Home

Maestro User Manual - Biophotonics Imaging Laboratory

Contents

1. Figure 62 Removal of Maestro and components from shipping crate 2 With the front and top of the shipping crate removed identify and remove the packages containing the cables user manual installation software specimen pad and the CRi business card with QDot spots 3 Carefully lift the illumination module from the top of the Maestro cabinet and set it aside Remove the plywood panel and the foam blocking that supports it 4 The Maestro imaging module can now be removed At least two people should remove the imaging module by carefully sliding it out of the crate Note The imaging module is shipped in a plastic bag which may cause it to feel A slippery Be careful not to drop the unit as it clears the crate supports 5 Remove the plastic bag and remove the shipping tape from the door Place the imaging module on the table or bench where it will be installed 6 Open the imaging module and carefully remove the foam blocks that are contained within the chamber These blocks are used during shipping to protect the illumination and stage components from shipping damage 98 System Setup amp Installation Setting Up the Maestro n Vivo Imaging System Figure 63 illustrates a typical setup configuration T POM Maestro Imaging Module LCD Monitor Illumination Module 36 INCH MINIMUM
2. subq2mice Hardware Status l Ready Protocol Figure 15 Maestro Work Area Control Panels To select a control panel Click the control panel s tab The following panels are available e The Acquire panel includes all the controls needed to acquire new spectral images and image cubes The Spectral Processing panel is used to create spectral libraries and unmix cubes The Measure panel is used to find measure and adjust measurement regions in component images The Display panel is used to change current display settings of images Thumbnails and Image Data Pages There are four pages with tabs across the bottom of the image viewing area You can change the viewable size of these pages by clicking the Shrink or Grow buttons at the far right see Figure 15 These pages display the following types of information Introduction to the Maestro In Vivo Fluorescence Imaging System 23 Window Layout e Thumbnails This page displays a thumbnail of each open image in the image viewing area You can click on any thumbnail to activate its image window If you have zoomed in on an image the zoom view rectangle within the thumbnail shows the zoomed region You can drag this rectangle with the mouse pointer to pan the zoomed image Thumbnails Measurements Log Cuso d subgemice Background Signal_3 Signal_4 Composite E3 L3 HI Figure 16 Thumbnails Page
3. 30 e Acquiring an Image Cube sess 31 e Unmixing the Image Cube sess 32 Quantifying Results ntn re E Ree e tere ib RI eade egi ade 36 Imaging the CRi Business Card with QDot Spots 29 First Steps l Before you begin verify each of the following connections are complete refer to Appendix B System Setup amp Installation if necessary e The imaging module is plugged into a surge protected electrical outlet e The USB interface cable is connected between the workstation computer and the imaging module e The illumination module is connected to the imaging module with its interface cable and its power cord is plugged into the accessory outlet on the back of the imaging module Switch on the imaging module the illumination module and the workstation computer and switch on the WHITE interior lights On the workstation computer launch the Maestro application The CRi business card test specimen has three QDot spots applied to its front surface 565 nm towards the left and 605 nm towards the right and a mix of both 565 and 605 nm dots in the center The excitation wavelength is 465 nm Therefore we will use the blue Maestro filter set for this exercise Insert the blue excitation filter in the illumination module The section Excitation Filter Slider on page 21 explains how to install the excitation filter slider Open the door of the imaging module and install the c
4. e Measurements This page displays the measurement data of measurement regions drawn on component images See also Chapter 7 Quantifying Results Thumbnails Measurements Log Cursor Y Location Total Signal Area mm 2 CubeID Spectrum ID Cube Time Sta T Total Signal Exp ms Max Signal Area Pixels s nn Minor Axis X Location 107 I33 7 105771 Urs UTITIUWIT ERE TI Major Axis Selected eor rumber Avg Signal GI zz gra JOU ISy UZ TO v Am 130 40 47 13 66 136 99 222 65 349 16 Unknown Exp 43 268 1 82 Signal 4 2006 May 02 10 v 5 112 24 78 14 03 104 02 68 76 252 70 Unknown Exp 24 225 153 Signal 4 2006 May 02 10 6 1 25 2578 15 61 118 60 216 55 245 36 Unknown Exp 285 197 Led Signal 4 2006 May 02 10 v gt Figure 17 Measurements Page Log This page contains an action history of the current Maestro session The log can be saved or cleared by clicking the buttons at the far right of the page Thumbnails Measurements Lod Cursor 2006 May 02 10 36 06 Created Log File C Program Flles CRi Maestro 2p00 MaestroSystemLag txt Loading Cube FileName C Maestro Data Images Sample Data Q dot mouse Q dot mouse stack subq2mice_580 tif Wavelength 580 FileName C Maestro Data Images Sample Datal Q dot mouse Q dot mouse stack subq2mice_590 tif Wavelength 590 a Figure 18 Log Page e Cursor This page provides information about
5. 5 Close and latch the lamp access door Introduction to the Maestro In Vivo Fluorescence Imaging System 19 6 Reset the hour counter by pressing the button directly beneath the counter To press the button first unscrew its cover Then press the button and replace the cover To replace fuses in the illumination module Two 250VAC 8A slow blow fuses in the illumination module prevent accidental circuit overloads Fuses should be replaced with Cooper Bussman MDL 8 fuses available from CRi and other sources 1 2 4 To replace a blown fuse switch off power to the illumination module and unplug it Use a screw driver to turn the black fuse holder one half turn counter clockwise and unseat it Remove and replace the old fuse in the holder Reinsert the fuse holder into its port and turn one half turn clockwise until firmly seated Plug in the illumination module To clean the cooling fan inlet filter The filter for the cooling fan inlet should be cleaned on a regular basis to ensure the free flow of dust free cooling air Figure 13 shows the removal of the inlet filter Bow om Cooling Fan Inlet Cover 1 c F Foam Inlet Filter Cooling Fan Inlet Figure 13 Removing the cooling fan inlet filter Switch off power to the illumination module and unplug it Pry off the cooling fan inlet cover Remove the foam inlet filter and rinse it with mild soap and clean water Dry the filter thoroughly by
6. If there is no Internet connection you can still activate the Maestro software by Email 1 In the InstallShield Wizard activation dialog box select I have a serial number and I want to activate Maestro and click the Next button Enter the serial number found on the Maestro software CD case and click Next to activate You will receive an error message stating that the Activation Request Code could not be sent Click OK When the Serial Number dialog box reappears make sure the serial number is correct and select the Activate by Email option Click Next A dialog box containing an Email link prompts you to Email an Activation Request Code to CRi Use any computer to Email the code to the address indicated When you receive the Activation Response Code enter it in the activation dialog box and click Next to activate the software System Setup amp Installation 103 Calibrating Pixel Size To take measurements using mm units the Maestro software needs to know the camera s pixel size This procedure requires that you have already acquired a clear and focused monochrome image of a metric scale or ruler at least 100 mm long with the stage height on setting 1 and the CCD binning set to 1x1 1 With the monochrome image of the metric scale or ruler loaded select Calibrate Pixel Size from the Hardware menu The Distance Calibration dialog box opens Distance Calibration Instructions ler100m
7. Make sure the measurement regions you want to save are selected on the Measurements data page Select Save Measurements from the File menu The Choose Measurement File dialog box opens Select a location and enter a name for the text file Click the Save button to save the new text file Appending Measurement Data You can append new measurement data to end of an existing measurement data text file 1 84 Quantifying Results Make sure the measurement regions you want to append to a saved text file are selected on the Measurements data page Select Append Measurements from the File menu The Choose Measurement File dialog box opens Select the file to which you want to append the measurement data and click the Save button Chapter 8 Customizing Spectral Displays This chapter explains how to use the Display panel to change display parameters to obtain a clear view of an image Topics in this chapter Page e Adjusting Brightness and Contrast Levels essen 85 Saturation Mask ecciesie eet sand aere iere th de dd S 86 e Changing How a Cube is Displayed essen 87 e Changing How a Composite is Displayed sss 88 Adjusting Brightness and Contrast Levels The controls in the Display Mapping box at the top of the Display panel are used to change brightness and contrast levels within images Display Mapping Brightness 0 00 Contrast 299 f min Ma
8. a e 2 a 5 e i 2A T7 Specimen Stage gt E m Figure 8 Stage Height Positions Introduction to the Maestro In Vivo Fluorescence Imaging System 15 To set the specimen stage height l Squeeze the stage release levers on the front edge of the stage then slide the stage up or down to the desired position Carefully release the tabs and ensure that the stage is locked at the desired position Move the fiber optic illuminator arms see below to a position that corresponds to the stage height Set the corresponding stage height in the Maestro application Select Edit Hardware Settings from the Hardware menu and select the Camera Setting tab in the Edit Protocol dialog box Select the correct height in the Stage Height drop down box Fiber Optic Illuminator Arms Four fiber optic illuminator arms provide adjustable and even illumination of the specimen Move each of the illuminator arms to a height that corresponds with the height of the specimen stage This will ensure consistent illumination of the specimen To position the illuminator arms 1 2 Press the tab on the side of the arm to release it Slide the arm up or down to the position that matches the height of the specimen stage Note that there is only one intermediate fiber optic position for every set of three intermediate stage heights You should choose the position that gives the best result for your application
9. im3 cubes and TIFF tif cubes An RGB color representation of the cube will display in the Maestro image viewing area If a Live Stream window is still open you may close it by eS PF EES clicking its Close box g E 2 Name Color If desired select a spectral display option from the t Autofuor white C a M Scaling combo box obom er NNE e M 3 5 M Click the Real Component Analysis RCA button near the 4 raw 14 A 1 H 5 Cyan E MI bottom of the Spectral Processing panel The RCA function i Cee automatically detects and separates each of the E MI component signals in the cube e m Eo M s NHH M If you prefer to unmix the cube by manually identifying OM the component signals populate the color library with spectra and click the Manual Compute Spectra button e Refer to the Maestro User s Manual for instructions pies Import Spectra From Library In the RCA dialog box you have the option of manually Autofluor 630 nm NES EX E subgzmice 630 n 5x Composite Hardware Status No Hardware Protocol DataFolder sampling the autofluorescence Click the Sample Spectrum button and use the mouse pointer to draw a sampling line on an area of the specimen that contains only autofluorescence Hold down the left mouse button and drag the pointer to draw Autofluor Click the Find Compone
10. 10 720 Fixed Filters Blue Figure 34 Cube Wavelength Selection 54 Acquiring Spectral Images Using Preset Filter Settings The Preset Acquisition Settings store fixed filter names with their corresponding acquisition wavelengths For example Blue 500 10 720 means these filter settings are to be used with the excitation filter and emission filter marked with a blue dot on its slider The starting wavelength for this filter set is 500 nm the ending wavelength is 720 nm and images are acquired in steps of 10 nm To select a predefined filter set Select an option from the Preset Filter Settings drop down box The wavelength setting for the Live image autoadjusts to within the wavelength range of the current filter selection Manually Entering Acquisition Settings If you don t see the filter settings you want among the Preset Filter Settings you can edit the current values in the Start Step and End wavelength text boxes When you edit any of these values a new Unnamed filter setting is automatically created using the new manual settings Modifying and Saving Preset Filter Settings If you don t see the filter settings you want in the Preset Acquisition Settings drop down box you can edit and save the preset filter settings for use again later To edit predefined acquisition settings 1 On the Acquire panel click the Define button below the Preset Filter Settings drop down box 2 The Edit Protoco
11. CRi recommends maintaining an up to date operating system using procedures recommended by Microsoft and the computer manufacturer If the Maestro imaging module has never been connected to the computer it is best to wait until after the software installation is finished before connecting the module Also make sure the imaging module is switched off If the Maestro imaging module is connected to the computer disconnect it now and switch its power off Do not skip this step After the software is installed Windows needs to detect the Maestro hardware to install the correct drivers In order for this to System Setup amp Installation 101 occur you need to connect the imaging module to the computer after the software installation is complete 5 Exitall other Windows programs that may be running 6 Insert the Maestro software installation CD into the computer s CD drive and proceed with one of the installation options listed below a To install the Maestro software from the CD follow the onscreen instructions if the installation wizard starts automatically If the installation wizard does not start automatically access the CD drive and double click the icon labeled Setup exe to start the installation Follow the onscreen instructions to install the software b If you received a software update from CRi by Email or by downloading it from CRi s FTP site and you do not intend to install from the CD cancel the installation wi
12. Emission Filter Slider Holder Wavelength Labels Filter Slider Wavelength Labels Figure 11 Emission Filter Slider Holder with Slider The emission filter slider holds two 50 mm diameter longpass filters Emission filter slider sets ordered from CRi are color coded to indicate their wavelengths For example the blue 515 nm filter is indicated by a blue dot on its end of the slider the green 580 nm filter is indicated by a green dot and so on See Appendix C Filter Selection Guide for detailed filter specifications To insert the slider 1 Select the emission filter slider that contains the filter you want to use 2 Insert the slider into the slot in the side of the emission filter slider holder shown in Figure 11 The slider can only be inserted one way the wavelength labels and color coded dots must be upright and facing toward you 3 Move the slider through the holder until the wavelength label for the filter you want to use is visible in the center window of the holder You should feel the slider click into place when it reaches the proper position 18 Introduction to the Maestro In Vivo Fluorescence Imaging System Illumination Module The illumination module includes a Xenon light source a fiber optic delivery system and power and interface cables y Power ON OFF Switch Access to Fiber Optic Guide Locking Screw Fiber Optic Guide Port Excitation Filter Slot Control Cable Connector Figure 12
13. For this exercise scaling should not be necessary because of the strong QDot signal Note that the computed spectrum looks more like a simple gaussian curve which is the shape of accurate quantum dot spectra The wings on either side in the original mixed spectrum which are due to the contribution of the paper autofluorescence have been removed 34 Imaging the CRi Business Card with QDot Spots 3 oF Unmixing the Image 1 2 3 f At the bottom of the dialog box change the name of the computed spectrum from C4 to Q565 For simplicity change its color to green to correspond with the green QDots g Click the Transfer to Library button You will see the new selection added to row 4 of the color palette Now separate the 605 nm QDot signal from the autofluorescence a In the Known Spectrum box the black Paper color marker should still be selected b In the Mixed Spectrum box click the red Q605 Paper color marker c If necessary adjust the Error Scaler or turn scaling off to accurately compute this spectrum d At the bottom change the name from C5 to Q605 Change its color to red to correspond with the red spot of QDots e Click the Transfer to Library button Close the Manual Compute Spectra dialog box Notice that the check boxes in the Select column for each of the mixed signals were automatically cleared when you added the pure spectra selections to the color palette Now
14. Workstation Computer Figure 63 Basic Maestro Site Requirements amp Configuration 1 Position the imaging module on the table or bench as shown in Figure 63 2 Position the illumination module on the left side of the imaging module so its rectangular projection is positioned toward the back of the imaging module Allow at least 36 inches of counter space from the left side of the illumination module to the right side of the imaging module 3 Connect the fiber optic light guide from to the illumination module The fiber optic guide is permanently attached to the imaging module and receives light from the illumination module a Onthe top surface of the rectangular projection of the illumination module is a small white plastic plug that covers the fiber optic clamp screw see Figure 64 Pry the plug up with your fingernail or other tool that will not scratch the paint to expose the locking screw b Usea phillips head screwdriver to loosen the screw several turns c Remove the fiber optic guide from its plastic cable clamps on the back of the imaging module Remove the protective cap from the end of the fiber optic guide System Setup amp Installation 99 d Insert the fiber optic guide into the guide port in the end of the rectangular projection see Figure 64 Push the guide in until it stops The metal end of the guide should seat completely within the housing e Tighten the clam
15. along with the Workstation Computer and LCD monitor for superior fluorescence in vivo imaging Each component requires interconnecting cables and electrical power Unpacking the Maestro n Vivo Imaging System The Maestro in vivo imaging system is transported in a wooden shipping crate that is built to protect the system during shipping Additional boxes containing the computer and monitor if purchased are also placed on the pallet The entire pallet and its contents are wrapped with plastic material to facilitate shipping the entire system as one unit Crate containing illumination cc module Maestro imaging module software and accessories LCD Monitor if purchased Workstation ee C ter if omputer i EY purchased Figure 61 Maestro Shipping Crate and Pallet 1 When you are ready to install the system remove the plastic wrapping material then remove the monitor and computer boxes if purchased from the pallet The shipping crate should be opened by removing the marked screws on the front side and top of the crate Note CRirecommends that you store the pallet and all parts of the crate including the P d Screws in case the system ever needs to be shipped again To protect against possible damage during transport the system should only be shipped in its original packaging System Setup amp Installation 97 Exploded view of crate Illumination Module Li Imaging Module
16. autofluorescence signal click the Sample Spectrum button and use the mouse pointer to specify the background signal in the image cube Unmixing Spectral Images 65 13 14 15 e Alternately you can import a known autofluorescence spectrum from the current spectral library by selecting from the Starting Spectrum drop down box Use this option if there is no region in the current sample that contains only autofluorescence as is often the case when using an injected fluorophore e These settings can be discarded by un checking the Use Selected Background box Click the Find Component Images button in the Component Images box A set of monochrome images of spectrally distinct signals will soon appear to the right of the cube display window Select component images for use in unmixing Look for images that appear to contain significant signals a sample specific judgement To select an image click it once A drop down box opens and lets you accept or change the default color of the spectral library member e fautofluorescence or other background signal is thought to be present the image typically the top left should be designated as Background by clicking twice on it Only one image can be designated as Background e Cycle through the choices by clicking once for Signal twice for Background and three times for Ignored Click the Find Spectra button in the Computed Spectra box e f no Background component is
17. line or area ROI Use the mouse pointer to remove pixels from existing regions To clear all regions Click the Clear button to remove all measurement regions To move or delete a region Right click on the region you want to move or delete and select from the pop up menu Quantifying Results 79 Cloning Regions The Maestro software s ability to clone regions makes it easy to directly compare control regions and fluorophore regions of an image Once you clone a region you can then move the clone to another area of the image to compare the fluorescence signals To clone a region 1 Right click on the region you want to clone and select Clone Region from the pop up menu A clone of the region is created Move the clone to the desired area of the image The clone will become the next sequentially numbered measurement region E subq2mice C2 Component Zoom To Full Image Zoom Pan Import Spectra Load Measurement Regions Save Measurement Regions Copy Measurement Regions To Clipboard Paste Measurement Regions From Clipboard Clone Region Move Region Hs Delete Region Figure 54 Cloning a Region Dragging a Copy of a Region to Another Image You can drag a copy of a region from one component image to another When you drag a region to another image the Maestro software automatically pastes the region at the exact same location in the new image This feature is useful for compar
18. m e e 40 60 20 300 400 500 600 700 Figure 4 Overlapping Excitation and Emission Spectra In this example of overlapping excitation and emission spectra green is MitoTracker Green Fn Blue is Acridine Orange and Red is Fluorescein isothiocyanate FITC The Cyan line at 488 represents the illumination light Dotted lines are excitation spectra and solid lines are emission spectra Illustration derived from Invitrogen Spectraviewer There are three points to note in the graph shown in Figure 4 e The excitation spectra overlap significantly with the emission spectra This is why one needs to carefully select excitation filters The goal is to prevent as much excitation light as possible from appearing in the emission spectra Even with distinct emission spectra there is significant overlap in the emission spectra of these three dyes Visual examination of such a mixture of fluorescence spectra would be unable to distinguish these three dyes as individual colors They would be seen as some combination of yellow and green by most observers 6 Introduction to Multispectral Imaging Theory and Concepts e The graphical display of spectra is normalized and in actual practice some fluorescent materials are much brighter than others As Figure 4 illustrates many labels of biological interest have emission spectra that are so similar that separation using narrow band filters is difficult or impossible Multispect
19. spectrum should look like a simple gaussian curve which is the shape of accurate quantum dot spectra When you are satisfied with the computed spectrum select a Spectrum Number Name and Color and click the Transfer to Library button When you are finished adding spectra to the library remember to save it as described in Saving Spectral Libraries on page 43 This feature is also available when using Real Component Analysis as explained in Computing and Unmixing Spectra Automatically on page 65 Unmixing Spectral Images 69 Changing the Spectral Graph Scale There are two places where you can specify spectral graph scaling at the top of the Spectral Processing panel and on the Manual Compute Spectra dialog box 1 Select an option from the Scaling drop down box above the spectral graph Scaling Normalized X Nr Normalized XN Scale To Max Un Normalized A Intensity So 9 F Wavelength 9 Figure 46 Spectral Graph Scaling Selector e Select the Normalized option to view the spectral curves as normalized The Y axis in the spectral graph is scaled 0 to 1 Each spectral curve is independently scaled to 1 at its maximum height e Select the Scale To Max option to scale the Y axis in the spectral graph from 0 to maximum height of the highest spectral curve in the image cube e Select the Un Normalized option to view the spectral curves
20. 8x10 7 3x10 42 1023 5 to 4x10 to 10 tois About the size of bi amp amp amp Buildings Grains Protozoans Bacferia Molecules Atoms er of sugar ucle Figure 1 The Electromagnetic Spectrum The Electromagnetic Spectrum is illustrated here from radio to gamma ray frequencies We are concerned with the visible wavelength range for purposes of this discussion although some models of the Maestro in vivo imaging system can operate out to 950 nm into the so called near infrared wavelength range Although this illustration of the electromagnetic spectrum suggests that the visible range of light covers approximately 400 nm to 800 nm most humans realistically are limited to the range of 400 nm deep violet to 700 nm deep red Visible light makes up a very small portion of the entire electromagnetic spectrum Light has energy Visible light has an energy level of about one electron volt per photon As one moves to shorter wavelengths of light the energy per photon increases In the shorter ultraviolet wavelengths which approach soft x rays the electron energy per photon increases to 50 to 100 electron volts This energy content of light is useful when one wishes to induce a change of energy state in a material 1 e cause a receptive molecule to undergo a series of energy additions and then relaxations resulting in fluorescence Human Perception of Light Intensity and of Color A Highly Adaptive Light Detector The huma
21. All Images Simultaneously Zoom in on an image then hold down the Shift or Control key and drag the zoom view rectangle on a thumbnail image All images pan simultaneously E subq2mice_Background c 2 E3 E subq2mice Signal 3 Com EX E subq2mice Signal 4 Com Bee Thumbnails Measurements Log Cursor subq2mice Background Signal 3 Signal 4 Composite ES E3 EN EHI EHI Figure 20 Simultaneous Panning Specifying Camera Hardware Settings The Camera Setting tab of the Edit Protocol dialog box lets you specify a variety of settings and parameters for the camera in your Maestro in vivo imaging system To access these controls select Edit Hardware Settings from the Hardware menu and select the Camera Setting tab A brief explanation of each control is described below e Filter Serial Port can be used to select an alternate connection port for the Maestro hardware The autodetect utility should select the correct port automatically but this feature lets you override the default setting 1f necessary e CCD Bit Depth lets you capture either 8 bit 256 level or 12 bit 4096 level grayscale images per wavelength 12 bit images are twice as large as 8 bit images and can only be opened using scientific imaging applications including Maestro In most instances it is best to use the default 12 bit selection as this gives higher quality images Many Microsoft Windows applications cannot open 12 bit images The
22. Images 61 To view cube info Right click on the image in the Cube window and select Cube Info from the pop up menu The Cube Info window opens Cube Info Height Pixels Sample id Timestamp 2006Jul 31 10 15 53 Wavelength nm seo Exposure Time ms Figure 40 Cube Information Extracting an Image Plane from a Cube You can extract and view individual wavelength images from a cube 1 Right click on the image in the Cube window and select Extract Channel from the pop up menu 2 Select the channel you want to extract by clicking a wavelength value in the list A monochrome image of the selected wavelength opens in a new window Figure 41 Extract Channel 62 Acquiring Spectral Images Chapter 6 Unmixing Spectral Images Whenever possible spectral processing or unmixing of image cubes should be done using previously established Maestro protocols that you have saved and validated with your Maestro in vivo imaging system for each of your specimen experiment types In most cases these protocols contain spectral libraries that will be used when unmixing images as explained in Chapter 4 Method Development This chapter explains how to create a spectral library and unmix an image cube using the spectral processing functions on the Spectral Processing panel Once you have created your spectral library save the protocol and spectral library
23. Mono Image Snap Shot sss 58 e Acquiring an Image Cube sss enne nnne 58 e Acquiring Timed Sequences of Image Cubes ssesssssseeeeee 59 e Saving Images and Image Cubes cccceccesssesseesecsseeeeceeeeeseeaeceaeenseseeeeseeeaees 60 Acquiring Spectral Images 49 Tips for Obtaining Quantitative Results e Setup all hardware identically each time you acquire images during the course of an experiment Use the same stage height and illuminator positions select the same f stop and filter set Orient the mouse or mice similarly e Use one spectral library for unmixing all datasets Make sure you ve done control samples first e Save resulting images as Unscaled Data so they can be exported to other quantitation software if desired Acquiring Images Using a Saved Maestro Protocol If a Maestro protocol has already been saved tested and validated for the type of specimen cube you want to acquire load the protocol first then acquire the cube using the settings from the protocol Protocols are recipes for collecting and measuring data By using a saved protocol to acquire images of similar specimens throughout an experiment you don t have to specify acquisition settings every time This helps ensure consistency throughout the experiment Protocols can be opened before or after loading an image cube Note Be aware that using a protocol from one specimen to acquire an ima
24. Set the corresponding lighting position in the Maestro application Select Edit Hardware Settings from the Hardware menu and select the Camera Settings tab in the Edit Protocol dialog box Select the correct position in the Lighting position drop down box Caution The fiber optic guides for the two illuminator arms closest to the operator are A routed on the front operator side of the arms The guides for the rear arms are routed on the far side of the arms Be careful not to displace these guides dur ing cleaning or other action Doing so could limit the travel of the illuminators or the stage during the fiber optic and stage setting procedure 16 Introduction to the Maestro In Vivo Fluorescence Imaging System Interior Lights On the right hand side near the top of the imaging module there are switches that control the interior lights The switch labeled WHITE illuminates the visible wavelength lights and the switch labeled IR illuminates the near infrared wavelength lights see Figure 9 INTERIOR LIGHTS WHITE IR ON ON OFF OFF Figure 9 Interior Light WHITE amp IR Switches To switch interior lights ON and OFF Depending on your application switch on either the WHITE or IR lights The corresponding LEDs will illuminate see Figure 10 for location of LEDs Later when you are ready to capture an image or image cube remember to switch off the interior lights and switc
25. There is also a larger pseudo colored composite image displayed beside the original 14 RGB image of the cube The colors used to create this image are library pseudo colors The result should be a clear display of the specific fluorescence signal with obvious differentiation from the autofluorescence Save the resulting images a You can save all images as displayed by selecting File gt Save Image gt Save All As Dis played Select or create a folder in the same directory as the original image cube Images are saved as TIFF images that can be opened in a variety of image display programs Component Images nr pepe EU CCS Click again to unselect Cursor x Avg Signal Computed Spectra Find Spectra Ignored a Ont offset U o6 6 oe ES Name Color Select Specta ur gms ilAuofur whte 2 630 rm rc NE 3 o EE Magenta lt LeLe LL LLL LLL 4 5 6 Pk iu NA 8 9 10 White a Transfer To Library Transfer To Library Unix and b You can save all images as unscaled data by selecting File gt Save Image gt Save All Images As Unscaled Data See the Maestro User s Manual for more about saving cubes and images C You can also save the entire workspace by selecting Save Result Set from the File menu Enter a file name to save all images and results in a single file Save the Protocol and or Spectral Li
26. and attempts to prevent saturation of pixels by restricting the exposure time to a percentage of the dynamic range of pixel values For example the default value of 7096 for 12 bit 4096 level images results in a maximum intensity value of 2867 Maximum Exposure lets you specify a maximum exposure time The default value is 5 seconds Using Low Screen Resolution Mode Maestro software only supports screen resolutions of 1280x1024 or better However many projectors do not support resolutions this high The Maestro software has a special mode to support projectors or screens at 1024x768 resolution Some of the Maestro software s functions will not be available in this mode Screen resolution is detected at time of startup A dialog box warns you if you are running in low resolution mode If the Maestro software is started and subsequently the screen resolution is changed the Maestro software will not detect it If the Maestro software is run with a screen height of 800 pixels or less it makes the following adjustments In low resolution mode spectral libraries can only have five elements This will effect the Spectral Processing and Display panels as well as the Batch Mode Import Spectral Library and RCA dialog boxes The toolbar is removed from beneath the main menu The Define button for Filter Selection is removed from the Acquire panel The Sample ID and Notes boxes are removed from the Acquire panel The Notes box is removed f
27. as un normalized The Y axis in the spectral graph is scaled 0 to 255 8 bit or 0 to 4095 12 bit 2 Move the cursor over the image to see how the white curve changes shape You are seeing the spectral curve associated with each pixel as you move the mouse pointer Saving An Unmixed Result Set After unmixing an image you can save all unmixing results without having to save each unmixed image independently The Save Result Set feature saves the entire workspace excluding the cube in a single file This file will contain the component and composite image the file name of the cube but not the cube itself an RGB representation of the cube and other data such as measurements and regions display parameters user notes and the spectral library You can then reload the saved result set later to review the unmixed results and conduct further analysis quantitation You cannot do additional unmixing however because there Is no cube associated with a saved result set unless you import the associated cube into the result set see Importing a Cube Into a Result Set on page 71 To save the current unmixed result set 1 With the unmixed images on your screen select Save Result Set from the File menu 2 The Choose Results Set dialog box opens 3 Entera name for the new result set and click Save Result set files are saved with a umx file name extension 70 Unmixing Spectral Images Working With Saved Result Sets If unmixe
28. be displayed in the image viewing area 11 Click the Save Cube button on the toolbar to save the new image cube In the Save Cube dialog box select a location and name for the image cube and click the Save button For this exercise name the cube gdotcard 12 You are now ready to unmix the fluorescence signals from the autofluorescence in the image cube Unmixing the Image Cube 1 Enlarge the cube view window by clicking once on the window to make sure it is active and then press Ctrl E two times 2 Switch to the Spectral Processing panel 3 Sample the autofluorescence signal a Click the Draw MJ button in the first row of the color palette and use the mouse pointer to sample by clicking and drawing a line through an area of autofluores cence on the business card see Figure 23 A check mark appears in the Select box for row 1 32 Imaging the CRi Business Card with QDot Spots b Choose the Black color marker from the Color drop down box in row 1 Notice the sampling line turns black Unmixing the autofluorescence into Black will cause it to disappear in the unmixed composite image c In the Name column of the color palette rename this spectrum from C7 E to Paper 4 Next use the Green color marker to sample the QDot spot that contains the 565 nm fluorescence plus the paper This is the dot on the far left side a Click the Draw MI button in the second row of the color palette and
29. cri inc com www cri inc com CRI 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 7924 P 1 781 935 9099 F 1 781 935 3388 sales cri inc com www cri inc com Contents Chapter 1 Introduction to Multispectral Imaging Theory and Concepts 1 DASH oes M 1 Human Perception of Light Intensity and of Color sss ener enne enne 2 A Highly Adaptive Light Deteetor eene tenter enini eate ease Sra E deb NET ave ESS 2 Response o Illumination ore rr AS RE ERE Pepe ARM Ri wane 3 Ability to Distinguish Colors sese eene enne ener enne nennen enne enne trennen ense en rennen nnns 3 ElIuorescence eere Open te E ERE EE PRENNE YER ERES E bag en Feld eta E EA E E E EE 3 SIGIN E nd MEE 4 Autotfluorescence dr eU ERU RON Deni nee E E ER IR HI E 5 Excitation and Emission Filters esses enne nennen netten etre 5 The Multispectral Solutionsissa ei referto eee teer Pe nete odes ovo d be ebd ed dp e REEL RENE ERE RASSE ede E TR 6 Overlapping Emission Spectra eec oe esee uten abe v Due eda ee ee ERE AERE TEGERE sa ERE ed adeo eeu daga aii 6 Multispectral Analysis iier qe rea es petere deese EE Xi goat dee celta sod endeledendadedulgavanendeoencsts 7 Solution for Imaging Small Animals sess eene ener nennen nnns 7 Chapter 2 Introduction to the Maestro In Vivo Fluorescence Imaging System
30. during the sequence 5 Enter a base filename for all the saved cubes Acquiring Spectral Images 59 Click the Acquire Cubes button to begin the acquisition sequence A progress indicator Time Sequence Progress N Time To Next Exposure 21 sec displays the amount of time until the next Current Exposure 10f2 exposure cube the current exposure cube COT Remaining time 0 00 26 number and the total time remaining until the acquisition sequence is complete The Maestro software automatically saves Figure 38 Timed Sequence Progress each cube in the specified directory When the acquisition sequence is Beacons Sele complete you can locate the cubes at Ele Edit View Favorites Tools Help ay the destination directory and open them A C3 c Waestro Data cubes v EJ Name Size Type in Maestro As seen in Figure 39 the 60Second_Intervals_2006 Jul 12_16_31_05 im3 16 876K8 IM3File cub e filenames begin with the base 60Second_Intervals__2006 Jul 12__16_31_38 im3 16 876KB IM3File name you specified and include the lt gt date and time of acquisition Figure 39 Cubes acquired during timed acquisition Saving Images and Image Cubes m Save Cube Tip Devise a consistent naming convention for your images and image cubes Use file and folder names that incorporate specimen names as well as dates and or times and acquisition parameters such as filter cube binning and exposure To sa
31. gently squeezing out the water and pressing it with dry paper towels Reinsert the filter at the top of the cooling fan inlet and install the inlet cover Plug in the illumination module and switch on its power 20 Introduction to the Maestro In Vivo Fluorescence Imaging System Excitation Filter Slider The excitation filter slider holds two 50 mm diameter bandpass filters Excitation filter slider sets ordered from CRi are color coded to indicate their wavelengths For example the blue 465 nm filter is indicated by a blue dot on its end of the slider the green 529 nm filter 1s indicated by a green dot and so on See Appendix C Filter Selection Guide for detailed filter specifications Note The illumination shutter cannot be opened without a slider and excitation filter in Z place This is to prevent the unfiltered light source from burning the end of the fiber optic guide To insert the slider 1 Select the excitation filter slider that contains the filter you want to use Remember the filters are color coded so if you installed the blue color coded emission filter use the blue color coded excitation filter if green use green etc 2 Insert the slider into the slot in the side of the illumination module shown in Figure 12 There is no front to back orientation requirement for the excitation filter slider but there is an up down orientation The filter keeper on the slider must be toward the top 3 You shou
32. has three bars colored red green and blue The x axis represents the wavelength range of the cube The wavelengths in the red bar are mapped to the red plane in the display image green to green and blue to blue You can move these bars by clicking in their interior and dragged to a new position The bars can be resized by dragging the handles on the ends of the bars To View Visual RGB Click the Visual RGB button to set the bars so they approximate the human eye To View Equal RGB Click the Equal RGB button to set the bars so they divide the cube s wavelength range equally among the red green and blue planes Customizing Spectral Displays 87 Changing How a Composite is Displayed The Maestro software displays a composite image by combining the displays not the raw data of the component images When a composite image is active the Component Selection box becomes visible in the Display panel Component Selection Color Display Name Background Black Bu M Q605 Red W Figure 60 Component Selector There is a row for each component image Use the Display column to control which components are used to create the composite image If you want to change the color used to display any of the component images select a color from the component s Color drop down box This feature is very useful when you want to make the fluorophore signal of interest more clearly visible in a composite image For example you cou
33. measuring 77 modify 79 move 79 save 81 saving measurements 84 threshold segmentation 77 transparency and color 82 reinitialize hardware 28 resolution low 27 result set saving 70 S Safety cautionary statements 10 operator and equipment 10 technical assistance contact 11 sample area specification 91 sample ID save with cube 59 saturation mask 86 Save cube 60 image 61 region 81 unmix results 70 snap shot 58 software license agreement 111 Specifications mechanical dimensions 93 operating 91 Specimen Stage height setting 81 positioning 15 Spectra computing manually 68 import from component image 44 import from composite image 45 import from library 44 sampling manually 67 subtracting from cube 72 Spectral Graph Scale normalized 70 scale to max 70 un normalized 70 Spectral Library open 64 save 43 Stoke s Shift 4 T Temperature operating specification 91 storage specification 91 third party computers using 105 threshold segmentation 77 thumbnails introduction to 23 timed acquisition 59 timed sequences 59 U UL test 12 unmix cube 32 user s manual about 11 Index 119 V visible wavelength discussion of 2 WwW wavelength and exposure 52 wavelength range specification 91 wavelength slider using 52 wavelength to RGB map 87 weight system specification 92 window layout description 24 windows resizing 25 Workstation Computer overview of 22 specifications 92 Z zooming images 25 12
34. of Interest eere Specifying the Current Wavelength and Exposure cccecccessesssesseceseceseeeseeeseesseceaeceeeseeecseecsaeneeneeeseeesseeseesaeees Adjusting the Current Wavelength sess ennt entren entere enne Adjusting the Exposure Time eene enne nennen entren rennen trei enne n nennen eren Specifying Custom Collection Settings esses eene enne eene Selecting a Filter and Cube Wavelengths ssssssssesseseseeeeenee eere nennen ener en trennen nnn entren Using Preset Filter Setting Sirise E oe e nein er Yee eer pea ed este Da eV Cres eee et eode ded obese oa bd vegeta Manually Entering Acquisition Settings cccccccesssscescessecssecsecsecseeeeseecseeaecaeceeeseneecaecaaecseseeeseeeesaessaes Modifying and Saving Preset Filter Settings ssssssssssseseeeeenee ener nennen nnns Acquisition Setup Using Multiple Filters e seesseseseseeeeeeeee ener ener enne nnne nere enne nnns Maintaining Compatible Exposure Time Ratios ener rnnt Saving Many Filter Protocols in One Maestro Protocol esses enne nennen Fl t Fielding M m Acquiring a Mono Image Snap Shot eene enne nennen nnn enn nnne nnne Permiso tenes Assigning a Sample ID to an Image Cube ssssssssssessesee eene nnne enne Associating Notes with an Image Cube ccceccecssesseceseces
35. operates in the 500 to 950 nm wavelength range Introduction to the Maestro In Vivo Fluorescence Imaging System 9 Operator and Equipment Safety It is the responsibility of the purchaser to ensure that all persons who will operate the Maestro in vivo imaging system are aware of the following cautionary statements As with any scientific instrument there are important safety considerations which are highlighted throughout this User s Manual General Cautionary Statements READ AND UNDERSTAND THIS USER S MANUAL BEFORE ATTEMPTING TO OPERATE TROUBLESHOOT OR MAINTAIN THE MAESTRO IN VIVO IMAGING SYSTEM READING THIS MANUAL FIRST MAKES IT EASIER AND SAFER TO OPERATE AND MAINTAIN THE SYSTEM Operate the system on a flat stable surface Do not drop the imaging module or the illumination module Do not expose the imaging module or illumination module to prolonged heat above 40 C Do not operate the system in an environment with explosive or flammable gases Do not subject the imaging module or its components to intense light from laser focused arc or Hg lamp sources Do not operate the system in places where it may be splashed with liquid The imaging module may be cleaned with a sanitizing solution Refer to Cleaning Your Maestro In Vivo Imaging System on page 22 for cleaning guidelines Use only the supplied power cables with three prong plugs Some cables and adapters supplied with the system have proprietary specification
36. saturated if they were saturated in the cube used to create the component In a composite image a pixel will be saturated if it was saturated in the component image If saturation is present in your image use the Saturation Mask controls to reduce or eliminate the effects of the saturated pixels These controls are found at the bottom of the Display panel Saturation Mask Color Display Red E M Oo Figure 58 Saturation Mask To apply a saturation mask 1 Select the image to which you want to apply the saturation mask 2 Select the color that matches the saturated pixel color 3 Click the Display check box to apply the saturation mask to the image 86 Customizing Spectral Displays Changing How a Cube is Displayed Computers display color through varying intensities of red green and blue In order for the Maestro software to display an image cube which has many more color planes the Maestro software maps all colors in the cube to red green and blue planes When an image cube is active the Wavelength to RGB Map box becomes visible in the Display panel Wavelength ranges in the image cube are mapped to red green or blue to create the display image Wavelength to RGB Map Visual RGB Equal RGB Figure 59 Wavelength to RGB Map The plot below the bars shows the current spectral library It will also show the spectrum underneath the cursor when it is moved over the display image The upper control
37. see Acquisition Setup Using Multiple Filters the system will prompt you to install the correct filter set Install the requested filter set before continuing 3 Maestro will calculate the exposure time s that should be used to acquire an image cube of the specimen The value will display in the Exposure text box To autoexpose at the current wavelength Click the Autoexpose Mono button Maestro will calculate the best exposure time for the current wavelength and display the value in the Exposure text box To use custom wavelengths and exposures If you have specified the custom settings see Specifying Custom Collection Settings select the Custom Wavelengths And Exposures check box You may now acquire the cube using the custom collection settings Specifying Custom Collection Settings Custom collection settings let you use unevenly spaced acquisition settings and varying exposure times to acquire an image cube You can specify new settings and save them or you can load a saved wavelength and exposure table To specify custom collection settings 1 Select Edit Hardware Settings from the Hardware menu or press Ctrl H The Edit Protocol dialog box opens Acquiring Spectral Images 53 2 Click the Custom Collection Settings tab 3 The Wavelength Exposure table defaults to the acquisition settings of the currently selected presets 4 To change the defaults specify new Start End Wavelengths and Step interval
38. tee ette te reed ee tee a e eee eee erri ee Econ ee Eee AATE RE soleo oleo Unmixing the Ima G6 ERIS Quantifying Results eie entre tenete eere dera ia ee nas edis iur e EE une Tee ed adea Eod ae Eee ad aa on ee bdo to Chapter 4 Method Development Methods for Building Spectral Libraries essen eene nnne ener enne Tips for Spectral Library Development eni itii tt e tree ero ete E ed e e EORR ERES E reb SE PAR eR Obtaining Autofluorescence and Label Spectra from a Single Animal sss Obtaining Autofluorescence and Label Spectra from Two Animals sse Saving Spectral Libraries ctii h acea teet e n prie deae caddesededdedescadadaabadedanchdevededdesedendesenetdesense Saving ProtOco P Importing Spectra Into a Library ete teni t e ot ra E EER EEEE OE EE REEE EE REEE From an Existing Library ERE From a Saved Component Image sess enne n tren net nnne nennen nnn nnnes From a Saved Composite Image simonie aeien oe n eene eene A nennen tenere nnne enne ucfec discus eee Chapter 5 Acquiring Spectral Images Tips for Obtaining Quantitative Results sese eene eene nnn neret nnne Acquiring Images Using a Saved Maestro Protocol essere enne Viewing Lave Image Stream reet dei e ve Se ee EE Re PER WERTE Ree ER eue odere es ear EUER THE de Selecting Camera Binning and a Region
39. the correct filter set s before continuing 4 After acquiring the reference image the Acquire Cube button is once again available Replace the paper with your specimen click the Autoexpose Cube button again and then click the Acquire Cube button Acquiring a Mono Image Snap Shot To take a snap shot at the current wavelength and exposure click the Acquire Mono button at the bottom of the Acquire panel This function acquires a single picture not an image cube Acquiring an Image Cube 1 Autoexpose the cube as described in Adjusting the Exposure Time on page 53 2 Ifyou are satisfied with the wavelength and exposure settings click the Acquire Cube button at the bottom of the Acquire panel to acquire the image cube 3 Ifyou are using the multiple filter feature see Acquisition Setup Using Multiple Filters on page 55 the system will prompt you to install the correct filter set Install the requested filter set and each subsequent filter set before continuing 4 When Maestro has finished taking the image cube a color representation of the data will display in the image viewing area 58 Acquiring Spectral Images Assigning a Sample ID to an Image Cube Use the Sample ID text box on the Acquire panel to assign an ID to the current cube When you save the cube it will be saved with its sample ID All measurements derived from the cube s component images will be saved with the cube s sample ID Cube IDs disp
40. the mouse A on the right was not injected with the fluorescent antibody and was not fasted prior to imaging The thigh of the left mouse exhibits specific binding of the antibody region 1 the rest of the skin of the mouse exhibits non specific quantum dot fluorescence The right mouse exhibits no quantum dot fluorescence Conversely the left mouse exhibits no food related autofluorescence signal due to having been fasted while the right mouse exhibits food autofluorescence in the region of its stomach a Switch to the Measure panel and click the Draw button b Draw a region around the tumor which represents specific binding of the label shown as region 1 in Figure 31 Refer to Manually Drawing and Modifying Regions on page 79 for help drawing regions El Maestro subq2mice File Edit Hardware Tools Window Help Load Cube Save Cube Zoom Ful Image Acquire Spectral Processing Measure Display Scaing Normalized XN gt 3 subg2mice Cube L efx 5 subq2mice_Unmixed_Composite Composite Ex Library 10 Name Color Select Draw E subq2mice_q dot signal Component Ee 1 fautofuore wis gt MJ 2ladctsond a NEM M a foods vekw ra E e NEM eos WIN sos NEN m 114 a NEM White Restore Defaults _ Clear at _Delete MM Shrink Real Component Analysis e sm RCA Thumbnails Measurements Log Cursor Specific Binding Non Specific Binding E subq2
41. their OFF positions Verify that the electrical power supply is the correct rating see Appendix A System Specifications amp Dimensions then plug each component s power cord into the electrical supply Installing Maestro Software Important If you purchased a computer from CRi to use with your Maestro in vivo A imaging system the Maestro software is pre installed on the computer by CRi There is no need to perform a software installation unless you are installing the software on your own computer or if the contents of your computer s hard drive have been erased or if the system software has been rolled back using Microsoft Windows system administration tools Important Before following this procedure CRi highly recommends that you A familiarize yourself with the basic operation of the Microsoft Windows operating system e g saving and deleting files and folders startup and shutdown procedures and navigating through the contents of your hard disk drive If you need to install or re install the Maestro software follow the procedure below 1 Verify that your computer s hardware is in good working order If using a computer not supplied by CRi make sure that CRi s minimum specifications have been satisfied Refer to Operating Specifications on page 91 If you are not sure call your local authorized distributor or CRi directly Verify that your computer s operating system is also in good working order
42. using a spectral library from one specimen on a specimen that P d contains different fluorophores will lead to incorrect results To open a spectral library 1 Select Open Spectral Library from the File menu The Choose Spectral Library dialog box opens 2 Select the location and filename of the library or protocol and click Open Tip You can also open a Maestro spectral library by double clicking the library csl Q file in a My Computer directory window If Maestro is not already open this action will open Maestro with the selected library Or you can drag and drop the library file into an open Maestro window to open the library 3 The color palette on the Spectral Processing panel will be populated with the parameters of the new spectral library 1f you opened a protocol all of the parameters saved in the protocol take effect Opening an Image Cube C3 Load Cube Click the Load Cube button on the toolbar or select Open Cube from the File menu 2 Inthe Choose Cube dialog box browse to the location where the image cube is saved e If opening a CRi format im3 image cube double click the file to open it e fopening a TIFF image cube open the folder containing the images and double click any one of the TIFF image files to open the cube 3 Thecube will open and a color representation of it opens in the Maestro work area Tip You can also open an image cube by double clicking the cube im3 file in
43. usually installed so that their part numbers and wavelength are visible through the hole in the bottom of the slider Filter Part Number and Wavelength Figure 68 Excitation Filter and Slider Removing Filters from a Slider To remove a filter from the slider loosen the screw holding the filter keeper in place no need to completely remove the screw and slide the keeper out of the way Remove the filter by either pushing it up from below with a tissue or by carefully turning the slider over and allowing the filter to drop out into your hand Be careful not to touch the glass with your hand 108 Filter Selection Guide Cleaning Filters and Other Optical Surfaces If you need to clean any optical surfaces apply lens cleaning fluid spectroscopic grade isopropyl alcohol distilled water or methanol to a lint free lens tissue and drag wipe the surface Do not rub the surface because anti reflection coatings can be scratched Repeat with a fresh tissue and fluid after one pass since contaminants need to be wicked away not spread over the optical surface Filter Selection Guide 109 110 Filter Selection Guide Appendix D CRi Software End User License Agreement The following is an agreement the Agreement between you and Cambridge Research amp Instrumentation Inc 35 B Cabot Road Woburn MA 01801 CRi for software known as Maestro and its accompanying documentation collectively the Software By install
44. will subtract the known spectra from the mixed spectra without any scaling When there is a weaker signal you can manually adjust the amount of autofluorescence that will get subtracted to ensure that the best pure spectrum is found This is done by checking the Scaling check box and sliding the Error Scaler 1 In the Computed Spectrum box see Figure 45 select Scale To Max in the Scaling drop down box This provides the best visual representation of the spectra 2 The mixed spectrum and computed spectrum are shown in their respective colors The dotted best fit line is a scaled representation of the known autofluorescence signal see Figure 45 3 With the Scaling box checked try sliding the Error Scaler to the right to increase the scaling or to the left to decrease it until the non overlapping portions of the known and mixed spectra line up as closely as possible as shown in Figure 45 4 When you are satisfied with the computed spectrum select a Spectrum Number Name and Color and click the Transfer to Library button 5 When you are finished adding spectra to the library remember to save it as described in Saving Spectral Libraries on page 43 Occasionally for weak signals there may be too much baseline offset in the data If you did not get the desired unmixing results select the Fit Offset option in the Manual Compute Spectra dialog box Figure 45 and see if the unmixing results improve The computed
45. work until you restart the computer 16 Software and Hardware installation is now complete 17 See Launching the Maestro Application on page 22 for how to launch and activate the Maestro software 102 System Setup amp Installation Activating Maestro Software The first time the Maestro software is launched an activation code must be entered to activate the software Connect the computer to the Internet if an internet connection is available Activation is quickest 1f the computer has an active Internet connection Then select one of the following options in the InstallShield Wizard activation dialog box Note Keep in mind that the serial number i e activation code that came with your VA Maestro software allows you to install and activate the software on a limited number of computers The actual number of computers depends on your purchase agreement with CRi To activate Maestro software via the Internet l In the InstallShield Wizard activation dialog box select I have a serial number and I want to activate Maestro and click the Next button Enter the serial number found on the Maestro software CD case and click Next to activate Important This is the serial on the software CD Do not enter the serial number found on the back of the Maestro imaging module If the computer has an Internet connection your Maestro software will be activated within a few seconds To activate Maestro software via Email
46. 0 Index Warranty CRi warrants its Maestro systems for a period of one 1 year from date of shipment against defects in material and or workmanship provided its installation application and maintenance are within specifications Normal wearing parts are excluded This warranty covers only items manufactured by CRI CRi will correct by repair or replacement at its option and its expense any proved defects in items of its manufacture subject to above provided immediate written notice of such defects 1s given A valid Return Materials Authorization RMA must be obtained by contacting CRi before items may be returned to the factory Repair or replacement will be provided F O B Freight On Board at CRi s factory The total financial obligation of CRi under this warranty does not exceed the purchase price of the items of its manufacture as set forth on normal pricing schedules We will not assume any expense or liability for repairs made by others without our prior written consent This warranty is void and CRi will be free from all obligations hereunder if the items of its manufacture have been misused reprocessed or reused repaired or modified without our written consent CRi assumes no liability for damages or injuries resulting from the misuse misapplication or unauthorized repair or modification of its equipment We assume no liability to users of our equipment or to any other person or persons for special or consequential damages
47. 9 Tit GUC EL OTN ET M 9 Operator and Equipment Safety t rrr rentrer ete re era d eee E dps RE ee Fons en E E ede erede eed 10 General Cautionary Statements sss eene eren ener erre nennen enne ntes nnne 10 Illumination Module Cautionary Statements esses 10 For Technical ASSIS anoe sean nr ERR hes priest tan nahme hs ense io Fecerat edam d ea bel eR de age reete co 11 About This Marivial MR 11 Design Change Disclaimer ERE 12 Reproduction Disclaimer ette erroe rr pierre e aS E NT EKE EREE ENEEIER 12 CE CSA and UL Testing and Certification sss eene enne enne nennt enne nennen tenes 12 Maestro Hardware Components essei este toot ede a a deb nnb eth oned ub ase e EEA EAE are d reni de 13 Imaging Module E M 13 DY SEM POWER arenario E EE LLL LU 14 Specimen Stage and Illumination Assembly esee eene 15 Fiber Optic Illuminator Arms eeessesseeeeeeeen eene nennen enne nennen entren nre n tenentes tenerte nennen nennen 16 nrostydl ble UE 17 Emission Filter Slider esee ennt trennen tentent entente nnne nent tente nenne 18 Ilhimination Module 3 0 BPO e ONERE UG DR nM i OMS Em redo eM iere uS 19 Excitation Filter Sit M E 21 Switching the Illumination Module ON and OFF sess ener enne nnne 21 Workstation Conip ltet cuu acea rece sakes e e dep eee nie edere iae PORE eva du eed xe e e ENa re du a eda na
48. Alexa Fluor 750 750 779 cy2 498 506 Cy3 554 568 Cy5 649 666 Cy5 5 675 695 Cy7 743 805 GFP 475 509 FITC 490 525 eGFP 498 515 YFP 520 532 Rhodamine 550 573 RFP dsRed 558 583 ICG 800 850 Color denotes usable filter set X denotes recommended filter set Maestro Filter Set Part 4 Excitation Filter Emission Filter Acquisition Settings Deep Red M MSI FLTR DEEP RED 671 to 705 nm 750 nm longpass 730 to 950 in 10 nm steps NIR M MSI FLTR NIR 710 to 760 nm 800 nm longpass 780 to 950 in 10 nm steps The ending wavelength can always be made longer if there is fluorescence beyond the suggested end point Filter Selection Guide 107 Removing amp Installing Filters in the Filter Slider Installing Emission Filters into an Emission Filter Slider Emission filters must be installed in the filter slider with the correct top to bottom orientation A small arrow on the side of each filter must be pointing downwards towards the specimen stage when installed in the imaging module After installing the filter slide the keeper back into place and tighten the screw Filter must be installed with this arrow pointing toward bottom of filter slider Bottom of Filter we A Slider Filter Wavelength Labels Color coded Dots Figure 67 Emission Filter and Slider Installing Excitation Filters into an Excitation Filter Slider Excitation filters have no orientation requirements but they are
49. Chapter 9 Frequently Asked Questions 89 Contents iii Appendix A System Specifications amp Dimensions Operating Specifications eee eripi tired ore HERE s se EP O Eee P Eee e Reese ue E PETERE eva Computer System Requirements sse ener enne nnne entrer n rese tenerse narii nre e nennen rennen nn Mechanical Dimensiofis drittes or HE RYE SOY KEEPER COBRE eR RUN Ur rR en neuer ERR ed eee aE PEE rn dn Imaging sil e S Illumination MERE Specimen Sta gesais ans Appendix B System Setup amp Installation Unpacking the Maestro n Vivo Imaging System sss ener enne nenne ener Setting Up the Maestro In Vivo Imaging System sessssesesesseeeeen eene enne nnne nnne Installing Maestro Software esses eene enne n trennen nen eren rr entries nennen Activating Maestro SOL Wares iios EE tetur etre res rare etra e re S EORR rid etre E deese eue ende Rr e eR ed eR ER Calibrating Pixel Size ecne Tests for Ensuring Proper Operatloni reto Ere EHE RT Lowe AAN E A OS Using Third Party Computers with the Maestro Software eee nnne ener Appendix C Filter Selection Guide Removing amp Installing Filters in the Filter Slider essere ener Installing Emission Filters into an Emission Filter Slider eee Installing Excitation Filters into
50. LARY INDIRECT OR DIRECT DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES FOR LOSS OF PROFITS LOSS OF DATA RE RUN TIME INACCURATE INPUT WORK DELAYS BUSINESS INTERRUPTION OR ANY OTHER COMMERCIAL DAMAGES OR LOSSES WHETHER IN AN ACTION IN CONTRACT TORT INCLUDING NEGLIGENCE AND STRICT LIABILITY OR OTHERWISE AND EVEN IF CRI HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES AND REGARDLESS OF WHETHER ANY REMEDY FAILS OF ITS ESSENTIAL PURPOSE U S Government End Users The Software qualifies as commercial computer software for purposes of the Federal Acquisition Regulations FAR 52 227 19 and the Department of Defense Supplement to the FAR DFARS 852 227 7013 If the Software is acquired by a civilian government agency it is furnished with only the minimum Restricted Rights provided by FAR 52 227 19 If the Software is acquired by a military agency it is furnished with only the minimum Restricted Rights provided by DFARS 52 227 7013 c 1 ii Miscellaneous This Agreement contains the entire agreement of the parties with respect to the subject matter hereof and supersedes any proposal or prior agreement written or oral and any other communications between the parties relating to the subject matter hereof No modification or waiver of any provision of this Agreement shall be effective unless in writing and signed by the parties No delay or failure on the part of any party in exercising any right under this Agreement shall impair any su
51. Maestro n Vivo Fluorescence Imaging System User s Manual for Maestro 2 2 August 2006 Detect multiple Remove Improve sensitivity dyes even when contrast robbing and achieve spatially colocal autofluorescence results in seconds ized Notice The information in this document is subject to change without notice and should not be construed as a commitment by Cambridge Research amp Instrumentation Inc CRi assumes no responsibility for any errors that may appear in this document This manual is believed to be complete and accurate at the time of publication In no event shall CRi be liable for incidental or consequential damages in connection with or arising from the use of this manual This manual describes system operation using Maestro version 2 2 software For more information contact Cambridge Research amp Instrumentation Inc CRi 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 792 4 Phone 1 781 935 9099 Fax 1 781 935 3388 Email techsupport cri inc com or sales cri inc com Web site http www cri inc com Document Part No MD15379 Rev B August 2006 CRi 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 7924 P 1 781 935 9099 F 1 781 935 3388 sales cri inc com www cri inc com Maestro n Vivo Fluorescence Imaging System CRI 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 7924 P 1 781 935 9099 F 1 781 935 3388 sales
52. Maestro Illumination Module The light source is a Cermax type 300 Watt Xenon lamp It provides 5600 K illumin ation with a typical lamp life of approximately 500 to 1000 hours After this time the lamp intensity will be reduced by about 50 If lamp brightness is important to your application you should consider replacing the lamp during this time An hour meter on the back panel of the illumination module indicates the number of hours the lamp has been illuminated The housing of the lamp cartridge in your illumination module is colored either black or blue and can only be replaced with a cartridge of the same color Contact CRi if you need to purchase a replacement and indicate that you need a black Part OP10156 or blue Part OP10157 replacement lamp cartridge To replace the lamp cartridge Caution Do not remove the lamp immediately after operation Allow the lamp to cool A for at least ten minutes To prevent overheating replace only with the same type and rating of lamp Read all instructions before replacing the lamp Do not switch the illumination module on without the lamp in place 1 Switch off the illumination module and allow the lamp to cool for at least ten minutes 2 Open the lamp access door see Figure 12 for location to expose the lamp cartridge 3 Grasp the handles of the cartridge and pull firmly to remove the lamp 4 Insert the new lamp pushing it firmly into place The lamp can only be inserted one way
53. Pao ra S F amp F 5 the color specified at the bottom of the Fit Adjustment Scale Fit Offset Error Scaler dialog box 2 6 Ifnecessary use the Scale functions to fine tune the fit adjustment See Using Fit Offset and Error Scaling When Manually me e Color Computing Spectra on page 69 for sw o ue W ins truc tions Transfer To Library Close 7 Select the row name and color for this Figure 45 Manual Compute Spectra spectrum and press the Transfer To Library button 8 Repeat this process for the other mixed spectra 9 When you are ready to unmix the cube close this dialog box and click the Unmix button on the Spectral Processing tab 10 Save the new spectral library as described in Saving Spectral Libraries on page 43 68 Unmixing Spectral Images Error Scaling When Manually Computing Spectra Using Fit Offset To obtain an accurate pure spectrum which is required for an accurate unmixing the correct amount of autofluorescence must be subtracted from the mixed spectrum To do this it is important that the non overlapping regions between the pure fluorophore and the autofluorescence line up closely The Maestro software attempts to determine the amount of autofluorescence to subtract automatically When there is a bright fluorophore signal relative to the autofluorescence scaling is usually not needed In that case clear the Scaling box so that Maestro
54. R interior lights are not working when in fact they are Review the section Interior Lights on page 17 for a detailed explanation of the white and IR LEDs and how to verify they are working What fluorescence filters sets do need Refer to Appendix C Filter Selection Guide or contact your local authorized distributor or CRi to discuss your needs The filters used in the Maestro in vivo imaging system are designed as matched sets for optimum performance l installed the Maestro software but when I launch the application it doesn t recognize the hardware If the imaging module was left on and connected to the computer during installation of the software Microsoft Windows may not have been able to install the correct drivers for the camera Disconnect the imaging module from the computer Make sure the installation CD is in the CD drive then reconnect the imaging module to the computer Windows should detect the new hardware and begin the Hardware Installation Wizard Follow the prompts to install the drivers See Installing Maestro Software on page 101 90 Frequently Asked Questions Appendix A System Specifications amp Dimensions Operating Specifications Note that these specifications represent typical Maestro in vivo imaging systems as of August 2006 and are subject to change Parameter Maestro VIS Maestro GNIR Wavelength range 420 to 720 nm 500 to 950 nm Bandwidth FWHM Op
55. a Protocols contain the following information e acquisition settings including region of interest and binning wavelength settings filter selections exposure times and hardware settings bit depth stage height etc associated spectral library e auto save options e measurements options including threshold level and minimum connected pixels Whenever you make changes to any hardware settings or acquisition settings or edit the current spectral library save those settings in the current protocol or create a new one By saving your protocols you make them available for use again later when acquiring image cubes of similar specimens Method Development 43 To save a protocol l 2 Select Save Protocol from the File menu In the Choose Protocol dialog box select the name of an existing protocol if you want to overwrite it or enter a new name in the File Name field Click Save to save the protocol Note When you exit the Maestro application you will be prompted to save the current protocol if you have changed any hardware or software settings during the current session Be sure the save the protocol if you want to use the new settings again Importing Spectra Into a Library From an Existing Library You can import individual spectra from other libraries 1 Ts Press the Import Spectra From Library button on the Spectral fms Processing panel The Choose Spectral Library dialog box will prompt for the
56. a My Q Computer directory window If Maestro is not already open this action will open Maestro with the selected cube Or you can drag and drop the cube or multiple cubes into an open Maestro window to open the cube s 64 Unmixing Spectral Images Computing and Unmixing Spectra Automatically The Real Component Analysis feature lets you do the following Explore how many different spectral features are present e Extract spectra for further analysis Calculate pure spectra even if autofluorescence is present To compute spectra using real component analysis 1 Open or acquire a cube 2 Select the Spectral Processing tab 3 Click the Real Component Analysis RCA button Real Component Analysis RCA 4 The Real Component Analysis dialog box opens Component Images Click on component images ep M Click again to unselect Background S 6 Spectrum Select i M 2 v s m Transfer To Library Unmix and Close Figure 42 Real Component Analysis 5 Ifyou want to select a smaller region of the image on which to perform the RCA computation click the Select Area button and use the mouse to draw a rectangular region on the Cube image If you don t draw a region the whole image will be used Clicking the Clear Area button clears any user selected area from the image 6 If you want to provide an initial guess of the background
57. ach region show the region s major and minor axis drawn through the center of gravity If there are too many or too few measurement regions you may need to adjust the minimum number of connected pixels see Ignoring Smaller Regions on page 82 6 You can adjust the region s transparency or change its color see Adjusting Region 78 Quantifying Results Transparency and Color on page 82 Manually Drawing and Modifying Regions The Maestro software provides tools for manually drawing and modifying measurement regions These tools are located in the Manual Draw Regions box on the Measure panel To draw a region l 2 Click the Draw button to enter draw mode Select the draw tool shape by selecting from the toolbar or right clicking the image and selecting from the pop up menu rectangle ellipse line or area ROT Use the mouse pointer to draw new regions of the image to measure Each region is assigned an ROI number and is listed in the Measurements page at the bottom of the screen Measurement regions are numbered and sorted according to their size with the largest region first and the smallest last E subq2mice C2 Component Figure 53 Manually Drawing a Region To erase part of a region 1 2 3 Click the Erase button to enter erase mode Select the erase tool shape by selecting from the toolbar or right clicking the image and selecting from the pop up menu rectangle ellipse
58. aeeseeeseecseneeeeeeeeaeesaes 68 Error Scaling When Manually Computing Spectra esses eene nennen 69 WS Fit OFfSC bie T 69 Changing the Spectral Graph Scale sss sese ener enne 70 Saving An Unmixed Result Set cet anten teret te sa eek ee deg ANTE A Rune aa Re ERR e eA aE Ue Maa E RU een 70 Working With Saved Result Sets esee a a ee tr ea E Rae ENA NEE ERAN ENNE aS 7 Importing a Cube Into a Result Set sse eene eene 7 Baseline S btractioi n itor a ARTE INE IH E C ELU IR ERE RE E Pn UR o SRM RR dS 72 Batch Data Processing E 72 Using Line Profiles to Analyze Signals cccccecscssscescsescescecseeeseceseceseesaecsaecnesseeeseeesaeesaeenseessessseseseseaeenseeeneeags 74 Working with Line Prohiles 1 53 2 ett eU ER e es reor tt e pire iei cette peti eia bep veo n unte 76 Chapter 7 Quantifying Results 77 Measuring Reglons decet esee reed exe eet eee epe e EEEE REEE TEREE ETE E RERNE T Finding Regions Automatically Using Threshold Segmentation eese 77 Manually Drawing and Modifying Regions cccecscccsessseesseeeceseeeseesseceaeceseseeeeseecauenaeeseeeeseeeseesseeeeseneeass 79 Cloning GTI ERIT aee REE RE E EA R E E R Ea NETE E E RENEE E ESEI E EES 80 Dragging a Copy of a Region to Another Image sess ener enne nnns 80 Copying all Regions to the Clipboard s
59. an Excitation Filter Slider eee Ionen ciini Nod m Cleaning Filters and Other Optical Surfaces sess eene enne Appendix D CRi Software End User License Agreement Notice to Purchaset i eret ete geo eA d FOR ed o rd grey eae UE EE HRK E eMe eso sd xeu daten RE ordered n Appendix E Maestro Quick Start Guide Index iv Contents 107 108 108 108 108 109 111 113 115 117 Light Chapter 1 Introduction to Multispectral Imaging Theory and Concepts This chapter provides an introduction to the theory and concepts that enable CRi s multispectral imaging systems to function as well as they do Topics in this chapter Page La bri 1 e Human Perception of Light Intensity and of Color sesssess 2 duteisidtsct 3 The Multispectral Solution essere enne 6 Light as used in this discussion means the portion of the electromagnetic spectrum that is visible to the human eye While the physical description of light can be highly complex we will restrict this discussion to the wavelengths of light and the interaction of that light with physical and biological materials Introduction to Multispectral Imaging Theory and Concepts 1 The Electromagnetic Spectrum Wavelength in meters puse umm ee cem mene Er Gene er 1 11019 10 010
60. ard Q565 component image to make it active 3 In the Millimeters Per Pixel box make sure Stage Height is set to 1B 4 Inthe Measure Objects box Min Connected Pixels should be 100 and the Draw Region Labels check box should be selected 36 Imaging the CRi Business Card with QDot Spots 5 Click the Auto Calculate Threshold button in the Threshold Segmentation box Auto Calculate Threshold 6 The Maestro software automatically creates measurement regions around the two QDot spots in the image Tip You can use the Zoom tool to draw a zoom area around the regions to view them D more clearly 7 Repeat the procedure to find the measurement regions around the QDot spots in the qdotcard Q605 component image Your screen should look similar to Figure 26 Tip Ifyou prefer to draw the regions manually click the Draw button to draw regions O See Manually Drawing and Modifying Regions on page 79 8 You can adjust the transparency level and the color of the threshold mask using the Transparency slider and color drop down box 9 Notice that the Measurements page at the bottom of the screen displays the measurements of the regions in the active image the Q565 image in Figure 26 The Total Signal column shows each region s relative measure of fluorescence This column and the Area Pixels column are useful for comparing the amount of fluorescence in each region Si Maestro OdotCard File Edit Hardware Too
61. ated by the lighting system What can do about this It is possible to accidentally set incompatible heights that can result in uneven or suboptimal illumination Make sure the proper height settings for the specimen stage Frequently Asked Questions 89 and fiber optic illuminators are used Also be sure to use Flat Fielding when acquiring cubes see Flat Fielding on page 58 Why are my exposure times so long It is possible that there is too little light and the Maestro in vivo imaging system needs to use a longer exposure time Make sure the fiber optic illuminators are positioned properly and that the illumination module is properly set up and connected Check that the illumination light source has not exceeded its useful life span The light source should be replaced after 500 to 1000 hours of use Why are my exposure times so short The liquid crystal LC tuning element may have been moved out of the light path To correct this make sure the thumb slider switch is in the far right position The thumb slider switch protrudes from the ceiling inside the imaging module at the top of the case near the base of the lens assembly When I switch on the near infrared NIR viewing lights inside the imaging module don t see anything appear to get brighter with my eyes How can I be sure that the NIR illumination system is working Our eyes do not see NIR wavelength light very well so it is easy to mistakenly think the NI
62. avelength Selection manual adjustment 55 presets editing 55 presets using 55 current wavelength specifying 52 D dark current 27 Disclaimers design change 12 reproduction 12 Display 85 brightness and contrast 85 component selection 88 wavelength to RGB map 87 distance calibration 104 distributors CRi in the EU 12 E Electrical Supply specification 91 electromagnetic spectrum 2 emission filter slider inserting 18 emission spectra 4 6 environment operating specification 91 error scaling 69 excitation filter slider inserting 21 Exposure Time adjusting 53 custom settings 53 exposure time ratios 57 extract image plane 62 Index 117 F f stop 30 field of view 15 filter selection guide 107 filter serial port 26 Filters choosing 5 54 cleaning 109 excitation and emission 5 installing removing in sliders 108 selection guide 107 fit offset 66 69 flat fielding 58 fluorescence defined 3 frames to average 27 frequently asked questions 89 Fuses replacing illumination module 20 imaging module 14 G gain 26 H human perception of light 2 Humidity operating specification 91 storage specification 91 I Illumination module fuse replacement 20 lamp replacement 19 on off switch 21 overview 19 illumination eye s response to 3 illuminator arms positioning 16 image cube defined 7 image data pages introduction 23 image plane extract 62 image sensor format specification 91 image sensor pixel count s
63. ber indicating the wavelength for each image in the cube Unmix the cube as explained on the reverse side of this Quick Start Guide MaestroW Ri Maestro Nothing Loaded File Edit Hardware Tools Window Help a FG ux QA Ss Load Cube Zoom Full Image Acquire Spectral Processing Measure Display Region Of Interest And Binning Binning ROI D2 5 M ru Wv Wavelength And Exposure Wavelength nm Exposure ms 570 63 400 720 Autoexpose Mono Autoexpose Cube Cube Wavelength Selection Start Step End so B io B i amp Preset Filter Settings Blue 500 10 720 Fixed Filters Blue Multi Filter Support Flat Fielding C Flat Field Sample ID Notes Acquire Mono Acquire Cube CRi 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 7924 P 1 781 935 9099 F 1 781 935 3388 sales cri inc com www cri inc com Maestro 2 2 Quick Start Guide MD15459 Rev B August 2006 10 11 12 13 Unmix Images In the Maestro application select the Spectral Processing tab El Maestro subq2mice Fie Edt Hardware Tools Window Help a a Q Load Cube Save Cube Zoom Ful Image Acquire Spectral Processing Measure Display Scalna Normatwed XN gt subg2mice Cube Open or acquire an image cube To open a cube click the Load Cube button on the toolbar Cube format types include CRi format
64. brary and display them in the Candidate Cubes list Select from the Candidate Cubes list the cubes you want to process and click the Add button This will add them to the Selected Cubes list on the right Click the Run Batch button This will iterate through the selected cubes unmix and measure any components selected for measurement Using Line Profiles to Analyze Signals The Line Profiles tool can be used to analyze signals in Line Profiles component images This tool can be used to analyze any single channel monochrome image including component images and live images in Maestro Cubes and composite images cannot be analyzed with the Line Profiles tool To use this tool you draw lines on component or any monochrome images and the pixels sampled by the line show up as a plot in the Spectral Graph in the Line Profiles dialog box 1 Tumor Signal M To use the Line Profiles tool 2 Food signal oem M 2 M 3 Tumor Signal c NE J M 1 Select or open the component image or images in the velow A M Maestro work area ow Eg M Magenta MJ 2 Select Line Profiles from the Tools menu The Line s NEC MJ ney m L 4 1 A Profiles dialog box opens 2 mu Wc M e Tenrows in the color library let you draw up to ten Jwe 4 O MJ profile lines on multiple monochrome images e Each line profile appears as a plot in the Spectral Figure 49 Line Profiles Graph e You can change the Spectral Gra
65. brary for use throughout your experiment a You can save the complete Maestro protocol which includes the current spectral library by selecting Save Protocol from the File menu b If you want to save the spectral library as a separate file select Save Spectral Library from the File menu CRI 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 7924 P 1 781 935 9099 F 1 781 935 3388 sales cri inc com www cri inc com Index A absorption spectrum 4 acquire cube 31 58 acquire mono 58 acquire time sequence 59 acquisition time typical specification 91 acquisition wavelength 52 altitude operating specification 91 autoexpose at current wavelength mono 53 autoexpose cube 53 autoexpose target of dynamic range 27 autofluorescence 5 automatic save options 61 B bandwidth specification 91 batch processing 72 binning 51 81 bit depth 26 brightness level 85 C calibrating pixel size 104 CCD sensor active area 89 CE test 12 Cleaning filters 109 system 22 clone region 80 colors eye s ability to distinguish 3 component selection 88 Compute Spectra manual 68 using RCA 65 contrast level 85 control panels introduction 23 cooling specification 91 CSA mark 12 Cube acquire 31 58 autoexpose 53 definition of 7 importing into result sets 71 information viewing 61 save 60 save notes with 59 save sample ID with 59 subtracting spectra 72 unmix 32 zooming in out 25 Cube W
66. can reduce the rate of fading and different fluorescent materials fade at different rates all fluorescent materials eventually fade and this effect is irreversible For this reason specimens should be illuminated only while aligning and focusing and during actual image collection At other times the excitation light should be closed off Excitation and Emission Filters Filters that are used for fluorescence excitation and emission are specifically constructed to have very narrow bandpasses They pass only a limited range of wavelengths of light Restricting the excitation light wavelengths may reduce the amount of autofluorescence Restricting the wavelength range of the emitted light helps minimize the amount of autofluorescence light that interferes with observing and measuring the desired specific fluorescence 100 100 E 500 nm short pass Broad band pass 300 400 500 600 700 nm 300 400 500 600 700 nm Narrow band pass 500 nm long pass 300 400 500 600 700 nm 300 400 500 600 700 nm Figure 3 Excitation Filter Types Excitation filters should be chosen to match the excitation maximum of the fluorescence label being used The emission filter should match the emission maximum In practical terms the filter maxima may be slightly different from the ideal case simply due to limitations of filter manufacturing or to assist with autofluorescence reduction Specific excitation and emission filter combinations are availab
67. ch right or any remedy of such party nor shall it be construed to be a waiver of any continuing breach or default under this Agreement In the event any provision of this Agreement is held to be unenforceable the remaining provisions of this Agreement will remain in full force and 112 CRi Software End User License Agreement effect This Agreement shall be governed by the laws of the State of New York without regard to principles of conflicts of laws Any disputes relating hereto shall be adjudicated only in the state or federal courts in New York County New York State and you hereby consent to the exclusive jurisdiction of those courts This Agreement shall not be governed by the United Nations Convention on Contracts for the International Sale of Goods the application of which is expressly excluded You may not assign or otherwise transfer this Agreement or any of your rights or obligations therein without the prior written consent of CRi You may not use the Software for any unlawful purpose nor export or re export the Software except as authorized by law Should you have any question concerning this Agreement you may contact CRi by writing to CRi 35 B Cabot Road Woburn MA 01801 You may also call 1 800 383 7924 in the US or 1 781 935 9099 elsewhere Notice to Purchaser The CRi Maestro in vivo imaging system has many applications involving a variety of materials such as probes cell lines animal specimens etc Certain of these a
68. d 22 Cleaning Your Maestro n Vivo Imaging System ccccecccescssssessecseecsseseceecceseecseceseceseseseeesecaueceaeeneeeeneeaes 22 Launching the Maestro Application go ertt teer epit oe i E rte e EpOere Heady aa NEE 22 Understanding the Maestro Work Area sss eene enne enne nnne nnne nennen nennen nnns 23 Control Panels sete Pee ed poseen reb queer E ERE EQ eR EYE RE KK REINE AERE Ee ep e da 23 Thumbnails and Image Data Pages eene eene enne nene nre en nennen nnns 23 Window vnm mLEEEUEDDES 24 Resizing an EET ABL E 25 ZOOMING In and OUt m RE 25 Panning a Zoomed Image rinik ee anke ooann Pese dye RR AEE v QE cdava da gent eon Enea e dab a ede 25 Contents i Panning All Images Simultaneously cccecccescesseessecscecseeecesceeseecaeceseceseseceeseecsaecaeseseeseeeessecaeeeeseeesaeeaaes Specifying Camera Hardware Settings sss eene ennt enne nennen nnne nn nennen en Using Low Screen Resolution Mode sese enne nnne entren nnne eren enne nnne Reinitializing Maestro Hardware ettet ei eise a P e tesis eU DER PER e Ted ve ie oe ed ara aee ie eed Chapter 3 Imaging the CRi Business Card with QDot Spots First Steps M eaS Acquiring a Image Cubes P dence U rnmixing the Image Cube ente HR RD In petes a i ea a aa ee Calculating the Pure Spectra erede ee
69. d result sets have been saved you can open them for further analysis and quantitation See Saving An Unmixed Result Set for instructions on saving unmixed result sets To open a result set 1 Select Open Result Set from the File menu 2 In the Choose Results Set dialog box locate and select the result set umx file you want to open and click Open Tip You can also open a Maestro result set by double clicking the result set umx file Q in a My Computer directory window If Maestro is not already open this action will open Maestro with the selected result set You can also drag and drop a result set into an open Maestro window 3 Theunmixed images will open in the image viewing area If the Maestro software can locate the cube associated with the result set it must be a CRi format im3 Image Cube in its original location on the computer the cube will open along with its unmixed images If the Maestro software cannot find the cube or it is a TIFF Cube an RGB representation of the cube labeled RGB Cube Is Not Loaded will open 4 Ifthe cube did not load automatically you can import the cube into the result set See Importing a Cube Into a Result Set for instructions Importing a Cube Into a Result Set When a result set is saved the original image cube is not saved as part of the result set umx file An RGB representation of the cube is saved instead When you open a result set the Maestro software a
70. d six intermediate positions The selected height position determines the field of view that will be available when taking images Lower the specimen stage for a larger field of view raise it for a smaller field of view Illumination Assembly 4 A Field of View 3 1 0 x 1 4 in Field of View 2 2 0 x 2 7 in Field of View 1 3 0 x 4 0 in Specimen Stage Stage Release Levers Figure 7 Specimen Stage with Field of View Outlines For reference the specimen stage is etched with three numbered field of view outlines shown in Figure 7 The height positions for the specimen stage are also numbered shown in Figure 8 The outlines correspond to the three primary stage heights the lowest stage position 1 gives the largest field of view 1 the middle position 2 gives the middle field of view 2 the highest position 3 gives the smallest field of view 3 For example to use the 3 0 by 4 0 inch field of view set the specimen stage to position 1 The intermediate stage positions between each primary position are labeled 1A 1B 1C 2A 2B and so on To maintain clarity these intermediates are not marked on the specimen stage Depending on your application you may achieve better results using an intermediate stage height 3 Primary and Intermediate e s e Stage Height Positions e x
71. d to sample the known and mixed spectra manually Use the color palette on the Spectral Processing panel to build your spectral library by selecting colors for separating mixed signals into pure signals Ifthe cube you want to unmix was acquired using multiple filters with overlapping acquisition wavelengths see Acquisition Setup Using Multiple Filters on page 55 you will notice that there are two or more overlapping curves in the spectral graph for each of the spectra in cube Although this may look unusual the overlapping curves are used together as one spectrum for all unmixing e we 148 MI 20 ie M 3 Q565 s M o D he To MI s ow Em M s zoa MT j oom NN M 7 Je Loy M i WEN M 10 white L 17 M Figure 43 Color Palette There are ten rows available that you can use to build a spectral library To sample spectra 1 Open or acquire a cube and select the Spectral Processing tab 2 Click the Draw button within the row where you want to place the sampled spectrum 3 Click and draw on the part ofthe cube you want to sample Holding down the Control or Shift key will add additional regions to the sample When you release the mouse button a check mark appears in the Select column Tip The Control and Shift keys also let you sample multiple cubes Sample the current Q cube then select another cube from the thumbnails and hold down the Shift or Control key while sampling to obtain an average of the spectra f
72. data has not yet been enabled Unmixing Spectral Images 75 Working with Line Profiles After drawing line profiles on an image you can copy clone move and delete lines by right clicking on them To copy line profiles to the clipboard 1 Right click on the image that contains the lines you want to copy and then select Copy Line Profiles To Clipboard from the pop up menu This feature copies all profile lines in the image onto the clipboard If you want to copy a single line to another image simply drag and drop the line onto the other image Be sure to deselect the Draw M tool before attempting to drag and drop the line Note When dragging a copy ofa line to another image the line will not move while you S drag the mouse pointer However the copy will appear on the new image when you release the mouse button at the destination Right click inside the image to which you want to paste the line profiles and select Paste Line Profiles From Clipboard All line profiles on the clipboard are pasted into the current image Each new line profile is assigned the next row color in the color library and all lines are plotted in the Spectral Graph To clone a line profile 1 Right click on the line you want to clone and select Clone Line Profile from the pop up menu Cloning creates an exact copy of the original that you can move to another area within the current image You cannot move a clone to another image Th
73. e 2 a MitoTracker Image 3 a Acridine Orange image and 4 a FITC image By removing the autofluorescence contribution to the image the actual signals from the applied labels MitoTracker Acridine Orange and FITC can be readily seen If these individual images are recombined using highly contrasting colors to represent the location of each of the labels a composite image of high contrast and readily observable colors can be generated Solution for Imaging Small Animals Multispectral analysis also solves an even larger impediment to the imaging of small animals Animal skin and fur is highly autofluorescent In addition to the natural autofluorescence of skin there is also distinct autofluorescence from the sebaceous glands sebum and from a variety of commensal organisms that may be present mites fungi etc Small animals may also demonstrate fluorescence from ingested food particularly if the food contained chlorophyll whose breakdown produces fluorescence strongly in the red Multispectral analysis is able to separate all of these autofluorescence signals away from the specific labels applied to the specimen and provides the ability to localize each material present and to detect weak specific labeling even in the presence of strong autofluorescence Introduction to Multispectral Imaging Theory and Concepts 7 8 Introduction to Multispectral Imaging Theory and Concepts Introduction Chapter 2 Introduction to t
74. e MI Restore Defaults Clear At Delete Grow Real Component Analysis rt IRCA Thumbnails Measurements Log _ Cursor Manual Compute Spectra Selected ROI Number Avg Signal Major Axis Minor Axis X Location Ytocatifn Total Signal Thtal Signal Exp ms Max Signal Area Pixels Import Spectra From Library I 1 425 94 51 76 41 66 346 04 157 81 707061 70 Unknown Exp 2 178 82 51 76 41 66 231 04 199 81 296836 30 Unknown Exp Unmix 3 12 58 51 76 41 66 340 04 373N1 2087751 Unknown Exp Hardware Status No Hardware Protocol DataFolder Cursor X s Avg Signal III IR Figure 28 Autofluorescence and Label Spectra from Two Animals Notes e nthis example the autofluorescence signal is named autofluore and the fluorescence signal is named AF680 e After unmixing this cube we zoom in on the alexa 680 in mouse t2 AF680 component image and use the manual Draw tool on the Measure panel to draw a measurement region around the tumor We then copy the region right click on the region and 42 Method Development select Clone Region and move the copies to other locations within the image to compare fluorescence signals e As you can see from the Measurements data page at the bottom of the screen the area of the tumor Region 1 where specific binding ha
75. e mouse pointer changes to a hand with a floating clone of the original line attached to it Move the clone and click to release it at the desired location To move a line profile 1 Right click on the line you want to move and select Move Line Profile from the pop up menu The mouse pointer changes to a hand with the floating line attached to it Move the line and click to release it at the desired location To delete a line profile 1 Right click on the line you want to delete and select Delete Line Profile from the pop up menu 2 The line is removed from the image the selection is removed from the color library and its profile plot is removed from the Spectral Graph 76 Unmixing Spectral Images Chapter 7 Quantifying Results The Measure panel is used to draw measurement regions and set measurement parameters The Measurements page at the bottom of the Maestro work area displays the measurements of regions drawn on component images This chapter explains each of the tools used for measuring regions on component images Topics in this chapter Page e Measuring Reglons iiie eec ren eee QU e e EUR y eR E EARN VR aee AENEAN ait T e Ignoring Smaller Regions sess ener 82 e Adjusting Region Transparency and Color sse 82 e Understanding Region Measurements sess 83 Measuring Regions Finding Regions Automatically Using Threshold Segmentation 1 Select the compone
76. ed States and other countries Invitrogen is a trademark of Invitrogen Corporation Microsoft Windows and Excel are trademarks of Microsoft Corporation in the United States and other countries QDot is a trademark of Invitrogen Corporation Xenogen is a trademark of Xenogen Corporation O 2006 Cambridge Research amp Instrumentation Inc All rights reserved Document Part No MD15379 Rev B August 2006 CRi 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 7924 P 1 781 935 9099 F 1 781 935 3388 sales cri inc com www cri inc com
77. eeeseeeeecsaeeseeseeeescecsaecseceseseneseseeaaecaecneeseneeseeessen Acquiring Timed Sequences of Image Cubes ssssssssssseseeeeee eene ener enne nennen nenne Saving Images and Image Cubes cccccccsccesscesseeseeescessecenenecesenecseecsaenseceseceseeessecsecaeceseseaecaaecaaeneeseseeessesseeeaeees ii Contents 29 30 31 32 34 35 36 39 40 40 41 42 43 43 44 44 44 45 45 Saving Image Cubes Automatically sese eene 61 Viewing Cube Information iine c Rope THER Ore UE ted oce HE Rear eter eite bee petes eure eria 61 Extracting an Image Plane from a Cube esee eene enne nnne nennen nnns 62 Chapter 6 Unmixing Spectral Images 63 Unmixing with a Saved Spectral Library sss eene 64 Opening an Image Cube cete cepe re tte eere URL ek eT Y e RD eee ud QU ER etl V ER depre dua tea eua eei 64 Computing and Unmixing Spectra Automatically esee enne ener nnne nnne nnne 65 Computing and Unmixing Spectra Manually sees ener nrene etre nne 67 Sampling Spectra from a Cube cccecceccccssecsecsteeseceseceseecseecsecaeceseseaeeesecsueceaeceeeseeeseaecaaecsaeeeeeeseeesseeseeeaeees 67 Selecting a Region of Interest Shape ccccscccssessecsseceeceeeeeseecsseeseeeeeceseecseeeseceeeseeeeesecaaecaaeeeeeeseeeseeeaeenaeens 68 Manually Computing Spectra cccccccsccsscessceeceescecseeeseceseceseeeseeesecaecseseeesececsaecsaecsess
78. evels does degrade in both very dim light and very bright light Response to Illumination The eye s response to illumination is not a linear response but more closely approximates a logarithmic function The result is that while the human eye interprets differences between gray levels as even steps to achieve a two fold brightening of the perceived gray level the actual illumination level would need to increase significantly more than a simple doubling Contrasting the eye with a digital electronic sensor system any sensor that has 8 bit resolution can detect 256 levels of gray As the number of bits of resolution increase the number of gray levels also increases A 10 bit system gives 1024 levels and 12 bits yields 4096 levels of gray Digital electronic sensors are linear in response to light levels Ability to Distinguish Colors While the eye is relatively poor at discriminating gray levels intensity it is very good at distinguishing colors Most individuals are estimated to be able to discriminate thousands of colors The problem is no two individuals see precisely the same color In other words each individual interprets colors slightly differently when viewing the same color The basis for this is the way in which color sensors are arranged in the eye The eye contains three different types of color sensors similar in response to the red green and blue detectors in color cameras Individual colors are composed of some combi
79. file name of a spectral library Spectral libraries have a csl file name extension Spectral libraries from older versions of the software with txt file extensions may also be opened Double click the name of the library to open it s or Spectrum The Import Spectra From Library dialog box opens which o J zA re lists all the spectra stored in that library ereen IB f MET Make sure there is a check mark in the Select column next to each spectrum you want to import then click the Transfer to Library button If you want to load spectra from another library click the Load button and select a library to load Transfer To Library Click Close when finished importing spectra Figure 29 Import Spectra From a Saved Component Image You can import spectra from component images 1 2 44 Method Development Open the Result Set umx file that includes the component image from which you want to import spectra Right click on the component image and select Import Spectra from the pop up menu 3 The Import Spectra From Library dialog box Figure 29 opens with the spectrum used to unmix the cube into that component 4 Make sure there is a check mark in the Select column next to each spectrum you want to import then click the Transfer to Library button 5 Ifyou want to load spectra from another library click the Load button and select a library to load 6 C
80. for repeated use throughout the experiment with the same or similar specimens Topics in this chapter Page e Unmixing with a Saved Spectral Library sse 64 e Opening an Image Cube ssssssssssseseseeeeeeneren eene nnne enne 64 e Computing and Unmixing Spectra Automatically esses 65 e Computing and Unmixing Spectra Manually eee 67 e Saving An Unmixed Result Set eese eee 69 e Working With Saved Result Sets sse 7 Baseline Subtraction sesei eere peer eerie eie ie e E enS 7 e Batch Data Processing ssessssseseeeeeeeeren enne nennen ener 72 e Using Line Profiles to Analyze Signals 74 Unmixing Spectral Images 63 Unmixing with a Saved Spectral Library Ifa spectral library has already been saved and validated for the type of specimen you want to unmix use that library to unmix the cube Or if an appropriate Maestro protocol is available which should include the spectral library then load the protocol instead see Acquiring Images Using a Saved Maestro Protocol on page 50 By using a saved protocol and or spectral library on similar specimens to unmix and perform analyses throughout an experiment you don t have to recreate the spectral library every time This helps ensure consistency throughout the experiment Protocols and spectral libraries can be opened before or after loading an image cube Note Beaware that
81. ftware i1 agree not to disclose to anyone or allow anyone access to the Software and iii agree not to disclose any confidential information of CRi regarding the Software or that is otherwise disclosed to you in connection with this Agreement Transfer Restrictions The Software is licensed only to you The Software may not be transferred to anyone without the prior written consent of CRi The terms and conditions of this Agreement shall bind any CRi authorized transferee of the Software You may not transfer assign rent lease lend sell grant a security interest in sublicense or otherwise dispose of the Software on a temporary or permanent basis except for a CRi authorized transfer as stated above Termination The license granted in Section 1 above is effective until terminated This Agreement is conditioned upon your continued compliance with the terms and conditions hereof and will terminate automatically without notice from CRi if you fail to comply with any term or condition of the Agreement Furthermore CRi may terminate this Agreement at any time upon thirty 30 days notice Upon termination of this Agreement you shall immediately destroy all copies of the Software including all accompanying documentation and any other confidential and proprietary information you have received during or in connection with this Agreement CRi Software End User License Agreement 111 Limited Warranty CRi warrants that the media on which
82. g area and saves the images as TIFF or JPEG images e Save Image As Unscaled Data prompts you to enter a name for the currently selected component image and saves unscaled image data as a TIFF component e Save All Images As Unscaled Data prompts you to enter a name for each of the component images and saves all unscaled image data as TIFF components When the dialog box opens navigate to where you want to save the image s the default location is in C Maestro Data Enter a file name and click the Save button Saving Image Cubes Automatically The Maestro system can save your image cubes automatically To specify automatic save options 1 CU OBS 7 Select Edit Hardware Settings from the Hardware menu or press Ctrl H In the Edit Protocol dialog box click the Autosave Options tab Select the Autosave Image Cubes option to enable the autosave fields To specify a default directory click the Browse button and select from the browse dialog box Type a base name in the Base Name field if desired In the Auto naming Options box specify a naming option A sample appears in the File Name field Click the OK button to save the changes and close the dialog box Viewing Cube Information Cube information includes its size in pixels the number of planes in the cube a timestamp any comments associated with the cube and the wavelengths and exposure times used to generate the cube Acquiring Spectral
83. ge 67 for detailed instructions If you completed the exercise in Chapter 3 then you are already familiar with manually computing pure spectra Adding spectra from other libraries component images or composite images of the same or similar specimens see Importing Spectra Into a Library on page 44 Also depending on the nature of your experiment and the types of dyes or fluorophores used you may have imaged a single animal or two animals from which you will obtain the positive and negative controls Use negative controls for the autofluorescence spectrum use positive controls for pure spectrum calculations After building the library save it as described in Saving Spectral Libraries on page 43 Tips for Spectral Library Development 40 Method Development There is no substitute for proper controls Use negative controls and or subjects that do not contain exogenous fluorophores for determining autofluorescence spectra Use positive controls for determining the spectra of fluorophores of interest using one or more animals for each fluorophore The computed pure spectrum should be similar to but not necessarily the same as published spectra of that fluorophore Variations in the label s physical environment or depth within tissue can cause spectral shifts Verify that your libraries work correctly by testing them on a few positive and negative controls Use critical judgement Unmix into white or pink for autofluo
84. ge of a V4 specimen that contains different fluorophores will lead to incorrect results To open a protocol 1 Select Open Protocol from the File menu 2 Ifaprotocol is already open or if you have made any changes to settings that normally get saved in a protocol Maestro will ask if you want to save the current changes 3 In the Choose Protocol dialog box select the protocol you want to open and click Open Tip You can also open a Maestro protocol by double clicking the protocol pro file D in a My Computer directory window If Maestro is not already open this action will open Maestro with the selected protocol You can also drag a protocol file and drop it into an open Maestro window to open the protocol 4 Maestro now contains all the settings of the selected protocol 50 Acquiring Spectral Images Viewing a Live Image Stream You can view a live grayscale image of the sample specimen To view a live image 1 Click the Live button at the top of the Acquire panel A live image of the sample specimen opens in a Live Stream window within the image viewing area e The frame rate of the live image varies according to the specified exposure time e Ifyou see an all black image the exposure setting may be too low Also make sure either the interior lights or the illumination light source is turned on and the illumination shutter is open e Saturated pixels appear red in the live image If some or all of the i
85. gned to each filter set in a multi filter acquisition the following parameters must be the same for all filter sets Any changes you make to these parameters apply to all the filter sets you may have selected e Camera ROI e Camera binning e Camera bit depth e Camera gain To configure multiple filter sets 1 Click the Add button in the Multi Filter Support box This enables the Multi Acq check box and adds a new item B to the filters drop down box Multi Filter Support Multi Acq Add m X A 500 720 B 500 720 k 2 A inthe drop down box represents the first filter that will be used during cube acquisition With A selected choose the first filter set you want to use to acquire the cube 3 Select B in the drop down box and choose the second filter set to use during acquisition 4 Ifyou want to use a third filter set click the Add button again The next letter C will be added to the drop down box Select C and choose the third filter set to use during acquisition Repeat this process for each filter set you want to use e f you want to remove a filter set from the list select its corresponding letter and click the Remove button The remaining filters will move up in the sequence e f you want to autoexpose or acquire a cube using just one of the filter sets in your multi acquisition list select the letter of the filter you want to use A B etc and clear the Multi Acq check bo
86. h on the excitation light I Illumination Assembly Specimen Stage Figure 10 Position of Interior Light WHITE amp IR LEDs Note When using the IR lights you may not be able to see the near infrared light V4 emitted from the LEDs This is normal because near infrared light is not easily visible by eye To verify the IR interior lights are working When you switch on the IR interior lights the IR indicator light above the switch should light up to indicate that the IR lights are on If you want to visually verify that the IR lights are working do the following Introduction to the Maestro In Vivo Fluorescence Imaging System 17 1 Locate the IR LEDs by switching on both the IR and WHITE interior lights and looking for the white LEDs The IR LEDs are located right next to the white ones see Figure 10 for location 2 Now switch off the WHITE lights If you look very closely you should see a faint red glow emitting from the IR LEDs You may even need to dim other ambient lights to see this If you see the faint red glow the NIR illumination system is working properly 3 Ifthere is no glow or if the IR indicator light is not lit then contact CRi for repair Emission Filter Slider The emission filter slider holder is located at the top interior of the imaging module and is shown in Figure 11 Color coded Dots CEN 515mm 580nm e gt
87. h the illumination module ON using the switch on the lower right exterior of the imaging module Switch the interior lights ON using the switches on the upper right side of the imaging module Remember that if you choose the infrared IR lights you will not see any noticeable illumination Power up the computer and start the Maestro application Place your specimen onto the specimen stage and set the stage and illuminator arms to the heights appropriate for the size of your specimen Use the field of view markings on the stage surface as a guide to determine the stage and illuminator arm settings In the Maestro application select the Acquire tab Click the Live button to see live streaming video of the image Select Binning and Region Of Interest ROI options according to the desired image quality The default is full field and 2x2 binning Image files will be smaller with smaller ROI and increased binning Binning increases sensitivity to light but reduces image resolution In the Wavelength and Exposure box enter the wavelength at which you expect to see an image of your specimen This should correspond to the installed emission filter Click the Autoexpose Mono button to improve the live image Open the door and focus the lens to get a sharp image of your specimen If the live image becomes saturated with light when the door is open it may be difficult to focus the image properly Try autoexposing the image again with the door
88. h third party documentation as supplemental material only In cases where CRI and third party documentation differ and you have any doubt as to which applies to your system contact an authorized CRI distributor or service representative Introduction to the Maestro In Vivo Fluorescence Imaging System 11 Design Change Disclaimer Due to design changes and product improvements information in this manual is subject to change without notice CRi reserves the right to change product design at any time without notice to anyone which may subsequently affect the content of this manual CRi will make every reasonable effort to ensure that this User s Manual is up to date and corresponds with the shipped Maestro in vivo imaging system Reproduction Disclaimer No part of this manual may be reproduced photocopied or electronically transmitted except for reference by a user of the Maestro in vivo imaging system without the advance written permission of CRI CE CSA and UL Testing and Certification The Maestro in vivo imaging system has been tested by an independent CE testing facility and bears the appropriate CE mark The Maestro in vivo imaging system has been awarded the right to display the CSA mark The Maestro in vivo imaging system has undergone tests to meet UL standards CE The following are CRi distributors in the EU region authorized to function as primary contacts for CE related matters concerning CRi p
89. he Maestro In Vivo Fluorescence Imaging System This chapter provides an introduction to the Maestro in vivo imaging system It includes a brief description of each of the system s hardware components and software features Topics in this chapter Page e Operator and Equipment Safety 10 e About This Mari al eese tient a E E ENE Ni 11 CE CSA and UL Testing and Certification 12 e Maestro Hardware Components essssssesee eren eene 13 e Launching the Maestro Application sessesssseseeeeeeeeenenen ne 22 e Reinitializing Maestro Hardware sese een 28 e Understanding the Maestro Work Area sssssssseeeeeeeeen 23 e Specifying Camera Hardware Settings sss 26 e Using Low Screen Resolution Mode sese 27 CRi s Maestro in vivo imaging system is a high performance multispectral imaging system designed for fluorescence macro imaging Maestro systems use an Imaging Module and an Illumination Module that enable fast and accurate imaging of specimens The patented liquid crystal LC tuning element performs like a high quality interference filter which enables the transmitted light to be electronically tunable Intuitive Maestro acquisition and analysis software runs on a powerful desktop or laptop computer There are two Maestro in vivo imaging system models e The Maestro VIS FL operates in the 420 to 720 nm wavelength range e The Maestro GNIR FL
90. he width of the minimum area bounding box enclosing the region X Location is the center of gravity s x coordinate Y Location is the center of gravity s y coordinate Total Signal is the sum of all the pixel values in the region Total Signal Exp ms is the Total Signal divided by exposure time in milliseconds Max Signal is the maximum pixel value included in the region Area Pixels is the number of pixels included in the region Area mm 2 is the size of the region in square millimeters This measurement will only be displayed if the camera s pixel size has been calibrated see Calibrating Pixel Size on page 104 Also the accuracy of this measurement depends on setting the correct stage height and binning see Obtaining Accurate Measurements on page 81 Cube ID is the ID entered in the Sample ID field on the Acquire panel when the cube was acquired Spectrum ID is the ID of the spectrum used to unmix this image entered from the Spectral Processing panel Cube Time Stamp represents the time the cube was created Quantifying Results 83 Copying Measurement Data to the Clipboard 1 Make sure the measurement regions you want to copy are selected on the Measurements data page Click the Copy Measurements to Clipboard button in the Measure panel Copy Measurements You can now switch to a program such as Microsoft Excel and paste the measurements into a worksheet Saving Measurement Data as a Text File 1
91. his diagram provides external dimensions of the illumination module in inches Dimensions are subject to change without notice E a o t cq ens O 5 E Lp zz Ez n az Os Ea c o lu Z jal J W of NEZ 58 a g 2 IE Ale 4 8 e S g P s e z ni i seb e z 95588 32 E jo lf J i J ga L 2g ge SZ 5 Bu S t rA Li 59 e ge ko 2a Z k 4 e L OO hee Oo O t o wo E lo a o J m 4 e 3 ro a lo o i ex J p E B E MORE 2 amp K 98 fgg OR o e XE i d zz Zo go E ue Sz L z 35 2 po z E z EJ a Oo 4 e 94 System Specifications amp Dimensions Specimen Stage This diagram provides a 1 1 scale drawing of the field of view outlines on the specimen stage It is intended to assist you in determining if a particular specimen meets the size requirements of the system 1 0 2 0 3 0 1 4 PN 4 0 System Specifications amp Dimensions 95 96 System Specifications amp Dimensions Appendix B System Setup amp Installation Maestro in vivo imaging systems consist of an Imaging Module and an Illumination Module configured at the factory for either 420 to 720 nm or 500 to 950 nm This chapter describes how to set up both modules
92. ick the Mixed Spectrum e g autofluorescence plus label black Paper color marker i Q605 Paper b In the Mixed Spectrum box click the green Q565 Paper color marker c In the Computed Spectrum box make sure Scale To Max is selected in the Scaling drop down box d You will see the mixed spectrum in green and the computed spectrum in yellow the color selected in the color drop down box eee at the bottom of this dialog box The dotted best fit line is a scaled representation of the known Scale Fit Offset Error Scaler Transfer To Name Color autofluorescence signal Spectrum Number E 5 Q565 Yellow L X i d e To obtain an accurate pure spectrum which is required for an accurate Blue Ea unmixing one must subtract the correct Figure 24 Manual Compute Spectra amount of autofluorescence from the mixed spectrum To do this it is important that the non overlapping regions between the pure fluorophore and the autofluorescence line up closely To do this First try sliding the Error Scaler to the right or left until the spectra are aligned as closely as possible The Scale option is discussed further in Error Scaling When Manually Computing Spectra on page 69 Ifthe non overlapping regions do not line up closely using scaling try clearing the Scale check box and observe the results which should be similar to the plot shown in Figure 24
93. ing and or using the Software you agree to the following terms and conditions If you do not agree to all of the terms and conditions in this Agreement you may not install or use the Software Single Use License The Software is licensed to you and not sold Subject to the terms and conditions of this Agreement CRi hereby grants to you a restricted nonexclusive nontransferable nonassignable nonsublicensable and revocable license to use for your internal purposes only the executable code version of the Software and the accompanying documentation in hard copy or electronic format CRI RESERVES ALL RIGHTS NOT EXPRESSLY GRANTED BY THIS AGREEMENT Specific Restrictions You may use the Software only on a single computer at a time You may not use the Software on more than one computer or computer terminal at the same time nor allow access to the Software from one computer to another over a network the Internet or by any other means You may make only one 1 copy of the Software solely for backup purposes You may not decompile reverse engineer disassemble alter modify translate adapt decipher or determine the source code or create derivative works of the Software Ownership of Software CRi and or its suppliers own all right title and interest including all copyrights in and to the Software The Software contains confidential information and trade secrets of CRi You i acknowledge and agree not to contest CRi s rights in the So
94. ing regions across two or more composite images To drag a region to another image l 2 Release the mouse button anywhere in the new image The copied region will Click on the region you want to copy and while holding down the mouse button drag it to the new image automatically snap into position corresponding to the position of the original Copying all Regions to the Clipboard 80 Quantifying Results The Maestro software can copy all existing regions from an image to the clipboard You can then paste the copied regions from the clipboard to other images All regions in the image are copied with this feature See Dragging a Copy of a Region to Another Image on page 80 if you want to copy a single region To copy all regions to the clipboard 1 Right click on the image that contains the regions you want to copy and then select Copy Measurement Regions To Clipboard from the pop up menu 2 Right click inside the image to which you want to paste the regions and select Paste Measurement Regions From Clipboard 3 All measurement regions on the clipboard are pasted into the current image Each new region is assigned a ROI number and its measurement data displays in a new row of the Measurements page at the bottom of the Maestro window Note The ROI numbers can change from image to image depending on what other V4 regions are present in each image Saving and Loading Regions If you save your measurement
95. isse enne enne nennen nnne nennen nnns 80 Saving and Loading Regions cese ener ace coca den ee cae ea RR aea Pea duae NEKEEN ae ea deg e nda aon ais 81 Obtaining Accurate Measurements ccccceccesssesseessecseceseceseesseesseceaeceaeseeeeseecseeeseceseseaeeeaeesaecaeseeseseessaeeaaes 81 Ignoring Smaller Re8I0ns 3 2 nee ERE Dir iv Petre edes ie eo eat bibe Di es UR EIUS PESO E eR US 82 Hiding Region Labels e M 82 Adjusting Region Transparency and Color ccccccesccesscessesseesseceneceseseceeeaeesaecaaeceecseeeseeecseecaaenaeeneeeseeessesseenaeens 82 Understanding Region Measurements esses eene eene enne nennen nnne nennen rene nnne 83 Copying Measurement Data to the Clipboard sse eere ener enne 84 Saving Measurement Data as a Text File sess eene enne nennen nnne 84 Appending Measurement Data tet tdeo e ce RR RENE TRA Eee TRE NER PEN TRE RUE REOR EN S 84 Chapter 8 Customizing Spectral Displays 85 Adjusting Brightness and Contrast Levels esses enne nennen ener 85 Returming to Display Default et tror eret dtd rideo reed e aeri Ne Een ee opea eee R aede 86 Saturation Mask ER E DRE PRESE eee EE EE e obe dle er iere renes Ua E S 86 Changing How a Cube is Displayed sess eene ener entere eren nennen nnne 87 Changing How a Composite is Displayed sss ener nnne erre enne nnns 88
96. l Filter Settings dialog box will open 3 Editthe settings as needed To edit existing settings select the filter setting name in the list and edit its tunable filter settings Click the OK button to save To create a new filter setting name with a new set of tunable filter settings click the New button Enter a label name and click OK to add the new filter setting name to the list Then specify its tunable filter settings and click the OK button to save To delete a currently selected filter setting name click the Delete button To restore all preset filter settings to their factory defaults click the Factory Defaults button This will also delete all new filter setting names you have saved Acquisition Setup Using Multiple Filters The Maestro system offers a Multi Filter Support feature that lets you acquire a cube while employing two or more filter sets For example if you wanted to acquire an image cube that includes the acquisition wavelengths of the blue filter set 500 nm to 720 nm as well as the red filter set 680 nm to 950 nm then you could use this feature The acquisition Acquiring Spectral Images 55 wavelengths of the different filter sets may overlap as with the blue and red filters but wavelength overlap is not required Note There is no need to select and use the Multi Filter Support option when acquiring V4 image cubes that require a single filter set While different exposure times can be assi
97. lay in the Cube ID column of the Measurements page near the bottom of the Maestro work area Associating Notes with an Image Cube Use the Notes text box on the Acquire panel to save notes with the current cube Whenever the cube is opened any notes previously saved with the cube will display in the Notes box Acquiring Timed Sequences of Image Cubes The Maestro system can acquire multiple cubes of a specimen using a timed acquisition interval that you specify This is useful for observing changes in a specimen over a specified time span 1 Select Acquire Time Sequence from the Tools menu The Acquire Time Sequence dialog box opens Figure 37 Enter the amount of time in seconds that you want to elapse between the start of each cube acquisition For example if you enter 60 seconds and it takes 15 seconds to acquire the first cube there will be a 45 second lapse until the start of the next cube acquisition Be sure to enter a time that is greater than the amount of time it will take to acquire each cube Acquire Time Sequence Collection Parameters Time sec 60 Num to acquire 5 Save Options Directory C Maestro Data Cubes Base Name 0Second_Intervals Acquire Cubes X Figure 37 Acquire Time Sequence Enter the number of cubes you want to acquire during this acquisition sequence Click the Browse button to select a destination directory where you want the Maestro software to save all the cubes acquired
98. ld exclude the Background component thereby removing the autofluorescence from the composite image Doing this in combination with adjusting the Brightness and Contrast of the unmixed components using the sliders in the Display Mapping box can result in a clear bright image of the tumor fluorophore etc 88 Customizing Spectral Displays Chapter 9 Frequently Asked Questions This chapter discusses FAQs and troubleshooting issues for the Maestro in vivo imaging system Can I use the multispectral camera in my Maestro Imaging Module on my microscope Maestro in vivo imaging systems are designed specifically for macro imaging Contact CRi to find out about our Nuance Multispectral Imaging Systems for microscopy What is the CCD sensor active area dimensions and the individual pixel size The active light sensitive area of the CCD is 1392 pixels in the horizontal direction and 1040 pixels in the vertical direction Each pixel is 6 45 um square Can I use my own lenses on a Maestro in vivo imaging system The lens is built into the Maestro in vivo imaging system Other lenses cannot be used Why do I need an emission filter with the Maestro in vivo imaging system Without an emission filter specifically a longpass filter excitation light will bleed through the Maestro optical system and cause artifacts See Chapter 1 Introduction to Multispectral Imaging Theory and Concepts My specimens are sometimes unevenly illumin
99. ld feel the slider click into place when the filter is properly located in the light path 4 Once the slider with its filter is properly seated the illumination shutter can be opened If the illumination module SHUTTER and POWER switches are already in the OPEN and ON positions the shutter will open automatically upon inserting the excitation filter Switching the Illumination Module ON and OFF On the right hand side near the bottom of the imaging module there are switches and an indicator light for the illumination module shutter and power see Figure 14 ILLUMINATION MODULE SHUTTER POWER Q OPEN CLOSED OFF ON 9j 4 Figure 14 Illumination Module Power amp Shutter Switches After switching on the illumination module it takes approximately fifteen to twenty minutes for the lamp output to stabilize to within 2 of its current maximum brightness Introduction to the Maestro In Vivo Fluorescence Imaging System 21 Therefore to achieve the most accurate and repeatable results you should consider switching on the illumination module power when you first switch on the system Then open the shutter when you are ready to acquire an image cube Remember to switch off the illumination module power when you will not use the system for a prolonged time period After 500 to 1000 hours of illumination the lamp intensity will be reduced by about 50 If lamp brightness is important to y
100. le for most commonly used fluorescence dyes or labels Nevertheless regardless of how carefully one matches the excitation and emission filters to a given label there will be some background autofluorescence and this will reduce the perceived contrast between the real or actual label fluorescence and the specimen background Introduction to Multispectral Imaging Theory and Concepts 5 The Multispectral Solution The Maestro in vivo imaging system offers a unique solution to the problem of autofluorescence and selection of emission filters Multispectral analysis is based on the fact that all fluorescent materials produce a unique spectral emission In other words if you excite a material and then examine the emitted fluorescence over a range of wavelengths and record the intensity of emission at each point along the plotted curve of those wavelengths you can generate an emission spectrum like the green emission spectrum shown in Figure 2 illustrating Stoke s Law This spectrum is different for each specific fluorescent material Overlapping Emission Spectra The complication is that for many fluorescent labels of biological interest the emission spectra overlap significantly and these emission spectra may also be obscured by autofluorescence from other constituents of the specimen Often autofluorescence is a strong bright broad signal that may obscure the specific fluorescence that the investigator wishes to see e e
101. lick Close when finished importing spectra From a Saved Composite Image You can import spectra from composite images 1 Open the Result Set umx file that includes the composite image from which you want to import spectra 2 Right click on the composite image and select Import Spectra from the pop up menu 3 The Import Spectra From Library dialog box Figure 29 opens with all the spectra used to unmix the cube 4 Make sure there is a check mark in the Select column next to each spectrum you want to import then click the Transfer to Library button 5 Ifyou want to load spectra from another library click the Load button and select a library to load 6 Click Close when finished importing spectra Practice Exercise In this exercise you will open and analyze an image cube representing a mouse with a quantum dot labeled tumor This exercise represents the common problem of a tumor signal that is difficult to see due to the presence of autofluorescence We will resolve this problem using Maestro multispectral imaging The sample is courtesy of Dr Shuming Nie Emory University 1 To open the sample image cube of the mouse with the quantum dot labeled tumor click the Load Cube button and navigate to the sample data folder C Maestro Data Images Sample Data Q dot mouse Q dot mouse stack 2 Select any one of the TIFF files and click the Open button to open the image cube Opening the Q dot mouse stack image cube
102. ls Window Help a is cy Load Cube Save Cube Zoom Ful Image Acquire Spectral Processing Measure Display Threshold Segmentation Threshold Level 52 59 o 579 Auto Calculate Threshold Transparency Transparency Color reo NE Manual Draw Regions Milimeters Per Pixel Binning Stage Height mm Pixel 22 Hz 8 v Measure Objects Min Connected Pixels 100 Draw Region Labels Copy Measurements m f 5 to Clipboard o fermen Thumbnaiis Measurements Log Cursor 1 244 04 33 01 32 03 365 08 1821 85 578 2 117 04 30 39 27 61 47227 178 678 04 20147 Hardware Status Wo Hardware Protocol DataFolder Cursor X 471 Figure 26 Measured QDot Spots Imaging the CRi Business Card with QDot Spots 37 38 Imaging the CRi Business Card with QDot Spots Chapter 4 Method Development Method development is a fundamental component of any good experiment and building spectral libraries is a key part of method development This chapter discusses methods for building accurate and reliable spectral libraries We will explain how to create and save libraries using a variety of sample specimens and we will discuss their unique characteristics Chapter 6 Unmixing Spectral Images discusses how to open libraries and use them to unmix images If you are new to the Maestro in vi
103. lts 81 TIFF cubes do not save binning and stage height specifications If you load and unmix a TIFF cube the binning value on the Measure panel defaults to 1x1 and the stage height defaults to the current stage height specified in the hardware settings For TIFF cubes you can change both of these values Millimeters Per Pixel Binning Stage Height mmjPixel oa iy 1 v Figure 55 Millimeters Per Pixel To change the binning or stage height Select from the Binning or Stage Height drop down boxes Binning can be changed for TIFF cubes only The Maestro software calculates and displays the new mm Pixel value automatically based on the calibrated pixel size see Calibrating Pixel Size on page 104 Ignoring Smaller Regions The Measure Objects box on the Measure panel is used to specify a minimum region size based on its number of pixels Only regions that are larger than the Min Connected Pixels value are considered The Maestro software ignores regions that have fewer than this number of pixels Measure Objects Min Connected Pixels 100 v Draw Region Labels Figure 56 Minimum Connected Pixels Use this control to change the size of the regions to filter out This setting does not effect manual draw regions Hiding Region Labels Numbered region labels correspond to the ROI Numbers on the Measurements page at the bottom of the Maestro work area Sometimes a region label obscures important data or makes i
104. ly supported operating systems System Setup amp Installation 105 106 System Setup amp Installation Appendix C Filter Selection Guide Select which filters are optimal for your application This table is current as of August 2006 Contact CRi or your authorized distributor for pricing and ordering information The CRi Maestro in vivo imaging system has many applications involving a variety of materials such as probes cell lines animal specimens etc Certain of these applications and or materials may require licensing under patents held by third parties See Notice to Purchaser on page 113 for more details Fluorophore Filter Set Name Excitation nm Emission nm Deep Red 525 nm QDot 500 525 545 nm QDot 520 545 565 nm QDot 540 565 605 nm QDot 580 605 655 nm QDot lt 620 655 705 nm QDot lt 650 705 800 nm QDot lt 750 800 Alexa Fluor 488 492 520 Alexa Fluor 500 500 530 Alexa Fluor 514 514 545 Alexa Fluor 532 532 554 Alexa Fluor 546 556 573 Alexa Fluor 555 555 580 Alexa Fluor 568 577 603 Alexa Fluor 594 590 618 Alexa Fluor 610 612 628 Alexa Fluor 633 632 650 Alexa Fluor 647 647 666 Alexa Fluor 660 668 698 Alexa Fluor 680 679 702 Alexa Fluor 700 700 719
105. m RGB Note This procedure requires that you have already acquired a clear and focused monochrome image of a metric scale or ruler with at least 100 mm length with the stage height on setting 1 and the CCD binning set to 1x1 Instructions 1 Push the Calibrate button below 2 Left click with the mouse on one the acquired image at one end of a 100 mm length 3 Move the mouse to the other end of the 100 mm length and left click again 4 A number should show up in the box below labeled Conversion Factor 5 The Check button can be used to confirm the operation was done properly This button requires left clicking on any two points in the image separated by a known distance The correct distance should be displayed Other stage heights and binning settings can be checked as well Find Calibration Calibrate Conversation Factor mm pixel 5 08237221 Check Calibration Measured Distance mm 100 0000 xi 1 Figure 66 Distance Calibration 2 Position this dialog box on the screen so the image of the ruler is visible bod Select 1x1 in the Binning drop down box and select 1 in the Stage Height drop down box Click the Calibrate button Click with the mouse on the acquired image at one end of a 100 mm length Move the mouse to the other end of the 100 mm length and click again The number of millimeters per pixel displays in the Conversion Factor field 2 d OO M B You can use the Check button t
106. mage is solid red the image is saturated The exposure setting may be too high Make sure the door of the imaging module is closed 2 Click the Autoexpose Mono button to automatically adjust the exposure time to the best setting for the current wavelength To freeze the live image Click the Live button a second time The image in the Live Stream window remains frozen until you click the Live button again Selecting Camera Binning and a Region of Interest Region Of Interest And Binning Binning ROI 2x2 tH ru Wv Figure 32 Region Of Interest and Binning Selectors Binning combines multiple pixels into a single pixel Higher binning reduces exposure time and image size However higher binning also results in a lower resolution image There are three camera binning options in the Binning selector The default setting is 2 pixel 2x2 binning To set camera binning Select one of the following e Ifyou want no binning for maximum image resolution and size select the 1x1 binning option e 2x2 binning combines each 2x2 square of pixels into one pixel thus reducing the resolution in each direction by a factor of 2 but requiring 1 4th the exposure time e 4x4 binning combines each 4x4 square of pixels into one pixel thus reducing the resolution in each direction by a factor of 4 but requiring 1 16th the exposure time Acquiring Spectral Images 51 The ROI Region of Interest selector sets the imaging area which may be
107. mice_autofl E subqzmice food s fox IKISISISISISISISIS Manual Compute Spectra Selected ROI Number Avg Signal Major Axis Minor Axis X Location Y Location Total Signal Yotal Signal Exp ms Max Signal Area Pixels Area mm 2 Import Spectra From Library ivl 1 4 15 37 04 24 06 88 73 67 42 3285 85 Unknown Exp 5 37 v 2 0 93 37 04 24 06 98 3 126 48 732 49 Unknown Exp 4 5 37 v 3 0 16 37 04 24 06 170 73 143 44 128 19 Unknown Exp 5 37 Unmix Hardware Status No Hardware Protocol DataFalder Cursor X Y aas Avg Signal 0 00 Figure 31 Images Showing Pure Unmixed Signals 2 Xiaohu Gao Yuanyuan Cui Richard M Levenson Leland W K Chung Shuming Nie In vivo cancer tar geting and imaging with semiconductor quantum dots Nature Biotechnology April 2004 Method Development 47 c Right click on the new region and select Clone Region from the pop up menu Drag the clone to another region of the same mouse to measure the strength of non specific binding The Measurements of region 2 shows that the signal strength in this region is approximately one fourth the strength of region 1 d You can clone the region again and drag this clone to a region within the negative control mouse Viewing the Measurements of region 3 confirms that there is no label present in the negative control mou
108. milar to the one shown here Warning Incompatible Exposure Times Incompatible Exposure Times The ratio between the exposure times for the filter sets used to acquire the current spectral library and the current image cube are different Using the current spectral library with the current image cube can lead to erroneous results Please adjust the exposure time so the ratios of the exposure times are the same for each filter set or use a different spectral library A 500 720 B 550 800 79 7 Library Spectrum Exposure ms Cube Acquisition Exposure ms n ad Ratio 1 0 1 2 Do not show this message again Figure 35 Incompatible Exposure Times Saving Many Filter Protocols in One Maestro Protocol You can use the Multi Filter Support feature to save multiple filter protocols in a single Maestro protocol This is useful when you need to take multiple cubes of a specimen using different filter sets For example you may want to take separate cubes of the same mouse using blue yellow and red filter sets Remember each filter set can have a different exposure time but all sets will have the same camera ROI binning bit depth and gain Follow the instructions earlier in this section to configure the filter settings blue yellow and red for example in the multi acquisition list and then save the protocol When you are ready to take multiple cubes of a specimen load the saved mul
109. most no fluorescence with only 15 counts Obtaining Autofluorescence and Label Spectra from Two Animals In this example the upper mouse was injected with Alexa Fluor 680 which dispersed throughout the mouse s body The lower mouse is an uninjected negative control Due to the presence of the dye everywhere in the mouse the use of an uninjected mouse was necessary in order to obtain a representative autofluorescence signal Using Manual Compute Spectra we obtain the autofluorescence signal from the negative control mouse and a mixed Alexa Fluor 680 plus autofluorescence yellow spectrum not shown from the area of the injected mouse that fluoresces brightly We ensure that the signals in the selected regions are not saturated The red AF680 spectrum was computed using the white autofluore spectrum and the yellow mixed spectrum El Maestro alexa 680 in mouse 12 ye Fie Edt Hardware Tools Window Heb a Q Load Cube Save Cube zoom Ful Image Acquire Spectral Processing Measure Display E alexa 680 in mouse t2 Cube Ex Zzalexa 680 in mouse t2_Unmixed Composite Composite C1 X Scano Normais XN Areas of Non Specific Binding Yt Library 10 Specific Binding TAS m Name E alexa 680 in mouse t2 JAF680 Component 1 jautofluore 2 AF680 z Autofluorescence of Negative Control Mouse l alexa 680 in mouse x vac E 0 os soc NEM B Je El Black MI whit
110. mplicate such measurements Fluorescence is emitted by a molecule in all directions and most imaging systems are designed to capture light coming from a particular direction only Therefore there is no way to capture all of the light emitted by a fluorescent molecule with such a system Additionally fluorescence emission 1s influenced by the local environment in particular by pH The total amount of fluorescence will therefore depend on these local conditions of pH as well as other surrounding molecules that may either enhance or quench some of the fluorescence energy There is also the problem of obtaining identical excitation of all fluorescence molecules in a specimen and this can be exceedingly difficult to achieve Fluorescence is an extremely sensitive technique as it is much easier to visually assess or measure light emission against a dark background than it is to see a decrease in light intensity from absorption by a dye Regardless of the sensitivity of fluorescence the difficult in establishing uniform excitation and controlling for local environment effects makes quantitation of fluorescence emission difficult in biological preparations Stoke s Shift Materials that fluoresce almost always emit light at a longer wavelength than the wavelength of the exciting light As an example rhodamine isothiocyanate can be excited by green light and then emits red light This difference between the wavelength of the exciting light and the emi
111. n eye is a highly adaptive light detector becoming significantly more sensitive in low light and less sensitive under bright light This adaptive change is not instantaneous and it takes some time for the eye to fully adjust to a new illumination level This is the reason one needs to dark adapt by being in a darkened room for some time before observing weak fluorescence through a microscope While the eye can be very sensitive to low light levels and can also see in very bright conditions it does not discriminate light levels very well An individual has no internal 1 The Electromagnetic Spectrum illustration was prepared by NASA under Contract NAS5 26555 for the Education Group of the Space Telescope Science Institute s Office of Public Outreach It is used here under public domain in accordance with NASA s contract 2 Introduction to Multispectral Imaging Theory and Concepts meter that indicates the current sensitivity level setting for the eye The eye also has a limited ability to discriminate levels of illumination ranging from the lowest level to the highest level at any given sensitivity US Department of Defense research indicates that while some people can distinguish as many as 500 levels of gray most humans can only distinguish approximately 30 to 35 levels of gray ranging from black to white This is relatively insensitive to the actual total illumination level although the ability to discriminate gray l
112. n steps Select Expand Window or press Ctrl E or Reduce Window or press Ctrl R from the Window menu Zooming In and Out You can zoom in to focus on details or zoom out to see more To zoom in Click the Zoom button on the toolbar The mouse pointer changes to a magnifying glass Use the magnifying glass to draw a box around the area you want to zoom Zoom You can also click the area of the image you want to zoom Each time you click the image with the Zoom tool the zoom increases In addition you can scroll the mouse wheel to zoom in To zoom out Click the Full Image button on the toolbar Full Image You can also right click on a zoomed image and select Zoom To Full Image from the pop up menu You can also scroll the mouse wheel to zoom out Panning a Zoomed Image When you zoom in on a particular area of an image you might not be able to see other areas of the image To pan a zoomed image Do one of the following e Right click on the image and select Pan from the pop up menu The mouse pointer changes to a hand icon Click and hold the mouse button while dragging or panning to view the desired area of the image Introduction to the Maestro In Vivo Fluorescence Imaging System 25 e Drag the zoom view rectangle that appears on the corresponding thumbnail image in the Thumbnails tab e Scroll the mouse wheel while holding down the Shift key to pan horizontally or the Control key to pan vertically Panning
113. nails Measurements Log Cursor Manual Sa Spectra subq2mice Skin Tumor Composite import Spectra From Library Spectra From Library Unmix Hardware Status No Hardware Protocol DataFolder Cursor X Avg Siga IKI amp I amp IKI amp IK amp IKISISIS Figure 50 Line Profiles drawn on component images Note the following attributes of the profile lines e The first profile line Red intersects the bright signal of the quantum dot labeled tumor Notice that the Red plot peeks for the pixels that are in the brightest area of the tumor e The second line Green intersects two less bright signals caused by food and general autofluorescence in the control mouse e The third line Blue is a copy of the first line Red and is positioned at exactly the same location as the first line See Working with Line Profiles on page 76 for how to copy lines By comparing the Red and Blue lines one can conclude that there is no tumor signal in the control mouse e When you move the mouse pointer across the Spectral Graph in the Line Profiles dialog box you will notice a black dot appear on each line These dots correspond with the dots on the profile lines on the images and they can help you identify peeks and dips in the images spectral intensity The Line Profiles tool is for data exploration purposes only The ability to export profile
114. nation of responses from these three different types of color sensors While the general arrangement of these color sensors cone cells in the eye is reasonably standard there are differences in the total number of each type of cone cell and in the actual physical arrangement within the detecting cell layer retina These minor variations lead to the differences in perceived color between individuals as does the actual way in which the individual s brain learned to interpret color s Fluorescence Many biological and natural materials give off light of a particular color when exposed to light of another color This property is called luminescence If the emitted light occurs rapidly after illumination around one millionth of a second the luminescence is called fluorescence If the light emission takes longer than one millionth of a second the luminescence is called phosphorescence Materials that exhibit fluorescence have proven extremely useful as labels or indicators in many biological systems Introduction to Multispectral Imaging Theory and Concepts 3 Fluorescence light emission is different than light absorption Each fluorescent molecule generates light We measure the total amount of light generated and are not dependent on the interaction of the light with another material such as a dye While it would seem that fluorescence is much more amenable to accurate measurement than absorbed light there are a number of factors that co
115. nt or any other single plane image to be measured and make sure the Measure panel is visible 2 Inthe Threshold Segmentation box click the Auto Calculate Threshold button The Maestro software will attempt to find a threshold level that is appropriate for the image Quantifying Results 77 Threshold Segmentation Threshold Level 1 0 511 Transparency Transparency Color M EHE Figure 51 Threshold Segmentation Pixels with intensity values below the specified threshold are ignored If you are not satisfied with the auto calculated threshold level you can enter a new value in the Threshold Level field by using the slider or manually typing in a value You will see the region s change as you edit the threshold level After a threshold is set the image will have an overlay showing the regions that were measured as shown in Figure 51 E QdotCard_Q605 Component E QdotCard_Backer Qdotcard Backar O X Thumbnails Measurements Log Cursor Selected ROI Number Avg Signal Major Axis Minor Axis X Location Y Location Total Signal Total Signal Exp ms Max Signal Area Pixels Area mm 2 1 322 00 54 02 52 03 607 70 146 02 716765 30 8532 92 511 01 2226 10519 92 2 173 43 35 45 33 94 485 94 145 13 163371 60 1944 90 213 16 942 4451 82 KE Figure 52 Measured Threshold Regions The dashed lines drawn on top of e
116. nt Images button Maestro identifies and displays the autofluorescence and fluorescence signals Select the signals you want to unmix a The autofluorescence is displayed in the upper left image Click two times anywhere in the image to select it as the autofluorescence Set its pseudo color to white User Selected Area Select Area Starting Spectrum v Use selected background meseumj eee cam b Single click on the other component image s you want to unmix and select a pseudo color for each Extract the pure fluorescence signal s from the autofluorescence by clicking the Find Spectra button The component signals display in the spectral graph Occasionally for weak signals there may be too much baseline offset in the data If you did not get the desired unmixing results select the Fit Offset option and see if the unmixing results improve Use the controls in the Computed Spectra box to specify each spectrum s name pseudo color and location within the spectral library When finished click the Transfer to Library Unmix and Close button This adds the spectra to the spectral library and unmixes the spectra in the cube If a library is open already you may be asked if you want to overwrite the existing spectra New images will display in the image viewing area a There is a small component image for each selected spectrum each with a colored border that corresponds to the pseudo color of the spectrum b
117. o confirm that the calibration is accurate To use the Check function obtain an image with points separated by known distances Then click the Check button and click on any two points in the image separated by a known distance The correct Measured Distance should be displayed Other stage heights and binning settings can be checked as well using this function 104 System Setup amp Installation Tests for Ensuring Proper Operation The best test to make sure everything is working properly is to take images of the CRi business card included with each Maestro in vivo imaging system The quantum dots on its front should show up clearly and you can test the Maestro software s unmixing feature See Chapter 5 Acquiring Spectral Images for detailed instructions Using Third Party Computers with the Maestro Software CRi supplies a computer with each Maestro in vivo imaging system If you intend to use a third party computer system for offline data processing please consider the following parameters for optimum performance e The faster the computer CPU the faster certain operations in software will be accomplished See Appendix A System Specifications amp Dimensions More hard drive space allows more and larger datasets to be acquired and stored e Atleast two GB of RAM is recommended for data processing e A USB 2 0 port is required e The US English versions of Windows XP Professional or Windows 2000 Professional are the on
118. of any kind and from any cause arising out of or in any way connected with the use of the equipment All warranty and post warranty service is nontransferable from the original shipping address to another without the prior written consent of CRi The CRi Maestro system may be used for research purposes It is not intended for clinical or diagnostic use at this time CRi 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 7924 P 1 781 935 9099 F 1 781 935 3388 sales cri inc com www cri inc com TO PLACE AN ORDER OR RECEIVE TECHNICAL ASSISTANCE For more information contact CRi or your local authorized CRi distributor Cambridge Research amp Instrumentation Inc CRi 35 B Cabot Road Woburn MA 01801 USA Toll Free US 1 800 383 7924 Phone 1 781 935 9099 Fax 1 781 935 3388 Email techsupport cri inc com or sales cri inc com C3l Maestro and Nuance are trademarks of Cambridge Research amp Instrumentation Incorporated Adobe and PhotoShop are trademarks of Adobe Systems Incorporated Alexa Fluor and MitoTracker are trademarks of Molecular Probes Inc AntiCancer is a trademark of AntiCancer Incorporated Cermax is a trademark of PerkinElmer Incorporated Cooper Bussman is a trademark of Cooper Industries Inc Cyanine dye Cy2 Cy3 Cy5 Cy5 5 and Cy7 is a trademark of Amersham Biosciences Intel Centrino and Pentium are trademarks of Intel Corporation or its subsidiaries in the Unit
119. onent g quem ham pres prex pns Ls Restore Defaults Clear all Delete emm Thumbnails Measurements Log Cursor Manual Compute Spectra QdotCard Import Spectra From Library Unmix Hardware Status No Hardware Protocol DataFolder Figure 25 Grayscale and Composite Images 4 Save this protocol in case you ever want to acquire and unmix this or a similar CRi business card with QDot spots again in the future Select Save Protocol from the File menu Select a location and enter a name such as qdotcard for this example As discussed in Chapter 4 Method Development protocols save cube acquisition settings the current spectral library and the current hardware settings so you don t have to set them again each time you unmix or analyze a similar specimen 5 Now you are ready to quantify the results by taking measurements of the unmixed QDot spots in the component images Quantifying Results After unmixing the cube use the controls on the Measure panel to quantify the results by measuring the QDot spot regions This section provides specific step by step instructions for measuring the QDot spots For more detailed information on quantifying results see Chapter 7 Quantifying Results To measure the QDot regions 1 With the unmixed cube displayed as shown in Figure 25 switch to the Measure panel 2 Click the qdotc
120. open Close the imaging module door Autoexpose the image again if you had to change the exposure setting while focusing In the Cube Wavelength Selection box select the Preset Filter Setting that corresponds to the installed filters If you don t find a preset that works for your specimen you can edit the Start Step and End settings manually Refer to the Maestro User s Manual for instructions Switch all interior lights OFF and OPEN the excitation light shutter to illuminate the specimen with the excitation light Click the Autoexpose Cube button to automatically calculate the exposure settings for the image cube You can also adjust the exposure time manually a If the image is too dark increase the exposure time in the Time milliseconds box b If the image is too bright or saturated indicated by solid red pixels reduce the exposure time Click the Acquire Cube button to generate an image cube When Maestro has finished generating the image cube a color representation of the cube displays in the image viewing area Click the Save Cube button on the toolbar to save the cube Select a location and enter a file name File name format suggestion project sample operator datetime Select a cube type option a Image Cubes saves the cube in CRi format which includes hardware and display set tings for the cube b TIFF Cubes saves the cube as a series of TIFF images with the assigned file name plus an appended num
121. or of the imaging module you may notice that much of the business card image turns red This is caused by excess light entering the imaging module and causing the image to saturate This will correct itself when you close the imaging module door If the image is so saturated that you are unable to focus try autoexposing the image while the door is open This will reduce the exposure time and the saturation Imaging the CRi Business Card with QDot Spots 31 4 Close the imaging module door Click the Autoexpose Mono button again if you had to change the exposure setting when focusing 5 Accept the default Binning 2x2 and Region Of Interest Full settings for now 6 In the Preset Filter Settings box select the Blue 500 10 720 filter set from the drop down box This automatically populates the Start Step and End wavelength settings 7 Switch off all interior lights The specimen stage should now be illuminated with blue light You can open the door of the imaging module to verify this 8 In the Wavelength and Exposure box click the Autoexpose Cube button to automatically calculate the exposure settings for the image cube 9 Now to acquire an image cube click the Acquire Cube button at the bottom of the Acquire panel Acquiring Cube Acquiring Cube COT Cancel Figure 22 Acquiring Cube 10 When the Maestro system has finished taking the image cube a color representation of the cube will
122. orresponding blue emission filter in the imaging module The section Emission Filter Slider on page 18 explains how to install the emission filter slider Set the f stop position for the camera lens at 2 8 The f stop selector is located near the top of the lens assembly just below the ceiling inside the imaging module Note For most applications the f stop should be set at 2 8 If there 1s a need for greater depth of focus then set the f stop to the smallest value that will give an acceptable depth of focus Remember to return the f stop to 2 8 when you are finished with an alternate setting Move the specimen stage and illuminator arms to position 1B Specimen Stage and Illumination Assembly on page 15 describes how to move the stage and illuminator arms In the Maestro application select Edit Hardware Settings from the Hardware menu and enter the new stage height and lighting settings on the Camera Setting tab Place the CRi business card on the center of the specimen stage within the marked viewing area 30 Imaging the CRi Business Card with QDot Spots Acquiring an Image Cube Select the Acquire panel 1f not selected already by clicking its tab Before you begin note the Hardware Status bar at the bottom of the work area Verify that Ready is displayed in the status box If the status box displays No Hardware verify that the interface and power cables are properly connected and that
123. ot Keep cooling vents free from obstructions Endoscopic instruments used for this device must be certified to IEC 601 1 for medical equipment and IEC 601 2 18 for endoscopic equipment This warning is not applicable to Maestro systems Caution Do not remove lamp immediately after operation Allow lamp to cool 10 minutes To prevent overheating replace only with same type and rating of lamp Read instructions before replacing lamp See Illumination Module on page 19 d did iid Do not operate light source without lamp in place For Technical Assistance If you experience any difficulty setting up operating or maintaining your Maestro in vivo imaging system please contact your CRi representative Office hours are 8 00 a m to 6 00 p m Eastern Standard Daylight Time Monday through Friday Telephone US Toll Free 1 800 383 7924 e Telephone Worldwide 1 781 935 9099 e Facsimile Worldwide 1 781 935 3388 e Email techsupport cri inc com About This Manual This manual is designed to serve users of both models of the CRi Maestro in vivo imaging system using Maestro version 2 2 software Operating instructions functional descriptions troubleshooting illustrations and other relevant information are contained in this manual Your Maestro system may include support documentation from third party vendors Bear in mind that components of the Maestro system may have been modified or custom designed so treat suc
124. our application you should consider replacing the lamp during this time Use the hour meter on the back of the illumination module to track the number of hours the lamp has been illuminated Refer to Illumination Module on page 19 for lamp replacement instructions Workstation Computer Maestro in vivo imaging systems require a computer with a level of performance appropriate for acquiring and analyzing digital multispectral data Computers purchased from CRi that will be used with the Maestro system meet the performance requirements listed in Computer System Requirements on page 92 If you decide to supply your own computer be sure it meets the performance requirements as well Cleaning Your Maestro n Vivo Imaging System It may be necessary to periodically clean the inside and outside of your Maestro in vivo imaging system Follow these guidelines when cleaning e Spray a clean cloth with a sanitizing solution such as 70 ethanol and wipe down the specimen table and specimen pad Wipe all surfaces below around and behind the specimen stage as well e Be careful never to spray directly into the imaging module to prevent spraying the lens the camera or the fiber optic tips e Wipe down all external surfaces with the same solution and a clean cloth Launching the Maestro Application If you have already purchased the Maestro system your Maestro software was installed and activated for you at the factory Double click the Maes
125. p screw and replace the plastic plug Plastic plug covers the access to the fiber optic guide locking screw Insert fiber optic guide into the guide port here Connect the interface cable to the imaging module here Figure 64 Maestro Illumination Module 4 Usethe supplied interface cable to connect the illumination module to the imaging module Plug the cable into its port on the illumination module see Figure 64 and the ILLUMINATOR CONTROL port on the back of the imaging module 5 Use the supplied USB cable to connect the imaging module to the workstation computer Plug the cable into the square USB 2 0 port on the back of the imaging module see Figure 65 and any high speed USB 2 0 port on the computer Figure 65 Maestro Imaging Module Back Panel Connections Note The USB port labeled Software Key is no longer used to connect a software key This port may be used as a general purpose USB port 100 System Setup amp Installation 6 Setup the LCD monitor keyboard and mouse on the table with the Maestro in vivo imaging system and connect them to the workstation computer Detailed instructions for these connections are available in the materials supplied with the computer Caution The imaging module workstation computer and monitor require connection to 7 4 a surge protected electrical supply Before connecting system components to the electrical supply verify that all power switches are in
126. pecification 91 Imaging Module dimensions specification 92 overview of 13 imaging theory 1 118 Index Installation Maestro software 101 Maestro system 97 L Lamp Cartridge lifetime specification 19 92 Lamp Cartridge replacing 19 light delivery system specification 92 light discussion of 1 lights interior on off switch 17 Line Profiles 74 clone 76 copy 76 delete 76 move 76 live image 51 luminescence defined 3 M Maestro in vivo imaging system introduction to 9 Maestro Work Area how to launch 22 understanding 23 maximum exposure 27 Measurements 77 appending to a text file 84 copy to clipboard 84 definitions 83 measuring regions 36 77 save as text 84 Mechanical Dimensions illumination module 94 imaging module 93 specimen stage fields of view 95 multi filter protocols 57 multi filter support 55 multispectral analysis introduction to 7 N notes save with cube 59 P Panning Images individual 25 together 26 phosphorescence defined 3 pixel size calibration 104 Power Supply on off switch 14 Protocols 43 open 50 save 43 pure spectra calculating 34 R ratio of exposure times 57 real component analysis RCA 65 Region clear all 79 region of interest ROI 52 region of interest shapes 68 Regions clone 80 copy all 80 copy measurements 84 delete 79 drag a copy 80 erase part of 79 find automatically 77 ignoring small 82 labels 82 load to an image 81 manual draw 79 measurements 83
127. ph Scale to either Normalized or Scale To Max e The Clear All button is used to remove all profile lines from the image s and empty the Spectral Graph To draw a profile line click the Draw MI button on the first row Red of the color library and then use the mouse pointer to click and draw a line across the area of interest in the image As you draw a line through bright portions of the image the bright portions show up as peeks on the Spectral Graph 74 Unmixing Spectral Images 4 Figure 50 shows three profile lines drawn on two component images of a mouse sample This is the same sample cube that was used during the Practice Exercise on page 45 ST Maestro subq2mice Fie Edit Hardware Tools Window Help Q a 4 Load Cube SaveCube Zoom Full mage Acquire Spectral Processing Measure Display ZImhe mien nhe 7T rix jsubgzmica Unmixed Composite Composite EEx Ubrary E subq2mice_Tumor Component EEx Scaling Normalized X Line Profiles x Scaling Scale To Max X M mx L 14 c NEN ue NNC aor L 14 foo NEN za E soc NENN Pink sa NN wre C e Restore Defaults clear all Delete pA 3 g Fi subq2mice Food Component KI amp IKISISISISISISI S 1 Tumor Signal 2 Food Signe Green IBN 3 Tumor signal sue MENO coe CE E E ss MN imc L o NN wwe T Real Component Analysis Shrink TS fa rini Thumb
128. pplications and or materials may require licensing under patents held by third parties For example while AntiCancer Incorporated and Xenogen Corporation do not have U S patent rights covering in vivo imaging of conjugated fluorescent labels a license from AntiCancer Incorporated 7917 Ostrow St San Diego CA 92111 and or Xenogen Corporation 860 Atlantic Avenue Alameda California 94501 may be required to practice imaging within animals of cells genetically engineered to produce light emitting compounds CRi s sale or other transfer of the Maestro in vivo imaging system does not convey any right or license under such third party patents It is suggested therefore that users of the Maestro in vivo imaging system consult with counsel to determine whether licensing of such third party patents is required CRi Software End User License Agreement 113 114 CRi Software End User License Agreement Maestro 2 2 Quick Start Guide MD15459 Rev B August 2006 A This guide presumes that the Maestro n Vivo Imaging System is properly installed and 10 11 12 13 14 15 16 17 18 19 20 21 Acquire Images configured Install the excitation and emission filters for the fluorescence you wish to see Refer to the Filter Selection Guide in the Maestro User s Manual Switch the main system power ON using the switch on the back of the imaging module With the SHUTTER switch in the CLOSED position switc
129. r indicates the current filter range The highlighted region on the slider represents the acquisition wavelength range The colors on the slider approximate how we would see the wavelengths within the range 52 Acquiring Spectral Images e Type a value or use the up down scrollers in the Wavelength text box When typing a value you can set the wavelength in 1 nm increments The up down scrollers snap to 10 nm intervals Adjusting the Exposure Time Use the autoexposure buttons to automatically determine the correct exposure setting or enter a value in milliseconds in the Exposure entry field You can also use the Custom Wavelengths And Exposures option This option is enabled only if custom collection settings have already been specified See Specifying Custom Collection Settings To manually enter an exposure time Type an exposure value into the Exposure ms text box or use the scrollers to increase or decrease the current value in increments of one millisecond If you are using the multiple filter feature see Acquisition Setup Using Multiple Filters on page 55 you can enter a different exposure time for each filter in the sequence Select the first filter letter A in the Multi Acq drop down box and enter an exposure time Repeat for each filter in the filter sequence To autoexpose a cube 1 Before you acquire an image cube click the Autoexpose Cube button 2 Ifyou are using the multiple filter feature
130. ral analysis provides the solution to this problem and reduces the need for multiple expensive narrow band emission filters A single long pass emission filter can replace a large collection of emission filters Multispectral Analysis The Maestro system s combination of unique hardware and sophisticated software make powerful multispectral analysis possible Multispectral analysis generates spectral curves for the various fluorescent dyes or materials in a specimen In addition it generates a spectral curve for the autofluorescence that almost always is present to some degree In a multispectral analysis a series of images are captured at specific wavelengths The range of wavelengths captured should cover the spectral emission range of the labels present in the specimen The number of images within that range should be chosen to adequately define the emission spectral curve for each label The result will be a series of images called an image cube taken at specific wavelengths The data within the image cube is used to define the individual spectra of both autofluor escence and specific labels Using sophisticated algorithms the contribution of autofluor escence to the image can be removed and the individual fluorescence spectra separated The result is a set of images representing each spectrum that contributes to the final image In other words as illustrated in Figure 4 multispectral analysis yields 1 an autofluorescence imag
131. re column 3 The Choose Algorithm Steps box selects the algorithm steps and parameters Use the radio buttons to select one of the following options Unmixonly Unmix and Measure using Auto Threshold If you select this option then specify the Min Connected Pixels parameter in the Measure Options box Unmix and Measure using Manual Threshold If you select this option then specify both the Min Connected Pixels and Threshold parameters in the Measure Options box Unmix and Measure using Precalculated regions If you select this option then select a Regions roi file that you want to use to measure the unmixed results 4 The parameters in the Save Options box control how the result files are named Results are saved in the same directory as the cube e The Basename parameter is the file prefix The unmixed results will be named basename timestamp umx For example if the Basename option is test batch an unmixed file would be called test batch 2006 August OS 1l 47 48 umx Unmixing Spectral Images 73 The Global Measure File parameter is a file that contains all the result measurements in a single file The Choose Cubes box at the far right lets you select the image cubes Click the Find Candidate Cubes button A dialog box will prompt for a starting directory The Maestro software will search this directory and the directories one level below for cubes that are compatible with the current spectral li
132. refore if you want to capture images that can be displayed using non scientific imaging applications then select the 8 bit option Gain affects image quality because it modifies the CCD readout process For images that have low intensities consider increasing the gain value slightly to achieve images with brighter pixel values Increasing the gain can also reduce the exposure time Note however that higher gain settings can result in increased signal noise 26 Introduction to the Maestro In Vivo Fluorescence Imaging System which reduces image quality Gain values of 1 2 or 3 should be suitable for most applications Check Dark Current is used to calculate the amount of background noise present in the camera This is calculated using a zero time exposure and no light reaching the camera Frames to Average is a useful feature for reducing noise in acquired images by increasing the signal to noise ratio This value specifies the number of images the camera will take at each acquisition wavelength The software then averages the values of those images to create a single image for each wavelength The signal to noise ratio increases approximately by the square root of the number of images averaged For example averaging four images per wavelength gives a 2x signal to noise ratio This should translate into approximately a 2x reduction in the limit of detection Autoexpose Target of Dynamic Range sets the autoexposure target values
133. regions you can easily load them to other images This is useful for comparing control regions and fluorophore regions across multiple images To save a region 1 Right click anywhere on the image but not within a region and select Save Measurement Regions from the pop menu 2 Inthe Choose Measurement Regions dialog box enter a descriptive name and click Save to save the measurement region s To load a region to an image 1 Right click anywhere on the image but not within a region and select Load Measurement Regions from the pop menu 2 Theregion s will be loaded to their exact pixel location when they were saved You can move the region s by right clicking them individually and selecting Move Region Obtaining Accurate Measurements The Maestro software calculates each region s area in pixels and in mm based on the current binning and stage height settings on the Measure panel Therefore these settings 2 must be correct for the mm measurement to be accurate If the current unmixed cube is a CRi format Image Cube the Maestro software auto matically displays on the Measure panel the binning used to acquire the cube This setting cannot be changed The stage height defaults to the stage height specified in the hardware settings prior to acquiring the cube The Maestro software lets you edit this value in case the corresponding stage height was not entered correctly in the hardware settings Quantifying Resu
134. rescence Unmix into red green and or blue for fluorophores of interest This will give you a nice outline of the mouse with colors superimposed If you want to make a fluorophore or autofluorescence disappear assign it to the black channel Make sure you save the library for later use Obtaining Autofluorescence and Label Spectra from a Single Animal In the following example sample courtesy of Dr Shuming Nie Emory University we use Manual Compute Spectra We obtain the autofluorescence signal of the mouse from an area of pure autofluorescence We obtain the mixed 630 nm quantum dot fluorescence plus autofluorescence yellow spectrum not shown from the area of the tumor In this instance we are able to use a single mouse because the dye is limited to the immediate area of the tumor The red 630 nm dye spectrum was computed using the white autofluore spectrum and the yellow mixed spectrum SI Maestro ad rca File Edit Hardware Tools Window Help a Q a Cc Load Cube SaveCube Zoom Full Image p Acquire Spectral Processing Measure Display Library Scaling Normalized XN gt qd rca Cube 1 0 08 06 04 e amp amp eS El qd rca_630nm dye Component qd rca_autof 0X pem jen pen Le Ls KI amp ISISISISISISII Restore Defaults clear all Delete RON Thumbnails Measurements Log Cursor Manual Compute Spec
135. reveals a mouse with a dimly labeled tumor you may be able to see the quantum dot glow in the left animal near its intestines and relatively abundant autofluorescence Note Inthe image the mouse on the left was injected with an antibody labeled with a 630 nm quantum dot The mouse on the right received no injection Method Development 45 3 Use RCA to unmix the cube a Click the Real Component Analysis RCA button on the Spectral Processing panel b Click the Sample Spectrum button and use the mouse pointer to sample the autofluorescence of the right negative control mouse The Use selected background spectrum check box is now checked c Click the Find Component Images button d In the Component Images box click the upper left image twice to select it as the background and set its pseudo color to white e Click once on the image below the background image which shows the quantum dot labeled tumor and set its pseudo color to red Its name will default to Signal 3 f The image to the right of the red quantum dot image shows the food autofluorescence in the non fasted control mouse Click once on this image to obtain its spectrum Its name will default to Signal 4 g Click the Find Spectra button The computed spectra will display on the graph and appear in the color palette If desired rename Signal 3 to something more descriptive like q dot signal Rename Signal 4 to food signal or something
136. roducts LOT Oriel GmbH amp Co KG Im Tiefen See 58 D 64293 Darmstadt Germany Tel 49 6151 88 060 Fax 49 6151 896667 Email info lot oriel de LOT Oriel UK 1 Mole Business Park Leatherhead Surrey KT22 7BA United Kingdom Tel 44 1372 378822 Fax 44 1372 375353 Email info lotoriel co uk 12 Introduction to the Maestro In Vivo Fluorescence Imaging System Maestro Hardware Components Imaging Module The Maestro Imaging Module contains all of the principal imaging components in a single enclosure e High resolution scientific grade CCD imaging sensor e Solid state liquid crystal LC wavelength tuning element e Spectrally optimized lens and internal optics e Emission filter assembly e Flexible fiber optic lighting system e Light tight enclosure with adjustable specimen stage and large door for easy access The imaging module contains the following interface ports and indicator lights e USB Type A rectangular port e USB 2 0 Type B square port for connection to the workstation computer e Power connectors and fuse holders e Switches for power and control of the flexible fiber optic delivery system Anesthesia input and outlet ports Figure 5 shows a front exterior view of the imaging module Figure 5 Maestro Imaging Module Introduction to the Maestro In Vivo Fluorescence Imaging System 13 System Power On the rear of the imaging module there is a POWER ON OFF switch that controls
137. rom the Spectral Processing panel Plots on the Manual Compute Spectra dialog box are 4 5 their normal size The spectral graph is removed from the Display panel Introduction to the Maestro In Vivo Fluorescence Imaging System 27 Reinitializing Maestro Hardware When you launch the Maestro application it automatically detects the Maestro hardware imaging module if it is attached to the computer via the USB 2 0 cable If the Maestro application loses its connection to the Maestro hardware make sure the USB 2 0 cable is properly connected to the imaging module then select Reinitialize from the Hardware menu The Maestro application should detect and initialize the hardware If this fails to reinitialize the connection try restarting the Maestro software 28 Introduction to the Maestro In Vivo Fluorescence Imaging System Chapter 3 Imaging the CRi Business Card with QDot Spots This chapter steps you through the process of acquiring an image cube of the CRi business card with three QDot quantum dot spots painted on its front surface This business card was included with your Maestro in vivo imaging system We will also practice saving unmixing and analyzing the component and composite images By following the instructions in this chapter you will quickly learn how to use the Maestro in vivo imaging system as well as verify that the system is working properly Topics in this chapter Page LEM SICUT
138. rom the cubes 4 The default line sampling tool can be changed by selecting another region of interest shape from the toolbar see Selecting a Region of Interest Shape on page 68 n Assign a helpful name to each spectrum by entering a name in the Name column 6 To change the color assigned to a spectrum select from its drop down color menu Unmixing Spectral Images 67 Selecting a Region of Interest Shape 1 Click the Draw button for the row where you want to place the sampled spectrum 2 Click the button for the shape you want to use on the toolbar e e RectROI Ellipse ROI Line ROI Area ROI Figure 44 ROI Shapes 3 Click and drag over the part of the cube you want to sample Manually Computing Spectra 1 Open or acquire a cube and select the BVESISSSINES Spectral Processing tab Known Spectrum e g autofluorescence 2 After loading or building a spectral library press the Manual Compute Spectra button to display the dialog box 3 Inthe Known Spectrum box select the known spectrum the spectrum of the autofluorescence Multiple known spectra may be selected by holding down the Control key while making selections e e s 4 Inthe Mixed Spectrum box select the spectrum computed spectrum that represents the selected known spectra Scaling Scale To Max o z plus any additional spectrum For example autofluorescence plus a label 5 The Computed Spectrum will be displayed in
139. s Do not connect components supplied by CRi using unqualified cables or adapters Doing so could result in damage and voids the Warranty Use only a properly grounded power outlet when connecting the system to power If you are using third party mechanical components for the Maestro in vivo imaging system consult Appendix A System Specifications amp Dimensions Follow the recommended maintenance procedures This will help ensure optimal performance over years of use Caution Servicing should be performed by CRi authorized and trained personnel only A Power must be disconnected from the system before servicing Illumination Module Cautionary Statements The following cautions and warnings are printed on the illumination module Possible explosion hazard if used in the presence of flammable substances Before disconnecting read instructions This warning pertains to the risk of explosion in the presence of flammable substances if the unit is unplugged from its electrical supply while the unit is on Always switch the unit OFF before unplugging its power cord 10 Introduction to the Maestro In Vivo Fluorescence Imaging System Caution Disconnect power supply cord before servicing to avoid electric shock To reduce the risk of electric shock do not remove cover Refer servicing to qualified personnel Light source can cause permanent eye damage if viewed directly with unprotected eye End of light guide may be h
140. s occurred fluoresces the brightest with a Total Signal of 707 061 This is the region s relative measure of fluorescence Region Z2 represents an area of non specific binding and exhibits a bright fluorescence signal as well Region 3 represents the signal from a mouse with no added fluorophore Notice the much lower relative measure of fluorescence Saving Spectral Libraries By saving your spectral libraries you make them available for use again later when performing analyses on similar specimens Instead of just saving the library you could also save the Maestro protocol which saves all of the Maestro acquisition and analysis settings see Saving Protocols on page 43 for how to save protocols To save the current spectral library 1 Select Save Spectral Library from the File menu The Choose Spectral Library dialog box opens 2 Navigate to the folder where you want to save the spectral library then type a name in the File Name field 3 Make sure Spectral Libraries 1s selected in the Save As Type box and click the Save button If you want to save the library as text files then select the Text Files option instead This format cannot save color or hardware settings but it can be opened in other applications For example these text files could be opened in Microsoft Excel for graphing etc Saving Protocols The Maestro software saves its settings in protocols which are recipes for collecting and measuring dat
141. se 48 Method Development Chapter 5 Acquiring Spectral Images Whenever possible acquisition of image cubes should be done using previously established Maestro protocols that have been saved and validated with your Maestro in vivo imaging system for each of your specimens experiments Protocols are recipes for collecting and measuring data See Tips for Obtaining Quantitative Results and Acquiring Images Using a Saved Maestro Protocol Protocols are discussed further in Saving Protocols on page 43 This chapter explains how to acquire an image cube using the cube acquisition functions on the Acquire panel Once you have specified the cube acquisition settings for a particular type of specimen save the protocol for repeated use throughout the experiment with the same or similar specimens Topics in this chapter Page e Tips for Obtaining Quantitative Results sess 50 e Acquiring Images Using a Saved Maestro Protocol ssssssss 50 e Viewing a Live Image Stream ssseesssssesseeeeeeeneeeen enn eene 51 e Selecting Camera Binning and a Region of Interest esses 5 e Specifying the Current Wavelength and Exposure sese 52 e Selecting a Filter and Cube Wavelengths essen 54 e Acquisition Setup Using Multiple Filters sese 55 Flat Erelding idee Get EE E EX ESSE Tree aeuo edu e ed bie prese PRG 58 e Acquiring a
142. selected then the purest spectra present in the cube will be found fa Background component was selected then all other signals will be used as inputs into a computation that removes the presence of the background spectrum to give the spectrally pure components Occasionally for weak signals there may be too much baseline offset in the data If you did not get the desired unmixing results select the Fit Offset option and see if the unmixing results improve You may now use the controls in the Computed Spectra box to change a spectrum s name color and where it should reside in the spectral library When finished click the Transfer to Library button to add the spectra to the main spectral library If a library 1s open already you may be asked if you want to overwrite the existing spectra Click the Close button to close the RCA dialog box When you are ready to unmix the image first verify that the spectra you want to unmix is selected in the library then click the Unmix button Alternately you may press the Transfer to Library Unmix and Close button to perform all these actions with a single click If the spectral library you have created will be used again for future unmixing operations save the protocol and or spectral library 66 Unmixing Spectral Images Computing and Unmixing Spectra Manually Sampling Spectra from a Cube If you want to compute pure spectra without using RCA you nee
143. set to include all or just a portion of the entire field of view The active area is always centered and there are five ROI options in the ROI selector Four options are predefined and one is user definable The default setting is Full frame To select a region of interest Do one of the following To capture the Full frame 2 3 frame 1 2 frame or 1 3 frame select from the predefined options e To select a custom region of interest select Custom and use the mouse pointer to draw an ROI box within the Live Stream window You can also create a custom region of interest by selecting Set Custom Camera ROI from the Hardware menu Specifying the Current Wavelength and Exposure The Wavelength and Exposure box is used to manually adjust the current Wavelength in nanometers nm and the Exposure in milliseconds ms Wavelength And Exposure Wavelength nm Exposure ms 570 B is E v Range Acquisition Wavelength Current Filter Range Autoexpose Mono Autoexpose Cube Figure 33 Wavelength and Exposure Adjusting the Current Wavelength Depending on the expected wavelength of the emission light you may want to adjust the current wavelength for the filter being used To manually set the current wavelength Do one of the following Drag the slider to the desired wavelength The slider snaps to the wavelength intervals set in the acquisition settings As shown in Figure 33 the wavelength slide
144. similar Your screen should look similar to Figure 30 Cube Component Images Computed Spectra Find Component Click on component images Find p Once for Signal Twice for Background Images Spectra Click again to unselect Ignored a Fit Offset autofluore 10 08 06 04 q dot signal food signal ee P B EF OS User Selected Area Spectrum Name Color Select Number 1 autofluore White 2 a dot signal Red 3 food signal Yellow Brown iv Starting Spectrum v use selected background Sample Spactrumij ccce re oF choose from library User 7 v v 2x m v s Ignored su hea ERE peso pes pest red Transfer To Library v Transfer To Library nri and ose Figure 30 Unmixing with Real Component Analysis RCA h Click Transfer to Library Unmix and Close i The Maestro software unmixes the cube and displays the component and composite images in the image viewing area Figure 31 46 Method Development 4 The spectral signals in the cube are now unmixed Switch to the Measure panel and use the region drawing tools to quantify the signal strength of the quantum dot labeled tumor vs areas where non specific binding has occurred Note In this example the mouse on the left was injected with a quantum dot labeled antibody and was fasted for three days prior to imaging By contrast
145. t difficult to see the region clearly You can have the Maestro software draw or hide numbered labels on regions within an image A check mark in the Draw Region Labels check box Figure 56 will show the region labels that are normally drawn on top of the region Clearing the check mark hides these labels Adjusting Region Transparency and Color 1 Use the Transparency slider in Figure 51 on the Measure panel to change how much measurement regions block the image behind it Moving this slider all the way to the 82 Quantifying Results left makes the region transparent except for its outline Moving the slider all the way to the left fills the region with the selected color If you want to change the transparency s color select a color from the Transparency Color drop down box Understanding Region Measurements Each region s measurements are displayed as rows on the Measurements data page near the bottom of the Maestro work area Figure 52 The following information is displayed The Selected column indicates which measurement regions are displayed on the image Clear the check box from a measurement row to remove its region from the image The ROI Number for each measurement region corresponds to the number displayed in the center of the region drawn on the image Average Signal is the average value of the pixels in the region Major Axis is the length of the minimum area bounding box enclosing the region Minor Axis is t
146. the imaging module s main power Figure 6 Located immediately above the power switch is the fuse holder which holds two 250VAC 8A fuses These fuses prevent accidental circuit overloads in the imaging module 250VAC Fuse Holder System Power ON OFF Switch Main Power Input AC INPUT Figure 6 Main Power Switch To switch power ON and OFF Switch the imaging module on by moving the POWER switch to the ON position The imaging module will take approximately 45 seconds to initialize To replace fuses in the imaging module Use 250VAC 8A slow blow Cooper Bussman MDL 8 fuses 29V 8A 1 Unplug the imaging module 2 Locate the small black panel that covers the system fuse holder It is located immediately above the main system power switch on the back of the imaging module 3 Usea small screw driver to open the black fuse cover from the top and open it downward 4 Usethe small screw driver to gently pry the red fuse holder out of its port 5 Replace both fuses in the fuse holder with 250VAC 8A slow blow Cooper Bussman MDL 8 fuses available from CRi and other sources 6 Replace the fuse holder into its port and close the cover 7 Plug in the imaging module 14 Introduction to the Maestro In Vivo Fluorescence Imaging System Specimen Stage and Illumination Assembly The specimen stage and illumination assembly inside the imaging module can be set to nine discrete height positions There are three primary an
147. the Software is provided will be free from defects in materials and faulty workmanship under normal use for a period of ninety 90 days from the date of delivery Your exclusive remedy under this Section 6 shall be at CRi s option a refund of the price paid for the Software or replacement of the media on which the Software was provided so long as that media has been returned to CRi under a CRi issued return authorization CRi shall have no responsibility to replace media damaged by accident abuse or misapplication No Other Warranties EXCEPT FOR THE LIMITED WARRANTY STATED IMMEDIATELY ABOVE THE SOFTWARE IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND AND CRI EXPRESSLY DISCLAIMS ANY AND ALL IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THIRD PARTY RIGHTS CRI DOES NOT REPRESENT OR WARRANT THAT THE RESULTS OR THE USE OF THE SOFTWARE WILL BE CORRECT ACCURATE OR RELIABLE OR THAT THE SOFTWARE WILL OPERATE UNINTERRUPTED OR ERROR FREE OR THAT DEFECTS IN THE SOFTWARE WILL BE CORRECTED YOU ASSUME ALL RISK ASSOCIATED WITH THE USE RESULTS AND PERFORMANCE OF THE SOFTWARE Limitation of Liability IN NO EVENT SHALL CRI ITS AFFILIATES OR SUPPLIERS OR THEIR RESPECTIVE EMPLOYEES OFFICERS OR AGENTS BE LIABLE FOR ANY DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE THE SOFTWARE INCLUDING WITHOUT LIMITATION INCIDENTAL SPECIAL CONSEQUENTIAL PUNITIVE EXEMP
148. the imaging module is turned on 1 Ifyou don t already see the Live Stream window in the image viewing area click the Live button at the top of the Acquire panel A live image of the business card opens in the Live Stream window wire Spectral Processing Measure Display v Live Stream jon OF Interest And Binning Binning ROI 22 EH Ful Wv Wavelength And Exposure Use Custom Wavelengths And Exposures Autoexpose Mono Autoexpose Cube Cube Wavelenath Selection Start step End g j v amp Preset Filter Settings Blue 500 10 720 Fixed Filters Blue Define Multi Filter Support Flat Fielding C Fiat Field Sample D Thumbnails Measurements Log Cursor Notes ae a E Hardware Status Ready Protocol DataFolder Cursor X Ye Avg Signal Figure 21 Acquire Panel with Live Grayscale Image 2 Inthe Wavelength and Exposure box set the Wavelength to 570 nm and click the Autoexpose Mono button to improve the live image The calculated exposure setting will display in the Exposure ms text box 3 To adjust the position of the business card on the stage and focus the image open the imaging module door and position the card so it is clearly visible in the Live Stream window Then focus the lens to achieve a sharp live image Note When you open the do
149. the pixel directly below the cursor For cubes it displays the average signal For composites it displays absolute and percent contribution from each component For other image types it displays the pixel values Thumbnails Measurements Lag Cursor Composite Component Name Component Signal Percent of Total Signal 96 X Y Position 212 218 Background 27 48 89 52 Signal 3 0 17 0 55 Signal 4 3 05 9 94 Total 30 70 Figure 19 Cursor Page Each window type has a home position on the screen The cube lives in the upper left corner the composite image lives in the upper right corner and the component images are positioned in a grid starting from the lower left and working up and to the right Pressing Ctrl L will force the windows to their home positions In addition many operations will force the windows to their home positions These include resizing the thumbnail view unmixing a cube or activating a window from the thumbnail view 24 Introduction to the Maestro In Vivo Fluorescence Imaging System Resizing an Image Window There are a few ways to change the size of an image window or windows To manually resize a window Click and drag its window border to the desired size The image inside the window resizes to fill the window while also maintaining its aspect ratio To maximize the current window Click the window s maximize button The window will fill the image viewing area To expand or reduce a window i
150. ti filter protocol that contains the appropriate filter sets Choose the filter set you want to use to acquire the cube and then clear the Multi Acq check box so only the selected filter set will be used Acquiring Spectral Images 57 Flat Fielding Flat Fielding makes it possible for the Maestro software to create better more evenly bright image cubes When you take cubes without Flat Fielding you may notice that the outer edges of acquired images are slightly darker than the rest of the image Flat Fielding creates more even image cubes by taking a reference image before acquiring the image cube Once you have taken a reference cube it will be saved with the current Maestro protocol if you save the protocol However if you change the binning or region of interest ROD the current reference cube becomes invalid You will have to take a new reference image before acquiring a cube with different binning or ROI 1 Check the Flat Field check box This activates the Acquire Ref Image button The Acquire Cube button is now disabled until you acquire the reference image Acquire et Image Acquire Reference Image Flat Fielding Flat Field Figure 36 Flat Fielding 2 Remove the current specimen if any from the Maestro imaging module and replace the specimen with a stack of about ten sheets of clean white paper 3 Click the Acquire Ref Image button If you have activated multi filter support you will be prompted to install
151. tic Set 1 1 0 in Input output port diameter Set 2 0 5 in Set 3 0 25 in Imaging module dimensions 18 6 in wide x 16 3 in deep x 37 4 in high Imaging module 13 Weight Illumination module 100 lbs 45 kg 20 Ibs 9 kg l Filter sets must be purchased separately Contact CRi or your authorized local distributor for pricing and availability For anesthesia delivery system not supplied Unused ports may be kept covered with the supplied plugs 3 Does not include workstation computer Computer System Requirements Processor 3 0 GHz or higher Intel Pentium 4 class or Intel Centrino RAM minimum 2 GB Hard disk drive minimum 160 GB CD ROM writable CD ROM drive Operating system Microsoft Windows XP Professional or Windows 2000 Professional English Language Version Display minimum 24 bit color 1280 by 1024 pixels Computer Interface One available USB 2 0 interface 92 System Specifications amp Dimensions Mechanical Dimensions Imaging Module This diagram provides external dimensions of the imaging module in inches Dimensions are subject to change without notice He PROVED MAESTRO DIMENSIONS TLE DWG NO SCALE NIS 3 B 37 4 System Specifications amp Dimensions 93 Illumination Module T
152. timized for in vivo fluorescence Image sensor format 2 3 inch CCD Image sensor pixel count 1 3 mega pixels Cooling 5 C above ambient Sample area 1 1 3 in x 1 in to 4 in x 3 in Typical acquisition time Electrical supply 15 frame image cube in 5 to 10 seconds 100 to 240VAC 800W 50 60Hz Operating environment Operating temperature Indoor 15 C to 40 C Operating humidity 65 non condensing Operating Altitude maximum 2000 meters Storage temperature Storage humidity 10 C to 55 C 100 non condensing Pollution degree 2 Acquisition time depends on a number of factors including bandwidth of the Maestro in vivo imaging system and the amount of light emitted reflecting off or transmitted through the specimen at each wavelength of interest The Maestro in vivo imaging system is designed to be able to dwell at individual wavelengths if it determines that more light is needed to produce images with adequate spatial and spectral detail The system is not limited by a need to acquire images using identical exposure times at every wavelength System Specifications amp Dimensions 91 Parameter Maestro VIS Maestro GNIR Filter sets See Filter Selection Guide Lamp cartridge lifetime estimated hours 500 hrs lamp will then give approximately 50 of original intensity Light delivery system Fiber op
153. tra Selected Avg Signal Major Axis Minor Axis X Location Y Location Total Signal Total Signal Exp ms i Area mm 2 Cube Import Spectra From Library 1 277 1604 1608 114 11 8526 626 42 Unknown Exp 153 E 2 2 08 16 04 1603 12711 10926 470 12 Unknown Exp 153 lt 3 O07 16 04 1603 4811 4426 15 42 Unknown Exp 153 Hardware Status No Hardware Protocol DataFolder Cursor X Y 156 Avg Signal 0 00 Figure 27 Autofluorescence and Label Spectra from a Single Sample Notes e In this example the autofluorescence signal is named autofluore and the fluorescence signal is named 630 nm dye e After unmixing this cube we zoom in on the qd rca 630nm dye component image and use the manual Draw tool on the Measure panel to draw a measurement region around the tumor We then copy the region right click on the region and select Clone Region and move the copies to other locations within the image to compare fluorescence signals e As you can see from the Measurements data page at the bottom of the screen the area of the tumor Region 1 where specific binding has occurred fluoresces the brightest with a Total Signal of 626 counts Region 2 which represents a dimmer Method Development 41 region of q dot binding in the tumor also fluoresces but not as brightly with 470 counts Region 3 away from the tumors shows al
154. tro icon on the workstation computer s desktop to launch the Maestro application If you need to install and or activate the Maestro software see Installing Maestro Software on page 101 To evaluate Maestro software If you have not yet purchased a Maestro system but you want to evaluate the Maestro software select the I want to evaluate Maestro option when the Activation dialog box opens You will be allowed to use the Maestro software for 30 days before being required to purchase and activate the software to continue using it 22 Introduction to the Maestro In Vivo Fluorescence Imaging System Understanding the Maestro Work Area The Maestro application s work area includes a menu bar a toolbar control panels for acquiring and analyzing images an image view area and thumbnails and data pages Figure 15 shows a sample of the work area with an open image cube window El Maestr Menu Bar File Edt Hardware Tools Window Help Tool Bar OU ER LoadCube Save Cube Zoom FullImage Control Panels Acquire Spectral Processing Measure Display Ce Region Of Interest And Binning Binning 22 Hz Wavelength And Exp Wavelength nm Exposure ms Image View Area Preset Filter Settings Blue 500 10 720 E Fixed Filters Blue Multi Filter Support Flat Fielding C Flt Field Thumbnails and xem ample ID Thumbnails Measurements Log Cursor Data Pages
155. tted light is called the Stoke s Shift and is based on Stoke s Law A range of excitation wavelengths will excite fluorescence This range of wavelengths is known as the absorption spectrum The emitted light also covers a range of wavelengths and this is known as the emission spectrum Since the Stoke s Shift for most materials is not that great there is generally some overlap between the excitation and the emission spectra We will return to this point shortly as it does impact choice of emission and excitation filters in fluorescence systems Stokes s Shift Excitation lt gt Emission Maximum Maximum 400 450 500 550 600 nm Figure 2 Stoke s Shift Figure 2 contains an example excitation and emission spectra showing Stoke s Shift and the overlap of the spectra 4 Introduction to Multispectral Imaging Theory and Concepts Autofluorescence Many biological materials are naturally fluorescent In particular many vitamins some hormones and a variety of biological enzymes and structural proteins are naturally fluorescent These materials often fluoresce strongly enough to interfere with specific fluorescence labeling studies Because of this unwanted background or Autofluorescence both excitation light sources and emitted light paths are highly filtered in fluorescence systems On continued stimulation illumination at the excitation wavelength most fluorescent materials fade While some specific preparation methods
156. ttempts to open the associated cube as well If the associated cube cannot be found the RGB proxy opens instead You can replace the RGB proxy with the actual cube To import a cube into a result set 1 Right click on the RGB image and select Import Cube from the pop up menu 2 The Choose Cube dialog box opens 3 Select the cube an Image Cube or TIFF Cube you want to import and click Open Unmixing Spectral Images 71 Baseline Subtraction If there is stray light which contributes to a baseline offset it can be subtracted from the whole cube using Baseline Subtract This will ensure that the background will be near Zero 1 Select Baseline Subtract from the Tools menu The Baseline Subtract dialog box opens Baseline Subtract Spectrum To Subtract 1 0 08 0 6 S P Subtract from cuse Figure 47 Baseline Subtract 2 This dialog box displays the spectra from the current spectral library 3 Select spectra you want to subtract and click the Subtract from Current Cube button 4 The Maestro software subtracts the selected spectrum from the cube and closes the dialog box The cube s filename is also appended with subt to remind you that a spectrum has been subtracted from the cube Batch Data Processing In some instances you might prefer to acquire a large number of cubes before analyzing them or you might want to re analyze a large set of images with new analysis settings The Batch Data Processing fea
157. ture is useful for unmixing and measuring multiple cubes or a large set of images all at once 1 Select Batch from the Tools menu The Batch Data Processing dialog box opens 72 Unmixing Spectral Images Batch Data Processing Choose Spectra Choose Algorithm Steps Choose Cubes Ounmix Only Ounmix and Candidate Cubes Selected Cubes Measure using Auto Thresh QdotCard im3 QdotCardixi im3 10 08 06 04 4 Name 1 Q605 Paper 2 Q565 Paper 3 Pure Q605 4 Pure Q565 5 White 2 Ounmix and QdotCardix1 im3 QdotCard2x2 im3 Measure using Manual Thresh QdotCard2x2 im3 QdotCard4x4 im3 QdotCard4x4 im3 unmix and n Measure using Precalculated Regions QdotCard4xs tif R Measure Options Threshold Min Connected Pixels e Regions File card paper roi Color Select Measure cm coon M frea NE foren feroun MIN Pink die H Black iil m E E Save Options Basename test batch Global Measure File test measurements bxt Find Clear Clear Candidate Cubes Candidate Cubes Selected Cubes Figure 48 Batch Data Processing 2 The Choose Spectra box at the far left contains the current spectral library Select the individual spectra you want to use for unmixing by checking the check box in the Select column Select the component images you wish to measure by checking the control in the Measu
158. use the mouse pointer to sample an area by drawing a line within the green QDot spot see Figure 23 b In the Name column of the color palette rename this mixed spectrum from C2 to Q565 Paper 5 Inthe third row of the color palette use the Red color marker to sample the QDot spot that contains the 605 nm fluorescence plus the paper This is the dot on the far right side Name this mixed spectrum Q605 Paper Your work area should look similar to Figure 23 Acquire Spectral Processing Measure Display QdotCard Cube ex Scaling Normalized XN gt Library I amp I amp ISISI amp I amp ISIS s clear an Delete Real Component Analysis RCA Manual Compute Spectra Thumbnails Measurements Log Cursor QdotCard Import Spectra From Library LT EJ Hardware Status No Hardware Protocol Avg Signal Figure 23 Image Cube with Sampled Spectra Note Notice the overlapping spectra in the spectral graph at the top of the Spectral PA Processing panel Imaging the CRi Business Card with QDot Spots 33 Calculating the Pure Spectra 1 Click the Manual Compute button located es below the color palette to open the Manual Known Spectrum e g autofluorescence Compute Spectra dialog box a 2 Separate the 565 nm QDot signal from the autofluorescence S e a In the Known Spectrum box cl
159. using the drop down boxes in the Fill Wavelengths area below the table Specify the exposure Time milliseconds in the Fill Exposures box Return to the Wavelength Exposure table and edit individual settings as necessary Click OK to save the settings and close the dialog box ge c Qv GA On the Acquire panel select the Custom Wavelengths And Exposures option to use the custom collection settings to acquire the image cube All fields in the Cube Wavelength Selection box become disabled To save custom collection settings 1 After specifying custom collection settings click the Save button The Choose Custom Table dialog box opens 2 Specify a directory and filename and click Save to save the new wavelength and exposure table To load a wavelength and exposure table 1 Click the Load button on the Custom Collection Settings tab The Choose Custom Table dialog box opens 2 Locate the table you want to load and click Open The collection settings appear in the Wavelength Exposure table Click OK to use the custom settings Selecting a Filter and Cube Wavelengths Filter settings define the wavelengths for which the filter is tuned when acquiring an image cube The Cube Wavelength Selection box on the Acquire panel has preset acquisition settings as well as entry fields for manually entering cube wavelengths Cube Wavelength Selection Start Step End so B zo g Y Y v Preset Filter Settings Blue 500
160. ve an image cube 1 To save the current image s and associated data click the Save Cube button on the toolbar or select Save Cube from the File menu The Choose Cube dialog box opens Navigate to where you want to save the cube In the Save as type box select a cube type option e Select Image Cubes if you want to save the cube as a single CRi image cube file A CRi cube can include hardware settings spectral libraries user comments and display settings Cubes saved in the CRi format cannot be opened in third party tools e Select TIFF Cubes if you want to save the cube as a series of TIFF images Maestro saves a TIFF image in the specified location for each of the wavelengths used to acquire the cube When saving a cube that was acquired using multiple filters filename_A_ will be used for the first filter set filename B will be used for the second filter set and so on TIFF Cubes contain only images none of the associated data or settings is saved with this format 4 Enter a file name for the cube and click the Save button 60 Acquiring Spectral Images To save images 1 2 3 Select Save Image from the File menu and select a save option to save the image s e Save As Displayed prompts you to enter a name for the currently selected image and saves the image as a TIFF or JPEG image e Save All As Displayed prompts you to enter a name for each of the current images in the image viewin
161. vo imaging system and have not already familiarized yourself with the rest of this manual we suggest that you do so now Then return to this chapter for further study Topics in this chapter Page e Methods for Building Spectral Libraries sese 40 e Saving Spectral Libraries cccccccescceseceseesceesseceeceseeeecseecessecseecseceseeneeseneeaaes 43 Saving Protocols Em 43 e Importing Spectra Into a Library 0 cece cesccecceeseeseceeeeeceeeceseeesecesecneeseeeesneeaaes 44 Practice EXerclse ias eivee t PE Meier bone Fes boa Per ede ete REPE ed Roda 45 Method Development 39 Methods for Building Spectral Libraries You should create a spectral library for each of your experiments or series of experiments Acquire or open the image cube you want to use to create the library Chapter 5 Acquiring Spectral Images describes in detail how to acquire image cubes of your specimens There are a number of ways to build a spectral library A few examples include Using the Real Component Analysis RCA feature to automatically compute and unmix spectra see Computing and Unmixing Spectra Automatically on page 65 for detailed instructions This process adds spectra to the library which you can modify as needed and then save Using the Manual Compute Spectra feature which lets you specifically sample spectral signatures from images and save them in a library see Computing and Unmixing Spectra Manually on pa
162. x Red j 0 00 E Clip Stretch con O Histogram Eq T JEEP Blue DNE Figure 57 Display Mapping Customizing Spectral Displays 85 e Four sliders allow you to adjust the following Brightness Contrast Brightness for the individual red green and blue color channels These sliders are enabled only when a cube or composite image is selected e The Min Max radio button maps the minimum value in the image to 0 the maximum value to 255 and linearly interpolates in between those values This stretches dark signals so they become visible e The Clip Stretch radio button maps the lowest 0 01 of the pixels to 0 the highest 0 01 to 255 and linearly interpolates in between those values This prevents a few bright or saturated pixels from skewing the display e The Histogram Eq radio button maps the pixels so the histogram of the pixels have approximately the same number of pixels in each bin This gives the best display of the whole dynamic range of dim and bright signals Returning to Display Defaults If you want to return the display mappings to the default values click the Set to Default button at the bottom of the Display panel Set to Default Saturation Mask Saturated pixels in your images reduce the accuracy of your unmixing and quantitation results The Maestro software considers a pixel in a cube saturated if the pixel is saturated at any of the cube s wavelengths Pixels in a component image remain
163. x The selected filter will be used See also Saving Many Filter Protocols in One Maestro Protocol on page 57 5 Save this protocol if it will be used again later see Saving Protocols on page 43 6 When you are ready to autoexpose or acquire the cube using multiple filters select the Multi Acq check box During multi filter cube autoexposure and acquisition the 56 Acquiring Spectral Images Maestro system will pause during the operation and will prompt you to install the next filter set in the sequence according to the filter line up you created in the Multi Acq drop down box Maintaining Compatible Exposure Time Ratios When you open a Maestro protocol that includes multiple filters and a spectral library obtained from a multi filter cube the ratios of the exposure times between the filter sets used for current cube acquisition and those of the saved spectral library must remain consistent Using a library with an incompatible ratio of exposure times to unmix a new image cube may lead to erroneous results When using a saved multi filter protocol to acquire a new multi filter cube it is recom mended not to perform any additional autoexposures of the specimen or manually change the exposure times If changes to exposure times are required make sure the ratios remain consistent such as 1 1 2 1 3 1 etc If the exposure time ratios are different by 1096 or more the Maestro system will warn you with an error message si
164. you are ready to unmix the image and compute the pure spectra Make sure only the Paper Q565 Q605 color markers are selected in the color palette Click the Unmix button at the bottom of the Spectral Processing panel The Maestro software calculates and displays images for the pure fluorescence QDot signals and the autofluorescence paper signal A new set of images appears in the image viewing area Figure 25 shows the grayscale images of each signal as well as a new composite color image created using the colors you selected e The original image cube is shown at the top left An RGB color composite image of the pure signals is shown in the top right We cannot see the autofluorescence because it was unmixed into black Images representing each of the pure signals are shown across the bottom in grayscale Their window borders are color coded to correspond to the colors you selected earlier Imaging the CRi Business Card with QDot Spots 35 ISI Maestro QdotCard File Edit Hardware Tools Window Help a Ho amp LoadCube SaveCube Zoom Full Image Acquire Spectral Processing Measure Display Scaling normalized XN z Library 1 0 Name Color 1 Paper bak 2 Q565 Paper Green M 3 Qe0s Paper Red Ls 4 uS Li S 3 E I amp I amp I amp IS SISISS88 LC Ls ER 40565 Green 5 9605 red E Pink White ics Ls m isi x E Qdotcard_Q565 Comp
165. zard but leave the CD in the drive Windows will need to copy drivers from it during the update Double click the installation icon and follow the onscreen instructions to install the downloaded software 7 During the installation you will be prompted to select the hardware configuration you own Select the Maestro in vivo imaging system option Note If you are upgrading from an earlier version of the Maestro software for a system VA with a USB 2 0 interface your current settings will be backed up 8 When the software installation is finished switch on the imaging module and plug its USB cable into the computer s USB port 9 Windows will automatically detect the new hardware The New Hardware Wizard will ask if you want Windows to connect to Windows Update to search for software 10 Select No not at this time and click Next 11 Make sure the installation CD is in the CD drive select the Install the software automatically recommended option and click Next to continue 12 A Hardware Installation message will alert you that the CR1 unconfigured device has not passed Windows logo testing to verify its compatibility with Windows XP You should ignore this message by clicking the Continue Anyway button 13 The New Hardware Wizard will install the drivers from the installation CD 14 When the installation is finished the wizard prompts you to restart the computer 15 Restart the computer The hardware will not

Download Pdf Manuals

Related Search

Related Contents

Manuel d`utilisation Optoma Theme-Scene HD81  CBA-MementoPorteursProjet V4.indd  SL-6300  Husqvarna LTH140 User's Manual    OM, Automower 320/330X, 2013  ATEN - KM0532_KM0932_KM0032_DS.ai  Mode d`emploi du module tutoriels  Whirlpool EL7ATRRMQ00 User's Manual  Dataram 32GB(2 x 16GB) DDR3-1066  

Copyright © All rights reserved. Failed to retrieve file