Nuance Quick Start Guide
Contents
1. this is a two step process saving cubes and images a Unmix the 605 nm q dot signal from the background Select the background spectrum in Black for the Known Spectrum Select the mixed 605 nm signal in Blue for the Mixed Spectrum The Computed Spectrum displays in Red Name it Pure 605 qdot and click Transfer to Library e You can also save the entire workspace by selecting File gt Save Result Set Enter a file name to save all images and results in a single file e You can save all images as displayed by selecting File gt Save Image gt Save All As Displayed You can save all images as unscaled data by selecting File gt Save Image gt Save All Images As Unscaled Data b Unmix the 655 nm q dot signal from the background Select the background spectrum in Black for the Known 15 Save the Protocol and or Spectral Library for use throughout your Spectrum Select the mixed 655 nm signal in Yellow for the experiment Mixed Spectrum The Computed Spectrum displays in Green Name it Pure 655 qdot and click Transfer to Library e You can save the complete Nuance protocol which includes the current Spectral Library by selecting File gt Save Protocol If you want to save the Spectral Library as a separate file select File gt Save Spectral Library Ee E i i iuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuususssusIsI5MsI5M l PerkinElmer Inc 68 Elm St Hopkinton MA 01748 USA 508 435 9500 ww2. Nuance 3 0 2 Quick Start Guide P N 130805 Rev 01 Acquire Brightfield Images Make sure that the Nuance Multispectral Imaging System is properly installed connected and configured and the microscope is aligned 1 Turn on the computer and connect the Nuance module s power cable During startup the Nuance module initializes The LEDs on the front panel scroll through the full wavelength range while the filter is exercising The CAMERA and STATUS LEDs illuminate steady green when initialization is complete Start the Nuance application 2 Position the sample and focus the image at the microscope Then divert all light to the camera port For multispectral imaging make sure the MONO MSI slider on the Nuance module is in the MSI position 3 Select the Acquire gt Brightfield tab If you don t see the Live Stream window click the Live button to see live streaming video of the image 4 In the Wavelength And Exposure group enter the wavelength at which you expect to see an image of the sample Use 500 nm or above for focusing Then click the AutoExpose Mono button to improve the live image You can also adjust the exposure time manually e If the live image is too dark increase the exposure time in the Exposure ms box e If the live image is too bright or saturated indicated by solid red pixels reduce the exposure time 5 You can maximize the live window or click the Zoom button and zoom in for higher magnific
3. Spectral Graph has disappeared and a Magenta line representing the pure brown stain appears Click Unmix A new Unmixed Composite image appears to the right of the original image A grayscale unmixed image for each spectrum appears below the original image The unmixed ki67 sample generates two grayscale images one outlined in Blue and one in Magenta 7 ij al Ja z r heal Component Anshan RCA L Maral Compala Spectre 4 a ae e oe _ I Pure DAE ma l l renser To Library hone Hardware Status Preia E Aahoan siare a Uem ss ie Ede hadam Tose Meram Windoe Hiep Go Gd T T j E Lai Cabet Tiot Cube Tass Bd jsa Ps Tisi Beet Acpare GPHG Mysore Dumpiry ikii de Cube Liorery 6 Restore Coteus clear at Coben teal Component Analysis RCA termporent Arahi BCA Harual Compule Spet _Maruel Compute Spectra ferent Mensurerreenih Lag Cursor impart Spectre From Library be Petts Tue riemen Para Dad gt Q BER Hademe Stes Freios Corse EY Saya i s ai po tn dato Arguing A ra 16 Save the resulting images e You can save all images as displayed by selecting File gt Save Image gt Save All As Displayed 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 e You can save
4. all images as unscaled data by selecting File gt Save Image gt Save All Images As Unscaled Data Enter a name for each of the component images This saves all unscaled image data as TIFF components with the data values multiplied by 10 You can also save the entire workspace by selecting File gt Save Result Set Enter a file name to save all images and results in a single file 17 Save the Protocol and or Spectral Library for use throughout your experiment e You can save the complete Nuance protocol which includes the current Spectral Library by selecting File gt Save Protocol e If you want to save the Spectral Library as a separate file select File gt Save Spectral Library SS Se eee PerkinElmer Inc 68 Elm St Hopkinton MA 01748 USA 508 435 9500 www perkinelmer com 2 Nuance 3 0 2 Quick Start Guide P N 130805 Rev 01 A 10 11 12 13 14 15 16 p gt PerkinElmer Acquire Fluorescence Images Make sure the Nuance Multispectral Imaging System is properly installed and configured Make sure the microscope is aligned and setup for fluorescence imaging Select the appropriate excitation and emission filter set for the specimen If a longpass emission filter is to be used make sure that no other filters will interfere with its operation Keep the excitation shutter closed as much as possible to avoid photo bleaching the specimen Turn on the computer and connect the Nuance mod
5. ation to fine tune the focus 6 Select Binning and Region Of Interest ROI options according to the desired image quality Image files will be smaller with smaller ROI and increased binning Binning increases sensitivity to light but reduces image resolution 1x1 binning is the default for brightfield 7 In the Filter Wavelength Selection group select the wavelength Start Step and End settings If your Nuance module is equipped with a Flex filter and you want to use its narrower band width select the Narrow check box Refer to the Nuance User s Manual for instructions 8 Click the Autoexpose Cube button to determine the exposure settings for the image cube When autoexposure is complete you may notice that the Use Custom Wavelengths and Exposures check box automatically becomes selected This occurs when different exposure times are required for the individual wavelengths 9 Inthe Optical Density box notice that the Convert To OD check box defaults to selected When this OD box is checked you must acquire a reference cube before acquiring the multispectral cube a Move the specimen out of the field of view b Click the Acquire Reference button to gather a reference image for each wavelength c Move the specimen back into the field of view 10 Click the Acquire Cube button to acquire a cube using the selected wavelength range If you prefer to take a grayscale snapshot of the image at the current wavelength clic
6. cube in PerkinElmer format which includes hardware and display settings for the cube e TIFF Cubes saves the cube as a series of TIFF images with the assigned file name plus an appended number indicating the wavelength for each image in the cube Unmix the cube as explained on the back of this page PerkinElmer Inc 68 Elm St Hopkinton MA 01748 USA 508 435 9500 www perkinelmer com Ci Nuance Image_Cube File Edit Hardware Tools Macros Window Help 5 ll q Load Cube Save Cube Zoom Full Image i Acquire Spectra Measure Display Brightfield Fluorescence Binning And Region Of Interest Binning ROT za EA ru Wavelength And Exposure Wavelength nm Exposure ms 550 33 83 a OOO 420 720 Filter Wavelength Selection Start Step End Narrow mois oe fms Multi Filter Support Flat Fielding Flat Field Acquire Ref Image seme ws o O Acquire Mono Acquire Cube Hardware Status Protocol Cursor X rr Nuance 3 0 2 Quick Start Guide p gt P N 130805 Rev 01 PerkinElmer Unmix Fluorescence Images S The Nuance imaging module does not have to be attached to r the computer for you to unmix and analyze cubes 1 On the left side of the screen select the Analyze tab 2 Ifa cube is already open it displays in the image viewing area To open a cube click the Load Cube toolbar icon Cube format types include PerkinElmer format im3 cubes an
7. d TIFF tif cubes L L 3 The sample used in this tutorial is included with the Nuance software and is located in the following sample data folder C Nuance Data Images Sample Data kappa lambda tonsil im3 L s a B 2 Ls Ls Ls Og f Ls 4 Ifa Live Stream window is still open you may close it by clicking its Close 3 box a 5 There are two methods of unmixing spectral signals e To use Manual Compute Spectra you must first select the pure and mixed spectra of the specimen Follow the steps beginning with Step 6 below to continue with this method e The RCA Real Component Analysis feature lets you avoid selecting the signals and lets the software detect the different spectral signals See the Nuance User s Manual for instructions kappa Marable bool 60 A Cube 6 Identify the pure and mixed spectra of the specimen To sample spectra click a Draw Ml button for the Library color you want to use Then use the pointer to draw a line over the pixels of interest within the cube 7 You will see a spectral curve in the same color appear on the spectral display for each line that you draw You may select other spectral display options from the Scaling drop down box E kappa a a AA E apa tame a ANA E a a AA e In this example use the Black marker to draw a line through the dark background circled in White in the top image to sample the autofluorescence haze You
8. itivity to light but reduces image resolution 2x2 binning is the default for fluorescence In the Filter Wavelength Selection group 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 Nuance User s Manual for instructions If your Nuance camera is equipped with a Flex filter and you want to use its narrower band width select the Narrow check box Click the Autoexpose Cube button to determine the exposure settings for the image cube For evenly illuminated results check the Flat Field check box Then move the specimen out of the field of view and insert a plastic fluorescence slide not included Click Acquire Ref Image to gather reference images for each wavelength Move the specimen back into the field of view Click the Acquire Cube button to acquire a cube using the selected wavelength range If you prefer to take a grayscale snapshot of the image at the current wavelength click the Acquire Mono button instead When cube acquisition is complete a color representation of the cube displays in the image viewing area Click the Save Cube toolbar icon or select File gt Save to save the acquired cube or image Select a location and enter a file name File name format suggestion project_sample_operator_ datetime Select a cube type option e Image Cubes saves the
9. k the Acquire Mono button instead Or to take an RGB snapshot at the current wavelength click AutoExpose RGB and then Acquire RGB 11 When cube acquisition is complete a color representation of the cube displays in the image viewing area 12 Click the Save Cube toolbar icon or select File gt Save to save the acquired cube or image Select a location and enter a file name File name format suggestion project_sample_operator_ datetime 13 Select a cube type option e Image Cubes saves the cube in PerkinElmer format which includes hardware and display settings for the cube e TIFF Cubes saves the cube as a series of TIFF images with the assigned file name plus an appended number indicating the wavelength for each image in the cube 14 Unmix the cube as explained on the back of this page PerkinElmer Inc 68 Elm St Hopkinton MA 01748 USA 508 435 9500 www perkinelmer com p gt PerkinElmer Ci Nuance Image_Cube File Edit Hardware Tools Macros Window Help Ll 2i Q Load Cube Save Cube Zoom Full Image Acquire Spectra Measure Display Brightfield Fluorescence Binning And Po Of Interest Binning va O F E Wavelength And Exposure Wavelength nm Exposure ms 550 89 00 Use Custom Wavelengths And Exposures Filter Wavelength Selection Start Step End Narrow jo E o E amp ees RGB Exposure Optical Density Conve
10. may want to use the Zoom tool to see the signals more clearly e Use the Blue marker to sample the 605 nm q dot signal where it is mixed with the background circled in Blue e Use the Yellow marker to sample the 655 nm q dot signal a po a where it is mixed with the background circled in yellow e Change the Labels in the Spectral Library to something ie COSE ig Manual compute Spectra didog Dox more descriptive if desired 12 Inthe bottom image notice that the Yellow and Blue lines in the Spectral 8 When finished click the Manual Compute Spectra button Graph have disappeared and Red and Green lines representing the pure 605 nm and 655 nm signals appear 9 When computing pure spectra observe the plots of the spectral curves in the Spectral Graph Each plot should have a uniform Gaussian curve similar to that shown For this sample both the Scale and Fit Offset options were used for the pure spectra computation 13 Click Unmix A new Unmixed Composite image appears to the right of the original image Grayscale unmixed images also appear below the original image The unmixed kappa lambda sample generates three grayscale images one outlined in Black autofluorescence one in Red 605 nm signal and one in Green 655 nm signal 10 In the dialog box select Known and Mixed spectra to compute i 14 Save the resulting images see the Nuance User s Manual for more about pure spectra For this example
11. rt To OD Acquire Acquire Mono RGB Acquire Acquire Reference Cube Hardware Status Protocol Cursor X Nuance 3 0 2 Quick Start Guide P N 130805 Rev 01 4 10 11 12 13 14 15 p gt PerkinElmer Unmix Brightfield Images The Nuance imaging module does not have to be attached to the computer for you to unmix and analyze cubes On the left side of the screen select the Spectra tab If a cube is already open it displays in the image viewing area To open a cube click the Load Cube toolbar icon Cube format types include PerkinElmer format im3 cubes and TIFF tif cubes The sample used in this tutorial is included with the Nuance software and is located in the following sample data folder C Nuance Data lmages Sample Data ki67 20x im3 If a Live Stream window is still open you can close it by clicking its Close box Before unmixing a brightfield cube make sure it is converted to optical density OD Select Tools gt Convert to Optical Density to convert to OD If this option is disabled the cube is already OD converted Cubes are usually converted automatically during acquisition There are two methods of unmixing spectral signals Manual Compute Spectra and Real Component Analysis Because this tutorial uses a brightfield image we will identify and unmix the pure and mixed spectra manually To sample spectra click a Draw Ml button for the Library color you want
12. to use Then use the pointer to draw a line over the pixels of interest within the cube You will see a spectral curve in the same color appear on the spectral display for each line that you draw You may select other spectral display options from the Scaling drop down box e In this example use the Blue marker to draw a line over the pure blue nuclei circled in Blue in the top image to sample the pure spectra You may want to use the Zoom tool to see the nuclei more clearly e Use the Green marker to draw a line over the brown over blue stained cells circled in Green in the top image to sample the mixed spectra e Change the Labels in the Spectral Library to something more descriptive if desired When finished click the Manual Compute Spectra button In the dialog box select the Known and Mixed spectra to compute the pure spectra For this example select the pure spectrum in Blue for the Known Spectrum Select the mixed spectrum in Green for the Mixed Spectrum The Computed Spectrum displays in Red Change its color to Magenta for this example Click Transfer to Library When computing pure spectra observe the plots in the Spectral Graph Each plot should have a uniform Gaussian curve similar to that shown above In this example the Scale and Fit Offset options are turned on for pure spectra computation Close the Manual Compute Spectra dialog box In the bottom image notice that the Green line in the
13. ule s power cable During startup the Nuance module initializes The LEDs on the front panel scroll through the full wavelength range while the filter is exercising The CAMERA and STATUS LEDs illuminate steady green when initialization is complete Start the Nuance application Position the sample and focus the image at the microscope Then divert all light to the camera port For multispectral imaging make sure the MONO MSI slider on the imaging module is in the MSI position Select the Acquire gt Fluorescence tab If you don t see the Live Stream window click the Live button to see live streaming video of the image In the Wavelength And Exposure group enter the wavelength at which you expect to see an image of the sample This should be within the range of the installed emission filter Use 500 nm or above for focusing Then click the AutoExpose Mono button to improve the live image You can also adjust the exposure time manually e If the live image is too dark increase the exposure time in the Exposure ms box e If the live image is too bright or saturated indicated by solid red pixels reduce the exposure time You can maximize the live window or click the Zoom button and zoom in for higher magnification to fine tune the focus Select Binning and Region Of Interest ROI options according to the desired image quality Image files will be smaller with smaller ROI and increased binning Binning increases sens
14. w perkinelmer com 4
Download Pdf Manuals
Related Search