 49 1524 064 151 283 0 152749 307 529 5 18 09 44 126 313 609 1168 469 1563 009 155 311 0 18768 432 529 6 18 10 00 142 337 471 1274 902 1618 142 156 304 0 188592 471 529 7 18 10 15 157 344 798 1320 493 1641 541 149 261 0 186341 469 529 8 18 10 31 173 325 764 1232 783 1569 025 150 267 0 185284 449 529 9 18 10 47 189 312 181 1196 042 1535 664 154 280 0 176044 436 529 10 18 11 03 205 326 45 1255 057 1688 936 141 236 0 183691 455 529 11 18 11 19 221 340 648 1320 662 1622 681 137 227 0 187705 467 529 12 18 11 35 237 339 289 1343 817 1591 9 140 230 0 180893 454 529 13 18 11 51 253 334 665 1339 098 1494 662 139 232 0 178979 437 529 14 18 12 06 268 297 382 1140 826 1522 478 129 205 0 182955 376 529 15 18 12 22 284 264 548 1009 849 1507 147 125 192 0 176145 329 529 16 1
65. rnal turret position fa H Emission wheel position Unmounted LCD BF filter position Unmounted Camera Any 2 8 SlideBook Ratio FRET Manual SlideBook Ratio FRET Manual 3 Real time Ratio Display In this chapter you will learn how to set parameters for capture and interactively view your data as it 1s captured 3 1 Setting Capture Parameters for Time lapse Ratio Imaging Ratio capture works much like other image capture paradigms in SlideBook For a more detailed explanation of image capture please see the SlideBook 4 2 User s Manual Chapters 6 and 7 To capture a new time lapse ratio image 1 Bring your sample into view and focus in the Focus Window Chapter 5 User s Manual 2 Select Image gt Capture New Image The Capture Dialog box will appear Chapter 3 Real time Ratio Display Extent Offset and Binning pixels Image Bin Factor Width 1394 Height 1040 Update Autofocus fixi a Offset Offset White Balance fo fo Full Chip Available 1 50 GB Required 0 00 KB 3D Capture Use current f position RETINA yo Use reference Planes E asition 7 i Step Size Cun fos po Use top and bottom positions Offset fumi o B Range around center Capture Settings Default T Current Default amp dvanced Capture Type 3p E So correct Timelapse Timelapse Capture of Time Points 1 Estimated Dur
66. roll over the main view and observe that calcium concentrations are displayed next to the ratio channel menu 7 To set your display back to ratio calculations enter O in the Kd field and click OK SlideBook Ratio FRET Manual 6 Ratio and Concentration Data Graphing and Export There are three ways to mark areas for graphing and exporting ratio data from an image single region export from a marquee or point and click selection multiple region export from ROIs and multiple region export from a mask created manually You may wish to use the ratiosample sld file to experiment with data graphing and export 6 1 Quick Graphing and Data Export To rapidly generate ratio data 1 Select a single cell region by selecting the Point Selection tool 2 Hold the CTRL key while clicking on a cell A graph similar to the following will appear jRatio Timelapse Data UN 640 00 56 00 512 00 445 00 294 00 E Li i a 320 00 Intensity E 256 00 192 00 123 00 0 00 16 00 32 00 48 00 64 00 80 00 96 00 time point Aoo H 0 time point 130 Range 231 84 to 236 89 Mean 226 525 SD 1774 056 Channel 340 E sport Radius f3 pixels Close You may select the data you wish to display using the drop down channel menu You may increase the radius to increase the area that is being graphed 3 To export the data press the Export button 6 1 Chapter 6 Ratio and Concentration
67. ross talk between channels is necessary for making quantitative measurements of Three Channel Corrected Direct FRET This feature is discussed in the chapter titled Protocol for Sensitized Emission Direct FRET 1 3 2 Post acquisition calculation of Sensitized Emission Direct FRET You may specify donor and acceptor channels corresponding bleed through coefficients and a background region in order to automatically calculate direct FRET These settings may be saved for each image A complete protocol tutorial for performing Direct FRET is provided 1 3 3 Post acquisition calculation of Acceptor Photobleaching FRET You may specify donor and acceptor channels and a background region in order to automatically calculate photobleaching FRET A complete protocol tutorial for performing Acceptor Photobleaching FRET is provided 1 2 SlideBook Ratio FRET Manual 2 Configuring your System for Ratio or FRET Imaging 2 1 Defining Ratio Channels In order for SlideBook to be able to capture ratio images the filter configurations must be set properly One filter configuration should be defined as a Ratio Numerator and one filter configuration should be defined as a Ratio Denominator These two filter configurations determine a ratio channel SlideBook lets you define two ratio channels ratio channel 1 and ratio channel 2 If you wish to view ratio values real time you must set ratio channel definitions before initiating capture You may also
68. sing a procedure similar to that discussed above Channel definitions for Direct FRET and Acceptor Photobleaching FRET will be discussed in this section 2 2 1 Direct Sensitized Emission FRET In order to perform Direct FRET the appropriate filter definitions must exist The common FRET pair CFP and YFP is used as an example For three channel corrected FRET three filter definitions are necessary CFP donor YFP acceptor and FRET For two channel corrected FRET two filter definitions are necessary either CFP or YFP and FRET CFP and YFP filter sets are configured to use the respective exciters and emitters The FRET channel must use a CFP exciter and YFP emitter The exact configuration of the filters will depend on your particular hardware configuration 2 2 SlideBook Ratio FRET Manual 2 2 1 1 Three Channel Corrected FRET To add FRET filters for performing Three Channel Corrected FRET 1 Select Edit gt Define Optics gt Filter Configurations The Filter Configurations Parameters dialog box will appear 2 Adda new channel by clicking Add and then entering all of the necessary parameters A detailed explanation of all the settings 1s found in the SlideBook User s Manual Chapter 4 Sample settings for CFP YFP and FRET are shown below This example shows typical settings when using a JP4 filter set where the dichroic is in filter position 1 the CFP and YFP exciters are in filter wheel positions 1 and 2 respective
69. th baseline jo to E Export Close You may choose to display all regions for a single channel or all channels for a single region using the radio buttons 5 To export the data press the Export button To export the data without first viewing the graph select Image gt Export gt Ratio Timelapse Data 6 Type in the desired name of the text file where your data will be written 7 Open the data file using Excel The output should look as shown above in the section Graphing and Exporting Data The object identity will be shown in parentheses 6 5 SlideBook Ratio FRET Manual 7 Protocol for Direct FRET Sensitized Emission FRET Direct FRET Sensitized Emission FRET can be performed with SlideBook 4 2 as shown in the following protocol This example protocol uses the common FRET pair CFP and YFP You must first configure your filter definitions as discussed in Defining FRET Channels for Direct Sensitized Emission FRET on page 2 2 This protocol specifically describes Three Channel Corrected FRET Two Channel Corrected FRET is a technique used when the donor and acceptor are present in a fixed concentration ratio e g Cameleon imaging Two Channel Corrected FRET can be viewed and analyzed as a ratio of FRET intensity to donor or acceptor intensity using the ratio tools discussed in Chapters 4 and 5 For further explanation on the theory and calculations for direct FRET please see Appendix A FRET Introduction and T
70. ues for each time point as well as update a graph of ratios for multiple regions of interest The Capture Status dialog box will appear and display the current single channel image in the Status tab The Image Window will display a pseudocolored ratio image 3 3 Chapter 3 Real time Ratio Display Capture Controls xj Status FRAP Notes Live Stages Fura 360 all O Mean 316 36365 O show Full Dynamic Range ULI Next Capture 00 00 00 Time Remaining 00 00 06 Elapsed Time 00 00 04 Capturing channel Fura 380 timepoint 3 of 10 i Graph Channels Individual Fura 340 Y By Type Regions Select All Set Background You may also select a channel and the Show button which allows you to display data background subtracted if you have chosen a background region for all channels for a single region of interest or for one channel including the ratio channel for all regions of interest NOTE In all display views you can mark events using the Notes buttons you created when setting capture parameters The buttons are displayed on the Notes tab of the Capture Status dialog 3 4 SlideBook Ratio FRET Manual Capture Controls Status FRAP Notes Live stages Note 2 add chelator Mote 3 FO Note 4 FO Mote 5 PO Mote 6 FO Note 7 FO Mote 8 FO MAA Next Capture 00 00 00 Time Remaining 00 00
71. x 234 6 0 range _ lt FRETCD gt 50 226 St EI si 5 Repeat steps 1 4 for Mutant In pseudocolor red denotes high ratios and blue denotes low ratios Therefore red regions have higher normalized FRET relative to blue regions 6 Compare the two images again Decide which sample is a positive FRET control and which is a negative FRET control Chapter 7 Protocol for Direct FRET Sensitized Emission FRET cai direct RES fa directFRETSA lO lx 644 700 0 range _SFRETc D 2 754 0 range _ lt FRETc D gt 50 224 rp 50 209 SEP 3018140 pros sone Si e 7 4 Obtaining FRETc Statistics You can now generate FRETc information using masks and statistics 1 Open a main view of your sample image 2 Go to Mask gt Segment and generate a mask on the regions you wish to measure FRETc see Chapter 10 of the SlideBook User s Manual for instructions on masking Alternatively you may generate a mask manually 3 Select Mask gt Statistics Select the mask you have just created from the drop down menu and click on the next to the Cross Channel category as shown below 7 12 SlideBook Ratio FRET Manual ee ee A a um 3 Features utout Grouped By Current 20 30 Image Compute Category Statistic Curent 20 Time Lapse or 40 Images I Morphometry none Cross Channel C Intensity none Er Cross Channel none Description Correlation O Two Chann

SlideBook 4.2 Ratio and FRET Manual

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