MetaFluor Analyst
Contents
1. G F F G H The advantage of using fritter for optical transfer function design is that the diagram of fitter See filter function design clearly shows that which sized image details are transmitted amplified or rejected A filter function for transmission of given sized image details is designed for a certain size and resolution image Using different size of images with the same filter function will different filtering The uniformity of the filter in different directions is satisfied only if the image is quadrate Since this is not always the case or if a filter function had been designed earlier for a larger quadrate image but now only a region of the CCD was read out with the same resolution and this sub image is intended to be MetaFluor Analyst 6 6 Version 1 0 93 Advanced Image Processing filtered similarly as the larger one then the image has to be mapped into a larger quadrate one for filtering The mapping happens by tiling the large image quadrate with the small one placing the neighbouring images mirrored The mirroring applies periodic features for the image in this way the edges will not be damaged during Fourier transformation Filter Function Design i Optimize Scaling of factor y axis e l Resetto unitary function by setting its maximal value Save by dragging of half value Load a B oo a DET Preferences F aya Ml arar FT Caloulsie sbsohteo voke alter DFT 7 Li orly phase2. 1 2 Or similar larger derivative kernels like at temporal filtering Filtering in Fourier domain This section of the user manual assumes the knowledge of image representation in complex Fourier domain and optical transfer functions See optics or system control textbooks This filter acts as a user defined optical transfer function H The software allows to design the Fourier domain representation of the optical transfer function with the following limitations for simplicity and manageability assuming that the image is quadrate and the origin is in the middle of the image The transfer function does not distinguish between directions The transfer function does not affect phase only amplitude thus the transfer function is real Thus the transfer function imagined in 3 dimensions as transmission factor function of x and y spatial frequencies wave numbers looks like an axial symmetric object a cone in the case of a lowpass filter Thus the H is represented by a real filter function Frirter K which describes the filters transmission factors as a function of wave numbers ffitter Q is at the origin and His built up by circumrotating fritter around the origin ensuring the same filtering in all spatial directions BO F per lke tky The filtering is done by the real multiplication of the complex Fourier transform of the image G and reverse Fourier transformation that is why affects only amplitude and not phase
3. Intensity pixel value at pointer Timing and number of current frame Image Window Features Intensity Scalebar Displays the intensity levels of the selected frame Right click within the Intensity Scalebar to choose from the following options eCopy ScaleBar to Clipboard copies a bitmap of the Intensity Scalebar to the clipboard eGrayscale displays the image in grayscale ePseudocolor displays the image with an arbitrarily assigned color display el MD color displays the image in Intensity Modulated Display IMD mode IMD color is only available with IMD images Play Pause Button Press to start playback of image sequence Press again to pause Playback is looped back at the end MetaFluor Analyst 2 1 Version 1 0 93 Image Window Set the playback frame rate in the Misc tab of the Preferences dialog box Trackbar To move between frames drag the slider on the trackbar use the scroll button on your mouse or press the arrows keys on your keyboard Image Right click within an image to choose from the following options eZoom Select from the following magnification levels 1x 2x 3x or 4x eGradient Select from the following image colors grayscale red blue green or IMD coloring IMD color is only available with IMD images eSet Scaling Opens Set Scale dialog box eAttach IMD Intensity Opens the Attach IMD Intensity dialog box eAttach overlay image Opens the Attach overlay
4. Creates a new Graph Window with ROls by their numbering on the X axis and plot values in the current pointer position on the original plot corresponding to each ROI e Copy Plot Data Copies plot data to clipboard in tab delimited ASCII format for use in spreadsheet editors e Save Plot Data Saves plot data to clipboard in tab delimited ASCII format for use in spreadsheet editors e Erase Active Plot And ROI Deletes plot below the pointer and deletes the corresponding ROI Plots are identically colored with the corresponding ROls Deletion of a ROI deletes the corresponding plot and deletion of a plot deletes the corresponding ROI A Graph Window remains linked with the Image Window used to create it so any change of ROls or pixel values refreshes the plot If the plot does not refresh after an image processing command click the ROI with the ROI arrow To force a refresh If this does not work close the graph and create a new one MetaFluor Analyst 4 1 Version 1 0 93 Graph Window The axes of the graph are scaled automatically between the extremities of the plot data The appearance of the graph cannot be changed except for sizing the Graph Window The Graph Window was designed only for a short look at the data and helping in the selection of correct ROls e g manual selection for healthy cells The plots has to copied into a spreadsheet program or plotting software for further processing This is done by righ
5. Em T Enlarge image paperto 2 3 using minored ting 13 729 wot 307 12 7 334 x 013 x coordinate ofpointer on the plot actual value of functioan at pointer The Set DFT Filter dialog box DFT Discrete Fourier Transform can be invoked by the Filter 2D Fourier Setup DFT menu command This dialog box is for designing filter function and setting some properties of the Fourier filtering On the editable graph the transmission 1 100 of filter appears as a function of spatial frequences Thus low frequency large image details are transmitted if the beginning of the plot is high lowpass while the high frequency small image details are transmitted if the end of the plot is high highpass The pointer position is given in the status bar of the window for the horizontal position in relative distance from origin percentage 100 half image size in spatial frequency v in half of spatial wavelength 1 2 The shown vertical position is the transmission factor at the actual spatial frequency In the background of the plot a set of hyperbolically decreasing bars are visible if the dialog box was invoked while an Image Window was active The bars are showing 1 2 values corresponding to size of the active Image Window for the spatial frequencies of the graph So high transmission factor will transmit amplify if gt 1 or suppress if 0 image details in the same size as indicated by the bars in each position along the spatial frequen
6. Grayscale Ls Ratio image Automatic scale at Percentile Mies ES IMD Color sor image Min ho Max s0 l Fixed Vaule LE concentran Mir poo a po Cancel Real image data fixedpoint factor Factor for determination of the place of the decimal at fixed point real storage on 16 bits Default Scaling Set the default values of Image Window intensity scaling in some common conditions Normal image Ratio image DF F image and Concentration Use the following procedure to set default scaling 1 Select the type of Image Window by left clicking on the list on the left 2 Set scaling properties 3 Set the coloring for the selected Image Window type MetaFluor Analyst 5 1 Version 1 0 93 Preferences Dialog Box Data Saving Tab Preferences Resample e Save Copy plot data resampled Select for resampleing plot data by default during saving or copying to clipboard See in details at Graph Windows section e X Data resample interval sec Gives the base of resampling each new data point will be in a time point divisible with this value ROI calculation Treat zeros as mask If selected than the software neglects assumes masked pixels with zero value during ROI average calculation Use for calibrated images to exclude out of calibration pixels Use mask If selected the pixels with the given value are masked at visualization appearing in black Use with pseudocoloring MetaFluo
7. decimal is determined by a factor given in the Preferences dialog box Linescan Linescan Windows contains a time space or linescan diagram This diagram consists of pixel values along a linear ROI projected onto the y axis in every time point x axis Linescan Window can be created from an Image Window which contains an image sequence and defined ROI s by using the Linescan command in the calculate menu The X axis of the diagram is the timing of the measurement in seconds the Y axis is the projected pixel intensities along the actual linear ROI Plot Graphs can be created from Image Windows that have defined ROIs using the Plot command in the Calculate menu The X axis of the graph represents the timing of the measurement in seconds the Y axis represents the average values of pixel intensities covered by the ROI Advanced Plot Pixelwise deviation Similar to Plot but plots not the average of pixel intensities over a ROI but the standard deviation of pixel intesities Can be used for determination of punctuate pattern versus smooth however working with highpass Fourier filters gives usually better results Correlation The correlation window compares the pixel values of the two wavelength channels by assigning a point in the graph at the intensity value B and intensity value A coordinate for each pixel of a given ROI Filter Menu Note See Section 6 Advanced Image Processing for more information about the Filter menu
8. eens 3 4 Absolute value cintia a oa 3 4 Custom Affin Transformation oocconnncinncccnnnccnnonannnonnnancccnnrn nano rra rca 3 4 Manual UnMovetacd canta tia nite ani iat iden tienen tian 3 4 2D OU datada 3 4 MetaFluor Analyst v Version 1 0 93 Table of Contents A O ett deere 3 4 2D Fixed Kernel Convolution oooooconnccconocococccnnonanononnnancccnnrn cnn non cc rana rn 3 4 2D Custom Kernel Convolution cooocccinoccconocaconcccnonnnononnnn nono non cn nano rana nn canaria 3 4 visualize Men rt cascada ea eee 3 4 Attach IMD Intensity ecirar iia aa e e a aee eiea 3 4 Attach overlay mage serrr iioe ea ie a e edee aaa aed eee aia 3 4 Overlay TO ii daa 3 4 Options Meir ii d 3 4 TO A 3 4 AO OO 3 5 WVINGOWe Metro aaron ate deverans A E E E 3 5 CAC A iS 3 5 Macri dano nee erences 3 5 MINIMIZOAll aa aca cerrereeeererrertrrareerrere rrr otra reece ct A A AE A 3 5 Restore Alla or taxa utente ba E e sages aa oot sagt aan a aa a R A 3 5 SMOW IMAGES MM Oi ze sts hii reey aiticsser cut Rito AA E AE A AEE E EE 3 5 SHOW ROMO re nda dada add Ad adds Cda 3 5 HeD MORU sis cuicos ato 3 5 ADO a leed cera terre err 3 5 Kela ol Naer A EAEE E DEAD Ae ea eee ea E 3 5 Graph IOWA aA iaa a A A ee a iads 4 1 COrrelation Graph arie ta EE e aE E E ba 4 2 Ljnescan WIN AO Westin ta A eaae dele tec A ea ia A ai iid eiaa Da tadaa 4 3 Preferences dialog BOX iii A iaa aiid ii ge aia ida oe iaa 5 1 Vis lization Tab riita A A a ee ee ea ee vee 5 1 Real i
9. image dialog box eOverlay Text Opens the Overlay Text dialog box eCopy image Copies bitmap of image to the clipboard eCopy image with all ROIs Copies bitmap of image to the clipboard with ROIs included eShow image info Shows image information of the active image The image information can be edited copied and pasted into ia files eErase ROI Deletes active ROIs eErase all ROIs Deletes all ROIs Working with the Image Window Opening an Image Image windows contain each frame of an image sequence in segencial order Each Image Window displays one wavelength of a measurement If an experiment file inf contains more than one wavelength each wavelink sequence is opened in a seperate image window These image windows are automatically linked together linked image windows share ROls and can be processed together for example parallel filtering truncating ratio calculation overlaying IMD coloring There can be multiple sets of linked Image Windows in the workplace The timing of the measurement is loaded from the inf file If this file contains zero clock then only the images after the last zero clock are loaded If only one wavelength is loaded the recording time saved in the image is taken as the timing of the measurement with the first frame as t 0s A timing of 1 frame per second is assumed during bitmap import New Image Windows can be created by duplicating an active one using the Dupl
10. 60 1 60 MetaFluor Analyst 6 5 Version 1 0 93 Advanced Image Processing Filtering in Image Plane Filtering in image plane is used for noise reduction however it can also be used for extracting or suppressing certain image details 2D median filter Filter 2D Median menu command works in image plane with the given window size and the result is placed in the middle of the rectangular filtering window 2D median filtering suppresses spots appearing the images Image plane kernel convolution filtering Filter 2D Fixed or Custom Kernel Convolution menu command can serve as averaging high and lowpass filtering spatial derivation according to the given kernel matrix Kernels can be designed in the Preferences dialog box Fourier domain filtering can apply a user defined optical transfer function on the images The result is similar to low or highpass kernel convolution but the filter design for filtering in Fourier domain is more versatile and the filtering spares the edges of the images Practically by appropriate filter function design in Fourier domain the desired sized objects image details can be selectively amplified or suppressed This can range from the correction for uneven background to extraction of fluorescence signal coming from small organelles like mitochondria Kernels for convolution filtering 1 9 1 9 1 9 1 2 Averaging 1 9 1 9 1 9 X derivation 1 2 0 1 2 Y derivation 0 1 9 1 9 1 9
11. IVIINI Vite MetaFluor Analyst MetaFluor Analyst User s Guide For Use with MetaFluor Version 7 0 for Microsoft Windows XP 1030 2203 02 Copyrights Notices and Trademarks O 2004 2006 Molecular Devices Corporation All rights reserved Printed in the U S A Information in this document is subject to change without notice and does not represent a commitment on the part of Molecular Devices Corporation The software described in this document including information contained in any databases is furnished under a license agreement and may be used or copied only in accordance with the terms of the agreement It is illegal to copy the software except as specifically allowed in the license agreement No part of this manual may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying and recording for any purpose without the express written permission of Molecular Devices Corporation MetaMorph and ImageXpress are registered trademarks and Discovery 1 MetaXpress MDCStore and ImageXpress Micro are trademarks of Molecular Devices Corporation All other trademarks are the property of their respective owners Disclaimer Molecular Devices Corporation reserves the right to change its products and services at any time to incorporate technological developments This user guide is subject to change without notice Although this user guide has been prepared wi
12. TT EENAA KERA 6 6 MetaFluor Analyst vi Version 1 0 93 Table of Contents Filter Function Desi iii a a aa 6 7 DET Preferences naci iii 6 8 Presove EdE S umi iria 6 8 Real DFT OUUU caia 6 8 Enlarge image paper to 2 using Mirrored tiling o oooonnnnnninnnnn nncnnnnncoccnnacccccnnnncarnnrnnnons 6 8 Calculate absolute value after DFT ooocccnnnncciniccononccnnonnnnncnnnnnoccnonnn nn nn cnn 6 8 Leave Only PASO oooooconcccccncconnociconcccno nociones 6 8 RU aio ad AN EE T 6 8 Optimization of Filter Function for Image Detail ExtracCtiOM ooonnnnicicnndinnnnnnnnccnnccconnorannnccncnrccnn 6 9 Signal Background ROI atraer eaan oa aa a a E A A AA O E Ta 6 10 OPIMIZO no e a e a E E nee ey pect rrcae 6 10 GS OMPU TS tar e aa ae a e a caine op a a a A 6 10 Method Frequency SCAN erai ninie era EEn Ee EEEE E E EI EE AER ESNEK E ra 6 10 BOSU EEn fies ante e E E A T AEE AEA 6 10 O e a colorea 6 11 MetaFluor Analyst vii Version 1 0 93 Chapter 1 MetaFluor Analyst Overview MetaFluor Analyst was designed for high throughput routine work with ratiometric and non ratiometric fluorescent dyes It enables fast loading of image sequences convenient region of interest ROI drawing and fast plot extraction Other features of MetaFluor Analyst include e Background subtraction e Ratio and DF F calculation with calibration e Bleach correction e Temporal smoothing e Image plane filters including Fourier domain filtering which e
13. XT file of current Region of Interest ROI 1D array Movie series of 001 bmp files for each frame Movie series of 001 bmp files for each frame Load ROls Opens the Load ROls dialog box Use to load previously saved Regions of Interest from disk The roi binary format contains corner coordinates of all ROI polygons Save ROIs Opens the Save ROls dialog box Use to save Regions of Interest to disk The roi binary format contains corner coordinates of all ROI polygons Save Plot Data Runs the Plot command and saves the plots from the resulting Graph window to ASCII file in tab deliminted format chart The first column contains the timing in seconds MetaFluor Analyst 3 1 Version 1 0 93 Menu Commands and Toolbar Exit Closes all open images on the desktop and quits MetaFluor Analyst Edit Menu Copy image Copies current frame of active Image Window to clipboard in the following formats As image As image with active ROls As image with all ROls As 3D graph ASCII tab delimited Copy plot data Copies plots from the active Graph Window to the clipboard in ASCII tab delimited format Copy all ROls Copies ROI polygons to clipboard in the roi binary format The following choices are available 4Copy active ROI 5Copy all ROls Paste ROls Pastes ROls from the clipboard to the active Image Window and all linked windows if applicable Delete ROIs Deletes active ROls Set linkage Opens the Se
14. ber parts Result New filters can be built either by taking the union of the Use the number most optimal bandpass filters or by building a proportional filter assigning 1 transmission to the best bandpass MetaFluor Analyst 6 10 Version 1 0 93 Advanced Image Processing Optimize Press Optimize button at the bottom to run optimization A series of bandpass filter will run through the filter function plot generating results in the output of Method box The Image Window content is left intact Build a new filter function by pressing the Build filter buttons of Result box Optimizing in situ mitochondrial calcium measurement Use a high resolution image sequence of rhod 2 fluorescence recorded during stimulation evoking calcium transient e Set the signal ROI for the mitochondrium rich peripheral area of the cell e Set the background ROI to the nucleus of the cell e Open Set DFT filter dialog Select Enlarge image if the recorded image is not quadrate Select Absolute value e Open Optimize filter dialog e Enter minotchondrial ROI for Signal and nuclear for Background e Select Signal Background for optimization e In the Compute box select Change of signal Enter a frame number from the baseline and one from the stimulus evoked fluorescence peak where both nuclear and mitochondrial fluorescence rise is present Select Use DF F e In the Method box scan range can be 0 0 5 dividing into 10 parts however ot
15. cence Imaging of Mitochondrial Ca Concentration Cell Calcium 2001 30 5 311 321 Optimization of Filter Function for Image Detail Extraction Image detail extraction like the discussed Mitochondrial Calcium measurement is based on the transmission of spatial frequencies carrying the important information while rejecting the rest For the best results the optimally designed filter function is essential Finding the best filter function is largely empiric manual work the Optimize filter dialog box gives help in this This routine scans a range of spatial frequencies by bandpass filters in the given range while determining the level of image detail extraction by comparing intensities in different ROIs on the filtered images A new filter function can be built from the result of the scan which probably needs also some further manual enhancement but gives the sketch of the filter function To start optimization dialog perform the following steps 1 Draw two ROls on the image one over an area rich in details to extract and one over an area which is rich in details to reject 2 Start Set DFT Filter dialog box Filter 2D Fourier Setup DFT menu command and set up preferences like enabling image enlargement and absolute value calculation The filter function will be deleted so save it if needed 3 Start Optimize filter dialog box by pressing optimize icon in the top right corner The Optimize filter dialog box opens as sho
16. cent dyes R a VR nin Fs C k mA VR max R Fi Where the concetration C is calculated from the dye dissociation constant Ka the maximal and minimal ratio values Rmax and Rmin the intracellular viscosity use 1 for water usually between 0 6 0 8 and from the free and bound dye fluorescence at the wavelength in the denominator Fi2 and Fp2 For non ratiometric calibration use Fmax and Fmin fluorescence intensities instead of Rmax and Rmin taking Fro and Fp2 equal to 1 Normalization Proportional scaling for general purposes F E Fain C k min Fina OE cay Where the concetration C is calculated from a arbitrary constant k and the maximal and minimal fluorescent intesities Fmax and Fmin MetaFluor Analyst 6 3 Version 1 0 93 Advanced Image Processing Entering values To acquire calibration values from Image Windows press the red arrow button at each field after the corresponding ROI has been selected in the active Image Window Output The order of magnitude of the calibrated values are highly affected by Ka and k The result can easily be out of the range of the used 16 bit data storage type Pay attention of output box where the data storage type is shown of the Image Window to be calibrated Set the data type of the calibrated Image Window real or integer here For example if calibration ratio real values to intracellular calcium is nM switch output type to integer Threshold min max If selecte
17. commands Running median Filters the image sequence by running a median filter in time over each pixel sequence Running average Filters the image sequence by running a average filter in time over each pixel sequence Places the average in the middle of the given window Deletes window 2 frames from the beginning and end Bleach correction Fixes photobleaching or leakage of fluorescent dye It does this by manipulating the data by the extrapolation of a fitted exponential function on a part baseline of the trace Temporal derivation Calculates temporal derivative by convolution of image in time with the given column vector kernel Can MetaFluor Analyst 3 3 Version 1 0 93 Menu Commands and Toolbar be used for any kind of convolution process in time temporal filtering The result is multiplied by the given time factor Subtract background Removes image backgroud data based on a number of variables Absolute value Takes the absolute value of pixels Custom Affin Transformation Manual UnMove Manually correct image movements that occured during measurements 2D Fourier Configure a filter that acts as a user defined optical transfer function e Setup DFT filter See DFT filter designing in chapter e Run DFT filter Runs the previously configured DFT filter on the active Image Window 2D Median Used for noise reductionand extracting or suppressing certain image details 2D Fixed Kernel Convolution Makes k
18. cy If the filter function is zero at zero spatial frequency then the average intensity of the image will be zero Edit the plot in the following way 1 Double click to add a new point to the plot 2 Click and drag point to move 3 Right click select Delete to delete a point Toolbar Icons e Optimization e Reset plot to unitary function MetaFluor Analyst 6 7 Version 1 0 93 Advanced Image Processing e Save saves into own binary format flt e Load loads previously saved flt format DFT Preferences Preserve Edges Maps the image with the mirrored tiling described at Filtering in Fourier domain into a double sized image during filtering in this way Fourier transformation does not cause artifactial stripes appearing at the edges of the images Double sized image means four times of calculation at DFT thus it slows down processing Real DFT output The result appears as fixed point real values Enlarge image paper to 2 using mirrored tiling Maps the image with the mirrored tiling described at Filtering in Fourier domain into a given sized window during filtering Select this option for filtering non quadrate images or smaller images than that for the filter function was designed For fast DFT transformation 2 n sized images are optimal Calculate absolute value after DFT Calculates absolute value of pixel intensities after filtering Select this option for filtering with highpass characteris
19. d pixel values out of the range between Rmin and Rmax Or Fmin and Fmax are masked by calibrating as zero Select Treat Zeros as Mask at Preferences dialog box Datasaving to enable masking during ROI calculation Also select Use mask 0 to show masking in the Image Windows Manual Unmove Manual Unmove was designed to correct movements that happened during measurements The stabile posistioning of structures for ROI calculation in the image sequence can be ensured in this way in the case of uniform dislocations Each frame can be freely dislocated or rotated by the numeric keypad arrows see popup menu The movement of frames s linearly interpolated between the set positions the ones after the last set position are remaining in the last set position If a frame position is set then an L sign Locked appears on the upper left corner of the image The first frame appears in green The current dislocated rotated frame appears in red so any dislocation can be easily noticed To apply the Manual Unmove command to an image select Manual Unmove from the Filter menu A sample Image Window after the Manual Unmove command has been applied is shwon below The L sign shows that ho wi 05A1 002 the current frame is locked The red color shows the actual dislocated rotated image COEN UE Are of rotation The green shows the original frame Vector of dislocation To unmove an image after apply the Manual Unmove command pe
20. d 2 or its derivatives loaded to monolayer cell cultures this dye preferentially loads mitochondria however not completely selective stains also the cytosol With the filter function design it is possible to design a highpass filter which transmits the high frequency small details of mitochondria while rejects the low frequency bulky details of cytosol Use the absolute value option for gaining images for ROI calculations MetaFluor Analyst 6 8 Version 1 0 93 Advanced Image Processing The theory is the following The fluorescent image G x y is assumed to consist of the sum of fluorescence deriving from the mitochondria Gm and the cytosol Ge G G G This can be done because the focal plane of wide field fluorescence microscopy is thick enough to image both mitochondria and cytosol together The Fourier transformation of the image keeps the sum structure due to its linear nature F G F G G F G FfG The goal of Fourier transformation is that F G and F G are separated in the wave number space i e mitochondrial fluorescence fine details are represented at high whereas that from the cytosol at low wave numbers Thus an appropriate optical transfer function separates mitochondrial derived fluorescence from the cytosolic one Use mitofilter flt for 512x512 pixel images acquired by a 100x optics 0 15 m pixel 1 Akos A Gerencs r and Vera Adam Vizi Selective High Resolution Fluores
21. e 3 1 Close Current image WiINKOW c cccceeeeeeeeeeeeeeeeeeeeeceaeeeeaaeeeeeeeecaeeeeaaeeeeaeeseeeeesaeeeseaeeteeeees 3 1 lose li Perey ere pre ere E e rere cen eee reece repeat rae 3 1 Export current image Series ccscceeeceeeeceeeeeneeeeeeeeceaeeeeaaeeeeaeeseaeeeseaeseeaeeseeeeessaeesseaeenenes 3 1 Load ROIS rae aa erence A N ereerereee prt emer ee A tree err er ere 3 1 SN O o O o 3 1 Sal Data CINE 3 1 Elia a deena ead ee eee ee 3 2 Edit Menu sic Aice blind AAA ede ae ed 3 2 Copy IMA ici A eee eit dee eet 3 2 Setlinkage w2ctcs cies AeA Aira Nevada 3 2 Truncate TrameSiie cciaii tiny cel ad intact gute Aa 3 2 Merge Image Series ii dik cei A Cd LA 3 2 Set Image Scaling ecc lenis clei eval end aen deia 3 2 Calctilate Menu aiii Ae eens 3 2 DR Eat oad ee ieee eel eed Ieee ee 3 2 MO A AE A A a 3 3 Calibre AA ia 3 3 CONVE O A tee E ee that dat he tae ies ees tal ass 3 3 A ae Bahama attate bias i leete ahd adit eel etd ata dente tata aaab ara iin cet ey 3 3 Pl A et ane Sadana A i eee 3 3 Advanced Plottier tdi gel E iis cela bear ieke egy 3 3 Correlation Anie an itil hha iain alate te hte inte i bn dis 3 3 FIER MON it A AA taste steht athe aoe tile ahd wate tant 3 3 PRUNING median ores c atten vv id idea 3 3 RUNNING average iiss waite tilts adel the ntdat igen telat tes 3 3 Bleach COrecton miii i 3 3 Temporal derivatiON gieciie t5lees veces ect a a evens dei nth E hats eevee ty 3 3 Subtract background tii ii A Ate Miata
22. ernel convolution filtering in image plane frame by frame with some predefined low and high pass filter kernels 2D Custom Kernel Convolution Makes kernel convolution filtering in image plane frame by frame with user defined kernel Visualize Menu Attach IMD Intensity Sets the color coding of actual Image Window to intensity modulated pseudocolor coding IMD by asking for a linked Image Window for intensity values The pseudocoloring hue shows the pixels values of the active image the brightness shows the pixel values of attached image IMD coloring can be switched off by setting the gradient in the popup menu of the Image Window to any other value Does not affect stored pixel values or ROI averages Attach overlay image Overlays the actual Image Window content with a chosen linked Image Window content always frame by frame The overlaid image shows in its original gradient and intensity values Change the original image settings to affect overlaid image Does not affect stored pixel values or ROI averages Overlay Text Enables you to print the frame number and frame time on the image The numbers are formatted using the Type and Justification combo boxes and the Font button The position of the numbers can be set in X and Y fields If the Image Window is resized then the absolute positions of the overlaid text do not change Options Menu Colors Stores the ROI coloring 16 colors are cycled during ROI drawing se
23. he scalebar linearly if the image is displayed in color These values are indicated above and below the scalebar and can be changed using the Set Scaling command in the edit menu Set Scaling Command Select type of scaling Automatic scale at Percentile Min 1 00 Max 89 00 Scale each frame independently Fixed Vaule in 245 000 Automatic scale at percentile calculates the minimum and maximum values of intensity scalebar from the histogram of image series taking intensities at the given percentiles Scale each frame independently the histogram and percentiles are calculated for each frame instead of the whole image series Fixed value Scales intensity between the entered minimum and maximum values Intensity scaling and coloring also have default values for the following conditions loaded image normal image ratio and DF F image These default values can be set in the Preferences dialog box For example setting the default intensity scaling and coloring of radiometric Image Windows to certain fixed values can assure that different image sequences will appear in comparable coloring in routine work The content of a linked Image Window can be overlaid onto the active window one using the Attach Overlay Image command in the Visualize menu The overlay frame from the linked image is always the same corresponding frame as the active window During overlaying the image shows its original gradient and
24. her setting should be also tried e Run optimization and build filter MetaFluor Analyst 6 11 Version 1 0 93
25. icate Image Window command in the Edit menu If the duplicate is linked to the original it will inherit any existing ROIs This command is useful to create a backup of an experiment before doing any image processing The contents of an Image Window with ROIs and image information can be saved into and loaded from the MetaFluor Analyst format ia This format does not support multiple wavelength each wavelength must be saved separately The pixel values of an image sequence are stored as 16 bit signed integer values range 32768 32767 Image Windows can be switched into fixed point real mode using the Convert to command in the Calculate menu This command stores images as 16 bit signed integer values but they display as real values in a range of 32768 factor 32767 factor with 1 factor precision The image is automatically switched to real mode during certain calculations like DF F ratio For best results the factor of fixed point real image storage has to be optimally set in the Preferences dialog box according to the dynamic range of calculations All functions work on both integer and real values MetaFluor Analyst 2 2 Version 1 0 93 Image Window Visualization The Image Window shows the first frame of the image sequence by default The intensity of the image is shown by the intensity scalebar at the left side of the image The pixel intensities between a minimum and maximum value are translated to the colors of t
26. ing the step sizes during manual unmovement e Move step X and Y direction step size in pixels for fine movements it has to be smaller than 1 e Rotate step Rotation step size in degrees should be smaller than 1 for fine movements Playback Set the playback rate of frames in the Image Window in frames per secona MetaFluor Analyst 5 4 Version 1 0 93 Chapter 6 Advanced Image Processing This section contains advanced image processing tecniques available in MetaFluor Analyst Background Subtraction Select Subtarct Background from the Filter menu to open the Background Subtraction dialog box as shown below Background Subtraction Subtract background at TBackground Image i th frame or Average all frames Calibrated W1 GROUP31 001 We GROUP32 001 Coros Subtract background at Options for type of background to subtract percentile the background is calculated for each frame from the histogram of the frame the intensity at the given percentile For subtract percentile calculated from the histogram of the whole image sequence set a minimum value at Scale Intensity dialog box of Image Window and subtract the value appearing below the scalebar as value value user defined value to subtract Use negative number here to add background ROI average by frames Subtracts the average of pixel intensities covered by the given ROI calculated frame by frame ROI average b
27. intensity values so it does not affect stored pixel values or ROI averages The Intensity Modified Display IMD technique helps discrimination of objects in the image after ratio or DF F calculation If IMD coloring is enabled the hue of the pixel is determined from the scaled pixel intensity value of the given Image Window and the brightness is determined by the attached IMD intensity image The result of a ratio calculation automatically appears IMD colored with the intensity brightness values derived from the average of the numerator and the denominator IMD coloring can also be set up by manually attaching an IMD intensity image using the Attach IMD Intensity command in the Visualize menu Use the following steps to produce an IMD colored DF F image 1Duplicate an image using the Duplicate command in the Edit menu 2Use the DF F command in the Calculate menu on the duplicated image 3Attach the original image as an IMD intensity using the Attach IMD Intensity command in the Visualize menu IMD coloring can be switched off by setting the Gradient in the popup menu of the Image Window to any other value IMD coloring does not affect stored pixel values or ROI averages MetaFluor Analyst 2 3 Version 1 0 93 Image Window Overlay Text Command Overlay Text Image time and frame V Show time Type Justification o E fen y Font At X 0 00 Y 2 0 00 position J Show frame number Justification fet
28. lculate Correlate Calculate Linescan Bg Filter Subtract background NA Subtract background with the active ROI set for subtraction frame by frame a File Save plot data a Edit Copy plot data MetaFluor Analyst 3 6 Version 1 0 93 Chapter 4 Graph Window Graphs can be created from Image Windows that have defined ROIs using the Plot command in the Calculate menu The X axis of the graph represents the timing of the measurement in seconds the Y axis represents the average values of pixel intensities covered by the ROI An F sign at the end of y axis indicates that if the plot was created from a normal intensity type non radiometric Image Window A R sign indicates it was created from a radiometric Image Window A sample Graph Window with created from a normal intensity type non radiometric Image Window is shown below 399 56 348 3 3 0 65 85 80 17095 X 0 650 sec v 470 518 Right click within Graph Window to choose from the following options e Show as DF F of intensity view calculates DF F normalization trace by trace If the signal to noise ratio of the image sequence is poor DF F normalization of plots rather than pixels is favourable This is an in plot calculation and does not affect image data A DF F sign at the end of the y axis indicates that this mode is active e Resample when saving or copying data resamples the data along the x axis during clipboard or file output e Plot Cross Section
29. mage Window can be changed using the Set Linkage command in the Edit menu The Set Image Windows linkage dialog box shows all the Image Windows already linked selected or matching the size criteria to be linked unselected Other sets of linked Image Windows that can also be linked together are listed in one row MetaFluor Analyst 2 5 Version 1 0 93 Chapter 3 Menu Commands and Toolbar Use the MetaFluor Analyst menu commands and toolbar to open manipulate and analysis experiment files created in MetaFluor Menu Commands The following menu commands are used available in MetaFluor Analyst File menu Open experiment Opens a previously saved Experiment file inf into an image window on the desktop for viewing processing or analysis If the image sequence descriptor inf file contains zero clock then only the images after the last zero clock are loaded The timing of the measurement has to be continously incrementing without zeroing The images in an image sequence must have the same format size and depth Save Saves a modified inf file to the ia Image Analyst format Close current image window Closes the selected window Dependent windows such as graphs are also closed with this command Close all Closes all open windows Export current image series Choose to exports pixel values of the current selected frame or whole image series to the follwing formats TXT file of current frame 1D array T
30. mage data fixedpoint factor ecceceeceeceeceneeeeeeeeeeneeceeeecaeeeeaaeseeeeeseaeeesaeeeeaeeesaees 5 1 Default Scaling is ic annie da Dd a ana Ne a La 5 1 Data Saving abyss tiie E de ae ieee i cae eae ad ses 5 2 PRROSAMPIG sedans Gere tee als ete cee a ae deve eee dae yeaa 5 2 ANA O AN 5 2 Use MASK siii A A A a ae lass Wee A Seeded eats Sud 5 2 Convolution kernel Tab isinisisi iiaii iie nn 5 3 IA A A 5 3 Size DIMENSIONS nati asadaliceio la llbcedlr tl rldicti trad 5 3 NOMAlIZO Cinta datado tias illa rd AE a 5 3 MISC Fado tiara ladilla 5 4 Manual Un Move SAn E A ro tt alerts alta 5 4 Playacar lll died mi 5 4 Advanced Image Processing seii hrr rE E shuces illes lali le rte lit 6 1 Background Subtract leccion ted torera rato atra ei ita 6 1 Subtract DackOrOUNG aiii trios 6 1 Background Mage latir lacas EE cie lao ben tia 6 1 BIGACH Corre cl carcasas 6 2 Make regression A ea ee Ee EER 6 2 Independent Vanable T setri aida nd pidan 6 2 Dependent variable aeie fu cies lt tae 6 2 Regres som POr 6 2 Regression method number of iterations precision oooccconococcconocnncconannnncnnnnnnnccnnnnnnnnnnn nn 6 3 A NO 6 3 A NN 6 3 DR rai rene pee ani E reer rere repre eee 6 3 Gal id eens deck ENAA E TE A AE AA E A 6 3 Manual O 6 4 Fikenngin TIMES se cseeccesteeece Ae AESA Tiaa T SATKAAT EVS TENE ARAE A EAV AASR 6 5 Filtering in Image Plane araiarraren ltd 6 6 Filtering in Fourier domam renerne iena i ATAA e ana Ei KARRA PSAE EE E
31. ming of the measurement in seconds the Y axis is the projected pixel intensities along the actual linear ROI A sample Linesscan window is shown below N W1 GROUP31 001 Linescan oj xj 563 329 0 1076 319 63 s 1 The content of a Linescan Window can be resampled by right clicking in a Linescan window and selecting Resample however in this case the appearing diagram is affected by the resampling Right click in a Linescan window to view additional options Note The Linescan command is useful for visualization of wave like propagation of signals together with temporal derivation On time space diagrams the stabile structures are drawn straight while the dislocating are drawn curved or sloped paths The characteristics or speed of movement can be determined in this way However further work or other software is needed to determine slopes The Edit Copy Image I as 3D graph menu command can be used for exporting data through the clipboard to other software MetaFluor Analyst 4 3 Version 1 0 93 Chapter 5 Preferences dialog box This section contains information about the Prefences dialog box To open the Prefences dialog box Select Prefences from the Options menu Visualization Tab Preferences Visualisation Data saving Convolution kemels Mise Real image data fixedpoint factor fi 000 Range 132 77 Accuracy 0 00100 Default Scaling Gradient E Norma image
32. n the given sized window from the beginning of the image sequence shifting the window position one by en frame to the end The result is placed back to the middle of the window The starting and ending window size 2 frames are discarded Temporal derivation Filter Temporal derivation menu command is a temporal 1 dimensional kernel convolution filter Capable for filtering any given column vector kernel thus can be used for not only temporal derivation but for lowpass or highpass filtering in time for data smoothing The calculated values are multiplied by timefactor to help data to remain in the dynamic range of the 16 bit data storage like temporal derivatives can be very small sometimes compared to the intensity values Kernels can be designed in the Preferences dialog box All filtering processes are in place operations Temporal derivation or DF F calculations are very sensitive for or amplifying noise That is why sometimes good derivative or DF F images can be obtained after thorough filtering in time but also in image plane Median filtering can be used for removing hairy spark like noise from the measurements Temporal derivation is used for enchanced visualization of propagating wave like intracellular Ca waves Drawing a linescan diagram of a temporally differentiated image sequence clearly shows the wavefront of the wave Kernels for derivation 11 60 7 1 12 9 60 8 12 45 60 0 0 8 12 45 60 11112 9
33. n the graph at the intensity value B and intensity value A coordinate for each pixel of a given ROI A sample correlation window is shown below la 73 00 273 00 400 83 528 67 656 50 784 33 912 17 B Correlation Graphs can be created using the Correlate command in the Calculate menu from a set of linked Image Windows where ROls are present The Correlation Graph is a kind of Graph Window so its usage and popup menu system is similar For statistical analysis the data has to be copied into the appropriate statistical software Note Correlation Graphs are useful for the investigation of colocalization of two different fluorescent dyes in the same subcellular structures If the two dyes are colocalizing then the same pixels are bright for both dyes Conversely the same pixels are dark for each dye This means there is some correlation between the pixel intensity values which appear in the graph as a line originating from the origin If there is no correlation the points are scattered in the graph MetaFluor Analyst 4 2 Version 1 0 93 Graph Window Linescan Window Linescan Windows contains a time space or linescan diagram This diagram consists of pixel values along a linear ROI projected onto the y axis in every time point x axis Linescan Windows can be created from an Image Window which contains an image sequence and defined ROI s by using the Linescan command in the calculate menu The X axis of the diagram is the ti
34. nables selective measurement of mitochondrial derived fluorescence The main window of MetaFluor Analyst contains the workplace the menu and the toolbar On the workplace unlimited number of Image Windows and graphs can be present simultaneously The software is controlled by the user through the menu system using the main menu and toolbar of the main window while certain functions can be accessed by right clicking active windows Experiments or image sequences created in MetaFluor are opened in image windows Image windows contain each frame of an image sequence in sequential order Each Image Window displays one wavelength of a measurement If an experiment file inf contains more than one wavelength each wavelink sequence is opened in a separate image window These image windows are automatically linked together linked image windows share ROls and can be processed together for example parallel filtering truncating ratio calculation overlaying IMD coloring Using this Guide The following sections provide information and instructions on using MetaFluor Analyst Section 1 Introduction provides an overview of the functionality of MetaFluor Analyst as well as hardware requirements Section 2 Image Window provides an overview of Image Window features Section 3 Menu Commands and Toolbar contains descriptions of the Menu and toolbar commands available in MetaFluor Analyst Section 4 Graph Window explains how to use the g
35. obleching of background dye fluorescence subtraction of ROI average calculated frame by frame can correct for these potential artifacts If the image contains a relatively large amount of background cell free areas then subtraction at given percentile can be also used as background subtraction Bleach Correction The Bleach Correction command fixes photobleaching or leakage of fluorescent dye It does this by manipulating the data by the extrapolation of a fitted exponential function on a part baseline of the trace Manipulation means that the first frame remains unchanged and in the following frames either the fitted function is subtracted or the frames are factorized by the relative change from the first fitted value In this way the drop in the trace due to photobleaching can be eliminated Select Bleach Correction from the Filter menu to open the Bleach Correction dialog box as shown below Age eon eee a0 irer pulini nimi oben eet raem jns E whale nag 7 Eure aba Fa f Papas pues ear ira i Bim mebi ah imore fan Famen 1 10 Faget ate pa al Sammie gustar i n 0 gubbe im rep C Parts ange t TL Goe U Ee wi GAOL ay DEI o tm Make regression between The range of the frames where to fit the exponential Independent variable The independent variable can be the time or frame number If the timing is irregular then frame number based bleach correction is suggested while if the decrease of flu
36. orescence is due to dye leakage the time based bleach correction is favorable Dependent variable The exponential function is fitted onto the values of the dependent variable this can be the average intensity of the whole frame or the average of a given ROI Regression type The two parameter exponential y a e b x decays to the zero It can be used with well subtracted MetaFluor Analyst 6 2 Version 1 0 93 Advanced Image Processing background The three parameter one y a e b x c serves more general purpose Regression method number of iterations precision The Simplex algorithm works the most reliably Regression method number of iterations precision should not be changed Action e The difference of fitted function from its first value is subtracted from the frames e Frames are factorized by the relative change of fitted function from its first value Show Fit Calculates the regression shows the parameters and displays the results on the graph in red OK Calculates the regression and process the images sequence as specified at Action filed Calibration Select Calibration from the Calculate menu to open the Calibration dialog box as shown below Grynkiewicz Normalize O ML Quiput vy Rmax R Fb2 Integer pie foo Beal V Threshold to min max Rmin 1071 3406 de Fi2 fioro Rmax 1071 9406 def Fb2 10719406 a Grynkiewicz Equation to calibrate ratiometric or non ratiometric fluores
37. r Analyst 5 2 Version 1 0 93 Preferences Dialog Box Convolution kernel Tab Visualisation Data saving Convolution kernels Misc Select kernel 1 00000 B amp Q Size Dimensions 3 y y Normalize Kernel file Select kernel Kernels are stored based on their names To browse kernels use the Select kernel field The selected kernel appears in the edit region of the dialog box Kernels are stored in a binary file ckr listed in the Kernel file field The changes are automatically written in the kernel file upon clicking OK button To open another kernel file or save in a new name use the icons below the Kernel file field Kernel icons below the Select kernel field e New kernel First enter a new name into the Select kernel field then click New Kernel button A new 3x3 nullmatrix kernel is created e Copy kernel First enter a new name into the Select kernel field then click Copy Kernel button The active kernel is copied and appearing in the new name in the list e Delete kernel Click Delete Kernel button to delete selected kernel Size Dimensions MetaFluor Analyst uses quadrate XY column Y and row X matrices as kernels with the size given at the Size field Normalize Divides the values of the kernel with the sum of absolute values in the kernel MetaFluor Analyst 5 3 Version 1 0 93 Preferences Dialog Box Misc Tab Preferences Manual Unmove For sett
38. raphing and charting features of MetaFluor Analyst Section 5 Preferences explains how to use set default preferences to use in MetaFluor Analyst Section 6 Advanced Image Processing details advanced filtering and processing techniques available in MetaFluor Analyst Hardware Requirements Operation Microsoft Windows 9X NT 2000 NT or 2000 recommended system Hardware e Hard drive space 10 MB for executable with all dlls e CPU Pentium class e Memory 256MB recommended Video True color 24 or 32 bit high resolution recommended MetaFluor Analyst 1 1 Version 1 0 93 Chapter 2 Image Window Experiments or image sequences created in MetaFluor are opened in image windows Image windows contain each frame of an image sequence in sequential order Each Image Window displays one wavelength of a measurement If an experiment file inf contains more than one wavelength each wavelink sequence is opened in a separate image window These image windows are automatically linked together linked image windows share ROls and can be processed together for example parallel filtering truncating ratio calculation overlaying IMD coloring A sample Image Window is shown below ad F Maximum value Intensity scalebar Minimum value li Play pause button um n pis if e A Magnification DE 34 13 0 656 113 64 5 57 15 2x Current position of mF 1x 2x 3x 4x pointer Trackbar for frame selection
39. rere At Xx 0 00 Y 4 0 00 position Use the Overlay Text command in the Visualize menu to print the frame number and frame time on the image The numbers are formatted using the Type and Justification combo boxes and the Font button The position of the numbers can be set in X and Y fields If the Image Window is resized then the absolute positions of the overlaid text do not change Image processing Image processing functions are accessed through menu commands and the Toolbar If an image processing command involves more than one wavelength of an experiment i e Multiple Image Windows then the Image Window to process can be chosen from the list of Image Windows linked to the active window The results of the command will overwrite the original image sequences except for ratio calculation where the result appears in a new window If the original image is needed later then duplicate it before processing using the Duplicate command in the Edit menu ROI drawing Use the ROI Region of Interest toolbar commands to edit regions of interest on the active image Select linear or area ROI editing from the toolbar then click and drag on the image to create a ROI Double click to set the ROI The ROIs are visible and can be edited in any linked Image Windows To move a ROI select the ROI arrow button on the toolbar then click and drag the ROI You can also use the ROI tools to copy paste or delete the active ROI Selec
40. rform the following steps 1 Drag the trackbar to the last correct position 2 Lock the last correct frame by pressing on Numeric keypad 3 Goto the first dislocated frame or if the dislocation is slow linear into any further position 4 Correct position by using the arrow keys in Numeric keypad for dislocation 1 and 3 for rotation The red image has overlaping the green one 5 Refine unmovement with repeating previous point in the interpolated frames By pressing on the numeric keypad the lock on a frame can be toggled if the frame is not locked MetaFluor Analyst 6 4 Version 1 0 93 Advanced Image Processing then its position is interpolated between the two neighboring ones The frame movement can be reset by pressing on the numeric keypad in this case it returns to the non dislocated position and becomes locked You can also right click in the Image Window to acess the Unmove commands The unmovement can be finalized by pressing Enter or cancelled with Esc Filtering in Time Filters acting in time are one dimensional filters filtering a sequence of pixels in the same position in image plane along the image sequence The running average Filter Running Average menu command and running median Filter Running Median menu command are for noise reduction Both are working with a window size in frames which can be entered in the appearing dialog box The average or median is calculated i
41. t Image Windows linkage dialog box Shows linked Image Widows and enables you to manually link Image Windows so they can be to be processed simultaneously Duplicate image windows Duplicates Image Window The new Image Window can be linked or unlinked to the original Truncate frames Opens the Truncate Frames dialog box for cutting or truncating frames Merge image series N A Set Image Scaling The intensity of the image is shown by the intensity scalebar at the left side of the image The pixel intensities between a minimum and maximum value are translated to the colors of the scalebar linearly if the image is displayed in color These values are indicated above and below the scalebar and can be changed using the Set Scaling command Calculate Menu DF F Calculates DF F normalization for each pixel normalized to the change from the first frame The result is stored as real values The Image Window intensity scaling and coloring changes to the DF F preset MetaFluor Analyst 3 2 Version 1 0 93 Menu Commands and Toolbar see Properties dialog box Ratio Runs dialog box for entering numerator and denominator for ratio calculation from the linked Image Windows to the active one The result is stored as real values Calibrate Enables you to run calibration equations on selected ROls Convert to Sets the pixel format elnteger default signed 16 bit 32768 32767 efixed point real 16 bit the place of the
42. t clicking a graph and selecting Copy plot data then pasting into a spreadsheet program Saving into ASCII files is also supported Right click Save plot data The output format is tab delimited ASCII the first column contains the time values in seconds and the remaining columns are the plot y values in the order of ROI numbering There is no header row Plots also can be copied as bitmaps The timing of a measurement is often affected by the limited speed of camera or data storage system so however the exact time of the exposition is recorded the interval between image acquisition can change This makes it difficult to average multiple measurements in a spreadsheet editor in a row by row base For this reason data resampling was implemented in the Image Analyst This can be enabled by right clicking the graph and selecting Resample when Saving or Copying If enabled the plotted data is resampled in time using linear interpolation between the recorded points yielding time points divisible by the given value in seconds at Preferences dialog box Data Saving Resample X Data resaple interval In the Preferences dialog box the default value of the Resample When Saving or Copying Data Popup menu point can be also set This option affects only the copied or saved data not the one appearing in the plots nor the image data Correlation Graph The correlation window compares the pixel values of the two wavelength channels by assigning a point i
43. t the custom colors to modify ROI coloring The color of an existing ROI cannot be changed only the newly created ones are affected MetaFluor Analyst 3 4 Version 1 0 93 Menu Commands and Toolbar Preferences Opens the Preferences dialog box Window Menu Cascade Displays image windows stacked and cascading from the top left to the bottom right of the screen Tile Displays image windows edge to edge Minimize All Minimizes all open image windows to the bottom of the workspace Restore All Restores all minimized images Show Image Info Shows image information of the active image The image information can be edited copied and pasted into ia files Show ROI Info Shows the parameters of the active ROI Help Menu About Opens splash screen with version and copyright information about MetaFluor Analyst Toolbar Tools available on the MetaFluor Analyst toolbar represent the most commonly used menu commands See the menu command description for more information about a specific tool The following tools are available Tool Menu Command E File Open Experiment a Edit Set linkage Edit Duplicate image window Edit Truncate frames N A Drawing of linear ROI N A Drawing of area ROI E A N A ROI selection and movement MetaFluor Analyst 3 5 Version 1 0 93 Menu Commands and Toolbar Tool Menu Command Edit Copy active ROI A Edit Paste ROI ie Edit Delete active ROI lh Calculate Plot du Ca
44. th every precaution to ensure accuracy Molecular Devices Corporation assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Table of Contents MetaFluor Analyst OVOSIVIO Wii di a taa 1 1 Using this GUID voir aida aE ne EA aaea E A a Decidir 1 1 Hardware Requirement iriiria tiiat ha narran 1 1 IE marri Tuia anana A A ETAETA tad A ETAR VAT EEAS SA i eA NAA EREIN 2 1 Image Window Features occccinnccinoninnnoccconccononnnn nan nnn nc rr 2 1 Intensity Scalebar cada ia Ra aE Tiaa 2 1 Play Pause BUON cisco tinte liado cae anita Diada VEIAS ATANN A ARAR KE VES NANETAS ARa 2 1 TaCD Aia td e iaa a aAa A Aveta ita a a SS 2 2 aE S e A E E E A A A AET T E ETN T E T 2 2 Working with the Image WindOW ooononcccnnnoccccnonoccccnononcncnanoncncnnnn cnc cnnnnnnnc cnn nn nncc naar nc cc nnnnnncnnns 2 2 OPENING an IMAGE miii ri ein 2 2 ViSUAQlIZ MON cia lit lie nii 2 3 IMAGE ProcEeSSINO oia cada 2 4 ROTdraWINg2 aia dai Ue noi Me ee 2 4 Data Evaluations id AA ae 2 4 Setting the linkage of Image WiNdOWS ooconccinnccnnnnconnconncccccocccnnnnnn naar can cnn arrancan 2 4 Menu Commands and Toolbar cccccccccceeceeceeeeeeeeeeeneeeceaeeeceaeeseaeeseeeeeseaeeseaaesseeeeseaeeesaeeseeneess 3 1 Menu Commands eina nar ino rei be etree eaten setae obra 3 1 HIM o errr rer n 3 1 Open SxpPenlin Slat anana oia Ae 3 1 A T IE EEEE TET EE A rer cere a reece eee E eee Cr ce
45. tics if the filtered image sequence is for ROI average calculations Loss of low and zero frequency components renders the image zero mean even during local ROI calculations However absolute value calculation applies halos around objects but makes ROI averaging feasible Leave only phase After Fourier transformation the complex amplitudes are set to unitary while the complex phase is left intact The filtering continues normally with the multiplication by the transfer function and reverse Fourier transformation Run Runs the filter on the active Image Window separately on each frame Same as the Filter 2D Fourier Run DFT menu command Sharpening images 2D blind deconvolution Microscope optics as every real optics has low pass optical transfer characteristics This phenomenon causes blurring of the image and can be considered as a limitation of the resolution Images can be sharpened by designing a filter function where the higher spatial frequencies are amplified for compensation of the loss of the high frequency components Very high spatial frequencies gt 50 of half image are carrying mostly noise that is why these frequencies should be suppressed rather than amplified Extraction of mitochondrial fluorescence for calcium imaging 1 Mitochondria are appearing as thin filamentous structures over the bulky cytosolic staining in high resolution fluorescent images of the calcium sensitive classical chelator dye rho
46. ting or deleting a ROI also selects or deletes the corresponding plot if a Graph Window is present deleting the plot deletes also the corresponding ROI Data Evaluation Use the Plot command in the Calculate menu to calculate a graph from the average of ROIs on a selected image Other data evaluation commands included Correlation plot Advanced plot pixelwise deviation and Linescan Window These commands are performed on the active ROI and running any of these commands will automatically refreshed any Image Window content including ROI settings The color of each ROI is represented by the same color on a graph Setting the linkage of Image Windows Opening a multi wavelength MetaFluor experiment in MetaFluor Analyst opens a set of linked Image Windows that are processed simultaneously You can also manually link Image Windows to enable them to be processed simultaneously using the Set Linkage command in the Edit menu You can link separately loaded or imported Image Windows if they have the same size and numbers of frames Image Windows can be duplicated with or without their links using the Duplicate command in the Edit menu There can be multiple sets of linked Image Windows on the Workplace MetaFluor Analyst 2 4 Version 1 0 93 Image Window Set image window linkage Dialog Box Set image windows linkage Current linkage of W2 GROUP32 001 Warning All ROIs will be deleted on affected images Cancel The linkage of an I
47. wn below MetaFluor Analyst 6 9 Version 1 0 93 Advanced Image Processing Denise her ipime onal ROL fi C GH reo feck ground Fill E i Giga beck ground Dogue thin the cunei a fi E Charge cl igual bawaan hanas 0 fn Use OFF A MON SEE 100 A 20S Pb 76 FEE HD TRESS 2S STE 40000 FRA TE HO Ee S071 SE 0000 625971 ATEEN abier Une the fi highest part foe srg Eud iid Progertena ilai ci Signal Background ROI Set the ROI number of details to extract in Signal ROI field and set the ROI number of details to reject in Background ROI field Optimize The routine will optimize the ratio of Signal over Background S N ratio or the difference Signal Background Compute In this field the input frames have to be entered There are two choices Calculate within the current frame Calculates the parameter given at Optimize within the actual frame in the actual Image Window Use this if there are no fluorescence increases in the image sequence Change of signal between frames Calculates the change of parameter given at Optimize between the given two frames Use this for extraction details brightening up upon a stimulus Select Use DF F to compare DF F normalized changes of Signal and Background Method Frequency scan The routine scans spatial frequencies in the range of Frequency range fields use numbers between 0 and 1 with bandpass filters dividing the range into Divide frequency range into num
48. y measurement Subtracts the average of pixel intensities covered by the given ROI calculated for the whole image sequence Image Subtracts the image s entered in the Background Image field Background Image Active if the Image option is set in the Subtract Background at field Enter the background file name here or click the browse button to locate the file Only images with same size can be subtracted If a MetaFluor measurement d inf file is given with more than one channels then images with the matching wavelength will be subtracted If individual image or image series is entered then it will be subtracted from any selected Image Window Since background measurements can contain image sequences the number of frame to subtract can be specified in th frame filed or all loaded frames can be averaged for subtraction Average all frames In all other aspects loading of measurements applies for background loading When using MetaFluor measurements for background subtraction all other Image Windows for background subtraction has to be selected in the list at the bottom of the dialog box MetaFluor Analyst 6 1 Version 1 0 93 Advanced Image Processing Note For elimination of uneven background illumination and CCD cold hot spots it is ideal to record and subtract a background image of a cell free area If there is temporal irregularity in background intensities like external scattered light entering the microscope or phot
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