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Image Intensity Processing

8,842 bytes added, 11:14, 22 March 2017
Name menu sidebars as such
= 4. Intensity vs Time analysis === 4.1 Brightness and Contrast ==To improve the visualisation of the image, the displayed brightness and contrast can be adjusted with “''Image/Adjust/Brightness/Contrast...''” (hotkey: Shift+C).__FORCETOC__ {{CookbookMenu}}
The “''Auto”'' applies an intelligent contrast stretch to the ''way in which the image is displayed''. The brightness == Brightness and contrast is adjusted based on an analysis of the image's (or selection’s) histogram. If pressed repeatedly, it allows a progressively increasing percentage of pixels to become saturated.Contrast ==
''Reset'' changes [[File:brightness_contrast_pic.png|thumb|right]]Brightness is the “maximum” and “minimum” back visual perception of reflected light. Increased brightness refers to 0 and 255 for 8-bit images and back to the maximum and minimum of the image’s histogram for 16-bit imagesan image's increased luminance.
If ''Auto'' does not produce a desirable result, select a region Contrast is the separation of the cell plus some background with a region-lightest and darkest parts of-interest (ROI) tool, then hit the “''Auto''” button againan image. It An increase in contrast will then do a stretch based on the intensities within the ROIdarken shadows and lighten highlights. Increasing contrast is generally used to make objects in an image more distinguishable.
'''Pressing Adjust the ''Apply'' button permanently changes the ''actual ''grey values brightness and contrast with {{bc | Image | Adjust | Brightness/Contrast...}} to make visualization of the imageeasier. Don’t press this button during while analysing image intensity!'''
If you prefer the image to be displayed as “black on white” rather than “white on black”, the image display can be “inverted” by Press the command “''Image/Lookup Tables/Invert LUTAuto''”. The command “button to apply an intelligent contrast stretch to the ''Edit/Invertthe image display''” inverts . Brightness and contrast is adjusted by taking into account the pixel image''values'' not just s histogram. If pressed repeatedly, the way button increases the image is displayedpercentage of saturated pixels.
== 4.2 Getting intensity values from single ROI ==If the movie has been opened as a stack, the ROI selected can be analysed with the command: “The ''Image/Stacks/Plot Z Axis ProfileReset''”. This generates a single column of numbers button makes the "maximum" 0 and the "minimum" 255 in 8-bit images and the "maximum" and "minimum" equal to the smallest and largest pixel values in the image’s histogram for 16- one slice intensity per rowbit images.
The top 6 rows of If the ''Auto'' button does not produce a desirable result, use the column are details region-of the -interest (ROI. This is useful ) tool to ensure select part of the same ROI isn’t analysed twice cell and allow relocation of any interesting ROIs. The details are comprised of area, x-coordinate, y-coordinatesome background, AR, roundness, and solidity of then hit the ROI. If the ROI is a polyline/freehand ROI rather than a square/oval, the details are given as if the ROI was an oval/square''Auto'' button again. The (oval) ROI can stretch will then be restored by entering based on the details prompted by intensities of the “''Edit/Selection/Restore Selection''” (hotkey: “Ctrl + Shift + E”) commandROI.
The results are displayed in a plot-window with Pressing the ROI details in the plot window title. The plot contains the buttons ''List, Save, Copy. ''The 'Apply'Copy'' button copies the data to the clipboard where it can be pasted into a waiting Excel sheet. The settings for permanently changes the copy button can be found under “''Edit/Options/Plot profile optionsactual''grey values of the image. Recommended settings are: ''Do If just analyzing image intensity do not save x-values ''(prevents slice number data being pasted into Excel) and ''Autoclose'' (prevents you having to close analysed plot each timepress this button.
== 4If you prefer the image to be displayed as "black on white" rather than "white on black", then use the "inverted" command: {{bc | Image | Lookup Tables | Invert LUT}}.3 Dynamic intensity vs Time analysis ==The plugin “''Plugins/Stacks – T-functions/Intensity v Time Monitor ''(this is command {{bc | Edit | Invert}} inverts the pixel ''Dynamic ZProfile fromvalues themselves''Kevin (Gali) Baler (gliblr at yahoo.com) and Wayne Rasband simply renamed) ” will update the plot window each time the ROI selection is moved. This may help locate initiation sites of calcium signals for examplepermanently.
==  4Getting intensity values from single ROI ==If working with a stack, the ROI selected can be analyzed with the command: {{bc | Image | Stacks | Plot Z Axis Profile}}.4 Getting This generates a single column of numbers - one slice intensity per row. The top 6 rows of the column are details of the ROI. This makes sure the same ROI is not analyzed twice and allows you to save any interesting ROIs. The details are comprised of area, x-coordinate, y-coordinate, AR, roundness, and solidity of the ROI. If the ROI is a polyline>freehand ROI rather than a square>oval, it acts as if the ROI is an oval>square. The (oval) ROI can be restored by entering the details prompted by the {{bc | Edit | Selection | Restore Selection}} (hotkey: {{key|Ctrl}}+{{key|Shift}}+{{key|E}}) command. The results are displayed in a plot-window with the ROI details in the plot window title. The plot contains the buttons ''List, Save, Copy.'' The ''Copy'' button puts the data in the clipboard so it can be pasted into an Excel sheet. The settings for the copy button can be found under {{bc | Edit | Options | Profile Plot Options}}. Recommended settings include: ''Do not save x-values ''(prevents slice number data being pasted into Excel) and ''Autoclose'' so that you don't have to close the analyzed plot each time. == Dynamic intensity vs Time analysis == The plugin ''Plot Z Axis Profile'' (this is the ''Z Profiler'' from Kevin (Gali) Baler (gliblr at yahoo.com) and {{Person|Rasband}} simply renamed) will monitor the intensity of a moving ROI using a particle tracking tool. This tool can be either manual or automatic. Use the {{bc | Image | Stacks | Plot Z Axis Profile}} command. == Getting intensity values from multiple ROIs ==Multiple You can analyze multiple ROIs can be analysed at once using with Bob Dougherty’s ''Multi Measure''plugin. There is a The native “ROI manager” "ROI manager" function which does a similar job except doesn't generate the results generated are not in sorted in to columns. Check Bob’s website for updates: [http://www.optinav.com/imagej.html http://www.optinav.com/imagejBob’s website] for updates.html]
The Multi Measure plugin that comes with the installation is v3.2.
1. # Open tconfocal-series.Remove and remove the background (See Background correction)# Generate a reference stack for the addition of ROIs. Use the {{bc | Image | Stacks | Z-project}} function and select the ''Average''.# Rename this image something memorable.# Open the ''ROI Manager'' plugin ({{bc | Analyze | Tools | Roi Manager}} or toolbar icon).# Select ROIs and "''Add''" to the ROI manager. Click the "''Show All''" button to help avoid analyzing the same cell twice.# After selecting ROIs to be analyzed in the reference image, you can draw them to the reference image by clicking the "''More>>''" button and selecting ''Draw''. Save the reference image to the experiment’s data folder and then click on the stack to be analyzed.# Click the "''More>>''" button in the ROI manager and select the ''Multi Measure'' button to measure all the ROIs. Click ''Ok''. This will put values from each slice in to a single row with multiple columns per slice. Clicking on "''Measure all 50 slices''" will put all values from all slices and each ROI in a single column.# Go to the ''Results'' window and select the menu item {{bc | Edit | Select All...}}. Then ''Edit/Copy''.# Go to Excel and paste in the data. Check that everything was pasted in correctly [[File: roi_select_all.jpg]] 10. To copy ROI coordinates into the Excel spreadsheet, there needs to be an empty row above the intensity data. Use the Multi Measure dialog and click the ''Copy list'' button. 14. In Excel, click the empty cell above the first data column and paste in the ROI coordinates.Save the ROIs with the Multi Measure button ''Save''. Put them in the experimental data folder. The ROIs can be opened later either individually with the button ''Open'' or all at once with the button ''Open All''. Oval and rectangular ROIs can be restored individually from x, y, l, h values with the {{bc | Plugins | ROI | Specify ROI...}} command. == Ratio Analysis == [[File:intensity_ratio_analysis.jpg|right]]Ratiometric imaging compares the recordings of two different signals to see if there are any similarities between them. It is done by dividing one channel by another channel to produce a third ratiometric channel. This technique is useful because it corrects for dye leakage, unequal dye loading, and photo-bleaching. An example application would be measuring intracellular ion, pH, and voltage dynamics in real time.  Background subtraction is needed before analysis of dual-channel ratio images. See also the [[#Background_correction|background correction]] section. The ''Ratio_Profiler'' plugin will perform ratiometric analysis of a single ROI on a dual-channel interleaved stack. The odd-slices are channel 1 images and the even slices are channel 2 images. If your two channels are opened as separate stacks, such as Zeiss, the two channels can be interleaved (mixed together by alternating between them) with the menu command {{bc | Plugins | Stacks - Shuffling | Stack Interleaver}}. The plugin will generate a green-plot of the ratio values. Ch1÷Ch2 is the default and you can get Ch2÷Ch1 if the plugin is run with the {{key|Alt}} key down. It will also generate a second plot of the intensities of the individual channels, Ch1 and Ch2, as well as a results table. The first row of the results table contains values for the x, y, width and height of the ROI. From the second row downward, the first column is the time (slice number), the second column is the Ch1 mean intensity, and the third channel is the Ch2 mean intensity and the ratio value. The stack must have its frame interval calibrated in order for the "Time" value to be in seconds. Otherwise, it is "Slices". The frame interval can be set for the stack via the menu command ''{{bc | Image | Properties}}. This table can be copied to the clipboard and pasted elsewhere with the "{{bc | Edit | Copy All}}" menu command. '''''Ratio Analysis Using ROI manager''''' 1.Subtract the background from the image. 2. Open ''ROI manager'' ({{bc | Analyze | Tools | ROI manager...}}) and click the "Show All" button. 3. Select the cells to be analyzed and add them to the ROI manager ("Add" button or keyboard {{key|T}} key). 4. Run the plugin. The results window contains the mean of ch1 and ch2 and their ratio. Each row is a timepoint (slice). The first row contains the ROI details. To generate a reference image: # Flatten the stack with the menu command ({{bc | Image | Stacks | Z-project}} with "Projection type: Maximum"),# Adjust the brightness and contrast if necessary.# Select the new image and click the "More" button in the ROI manager. After that select "Label". == Obtaining timestamp data ==  === Zeiss LSM === The [[LSM Toolbox]] is a project aiming at the integration of common useful functions around the Zeiss LSM file format, that should enhance usability of confocal LSM files kept in their native format, thus preserving all available metadata. In Fiji, corresponding commands are: "{{bc | File | Import | Show LSMToolbox}}" which displays the toolbox, from which all commands can be called and "{{bc | Help | About Plugins | LSMToolbox...}}" which displays information about the plugin. === Biorad === This reading can be found by using the menu command {{bc | Image | Show Info...}}. Scroll down to get the time each slice was acquired. Select this time, copy it into Excel, and find the time number obtained by using the Excel menu command {{bc | Edit | Replace}}. This will leave only the time data. The "elapsed" time can then be calculated by subtracting row 1 from all subsequent rows. == Pseudo-linescan ==
2Linescanning involves acquiring a single line, one pixel in width, from a common confocal microscope instead of a standard 2D image. It’s worthwhile generating This is usually a reference stack to add the ROIs faster way totake an image. Use All the “''Image/Stacks/Zsingle pixel-project''”function (hotkey: Shift+Z)[http://fiji.sc/mbf/intensity_vs_time_ana.htm#_ftn1 §]. Select wide images are then stacked to recreate the “Average” option2D image.
A pseudo-linescan generation of a 3. Rename this image “Ref -D (''expt namex, y, t''” or something memorable) image. It is useful for displaying 3-D data in 2 dimensions.
4A line of interest is drawn followed by the command: {{bc | Image | Stacks | Reslice}} or with the keyboard button {{key|/}}. It will ask you for the line width that you wish to be averaged. It will generate a pseudo-linescan "stack" with each slice representing the pseudo-linescan of a single-pixel wide line along the line of interest. Average the pseudo-linescan "stack" by selecting {{bc | Image | Stacks | Z-Project... Open “}} and use the ''ROI ManagerAverage''” plugin (“''Analyze>Tools>Roi Manager'' ” or toolbar icon )command. A poly-line can be utilized, but this will only generate a single pixel slice.
5. Select ROIs and "Fiji's default settings assume that stacks are ''z''Add-series rather than ''" to ROI manager (hotkey: t)''-series. Clicking "This means that many functions related to the third-dimension of an image stack are referred to with a ''Show Allz-''" helps avoid analysing the same cell twice. Just keep this in mind.
6. Once finished selecting ROIs to be analysed in the reference image, you can draw them to the reference image by clicking the "''More>>''" button and selecting ''Draw''. Save the reference image to the experiment’s data folder and then select the stack to be analysed by clicking on it.== FRAP (Fluorescence Recovery After Photobleaching) Analysis ==
7. Click "''More>>''" button in The FRAP profiler plugin will analyze the intensity of a bleached ROI manager over time and select “''Multi''” button to measure all normalize it against the intensity of the ROIs. Click “Yes” on “''Process stack?''” dialogwhole cell. This After that it will put values from each slice find the minimum intensity in to a single row (multiple columns per slice). Clicking on "''Measure''" will put all values from all slices the bleached ROI and each ROI fit the recovery with this point in a  single columnmind.
8. Go to “Results” window. Select menu item “''Edit/Select All…''”. Then “''Edit/Copy All''”.To use:
9# Open the ROI manager.# Draw around the bleached ROI and add it to the ROI manager. Go # Draw around the whole cell and add that to Excel the ROI manager. The normalization corrects for the bleaching that occurs during image acquisition and assumes the whole cell is in the field of view. '''The plugin assumes the larger of the two ROIs in the ROI manager is the whole cell ROI and paste datathat the smaller ROI is the bleached part. With large data sets this can take some '''# Run the FRAP profiler plugin.# The plugin will return the intensity vs time so check you’re pasting new data in plot, the normalized intensity vs time plot of the bleached area, and not the last dataset copied from Multi Measurecurve fit.
[[Image:intens4.jpg]]10. To copy ROI co== Non-ordinates in to the Excel spreadsheet, ensure there is an empty row above the intensity data. Got to Multi Measure dialog and click “Copy list” button.linear contrast stretching ==
14. Go to Excel, select the empty cell above the first data column and then paste the ROI co-ordinates.=== Equalization ===
The ROIs {| style="border-spacing:0;"| style="border:none;padding:0in;"| You can be stored have more control over brightness and re-opened later using contrast adjustments with the Multi Measure dialog button “''Save''”{{bc | Process | Enhance contrast}} menu command. Save them in With a stack, it analyzes the experimental data folder. The ROIs can be opened later either individually (Multi Measure dialog button “''Open''”) or all at once (Multi Measure dialog button “''Open All''”) which opens all each slice’s histogram to make the ROIs in a folderadjustment.
Oval and rectangular ROIs can also be restored individually from x, y, l, h values using “The ''Plugins/ROI/SpecifyROI…''” (hotkey: Shift + G)Equalize contrast''command applies a non-linear stretch of the histogram based on the square root of its intensity.''
== 4.5. Ratio Analysi|}[[ImageFile:intens1equalize_histrogram.jpg]]s ==Analysis of dual-channel ratio images requires careful background subtraction prior to analysis. See section 
'''7.5 Background correction. '''The''"Plugins/Stacks - T-functions/Ratio_Profiler"'' plugin will perform ratiometric analysis of a single ROI on a dual-channel interleaved stack, i.e. the odd-slices are channel 1 images, the even slices channel 2. Perkin Elmer ''Ultraview'' and Leica SP dual channel experiments can be directly imported as an interleaved stack using the menu command "''File/Import/Image Sequence''". If your two channels are opened as separate stacks, e.g. Zeiss, the two channels can be interleaved with the menu command "''Plugins/Stacks - Shuffling/Stack Interleaver''".=== Gamma ===
The plugin will generate <nowiki>Gamma performs a greennon-plot of the ratio values linear histogram adjustment. Faint objects become more intense while bright objects do not (Ch1÷Ch2 by default; Ch2÷Ch1 if the plugin is run with the Altgamma <1). Also, medium-key downintensity objects become fainter while bright objects do not (gamma > 1); a second plot . The intensity of each pixel is "raised to the intensities power" of the individual channels (Ch1 gamma value and Ch2); then scaled to 8-bits or the min and a results tablemax of 16-bit images.</nowiki>
The first row of the results table is the x, y, width and height of the ROI.For 8 bit images; New intensity = 255 × ''<nowiki>[(old intensity÷255)</nowiki> gamma'']
From Gamma can be adjusted via the second row downward, {{bc | Process | Math | Gamma}} command. It will allow you to adjust the first column is gamma with the time&nbsp;or slice number; the second column scroll bar. Click on ''Ok'' when you are finished. You can use the Ch1 mean intensity, Ch2 mean intensity and Scroll-bar to determine the ratio desired gamma valueon one slice of your stack. If the stack has its frame interval calibrated, the "Time" value will be in seconds otherwise it There is "Slices". The frame interval can be set for also an option to preview the stack via the menu command&nbsp;''"Image/Properties"''&nbsp;dialogresults.
This table can be copied to the clipboard for pasting to another program by using the "''Edit/Copy All''" menu command[[File:gamma_pic.jpg]]
&nbsp;== Filtering ==
'''''4See the [http://homepages.5inf.ed.ac.uk/rbf/HIPR2/filtops.htm online reference] for an explanation of digital filters and how they work.1&nbsp;Ratio Analysis Using ROI manager'''''
1Filters can be found using the menu command {{bc | Process | Filters...&nbsp;BG subtract&nbsp;the image}}.
2. Open&nbsp;''ROI managerMean filter''&nbsp;(''Analyze/Tools/ROI manager...'') : the pixel is replaced with the average of itself and click its neighbors within the "Show All" buttonspecified radius. The menu item {{bc | Process | Smooth}} is a 3×3 mean filter.
3. Select ''Gaussian filter'': This is similar to a smoothing filter but instead replaces the cells pixel value with a value proportional to be analysed and add to the ROI manager ("Add" button or keyboard 't' key)a normal distribution of its neighbors.
4''Median filter'': The pixel value is replaced with the median of itself and its adjacent neighbors. Run the plugin "This removes noise and ''Plugins/Stacks - T-functions/Ratio ROI Managerpreserves boundaries''"better than simple average filtering. The menu item {{bc | Process | Noise | Despeckle}} is a 3×3 median filter.
"Convolve filter": This allows two arrays of numbers to be multiplied together. The results window contains arrays can be different sizes but must be of the mean&nbsp; of ch1 and ch2 and their ratiosame dimension. Each row In image analysis this process is a timepoint (slice). The first row contains generally used to produce an output image where the ROI detailspixel values are linear combinations of certain input values.
"Minimum": This filter, also known as an erosion filter, is a morphological filter that considers the neighborhood around each pixel and, from this list of neighbors, determines the minimum value. Each pixel in the image is then replaced with the resulting value generated by each neighborhood. "Maximum": This filter, also known as a dilation filter, is a morphological filter that considers the neighborhood around each pixel and, from this list of neighbors, determines the maximum value. Each pixel in the image is then replaced with the resulting value generated by each neighborhood. ''Kalman filter'': This filter, also known as the Linear Quadratic Estimation, recursively operates on noisy inputs to compute a statistically optimal estimate of the underlying system state. == Background correction == Background correction can be done in multiple ways. A simple method is to use the {{bc | Image | Lookup Tables | HiLo}} LUT to display zero values as blue and white values (pixel value 255) as red. With a background that is relatively even across the image, remove it with the ''Brightness/Contrast'' command by slowly raising the ''Minimum'' value until most of the background is displayed blue. Press the ''Apply ''button to make a permanent change. === Rolling-Ball background correction === To generate fix an uneven background use the menu command {{bc | Process | Subtract background}}. This will use a ''rolling ball'' algorithm on the uneven background. The radius should be set to at least the size of the largest object that is ''not'' part of the background. It can also be used to remove background from gels where the background is white. Running the command several times may produce better results. The user can choose whether or not to have a light background, create a background with no subtraction, have a reference imagesliding paraboloid, disable smoothing, or preview the results. The default value for the rolling ball radius is 50 pixels.  {| style="border-spacing:0;"| style="border:none;padding:0.0194in;"| ''RAW''| style="border:none;padding:0.0194in;"| | style="border:none;padding:0.0194in;"| {{bc | Process | Subtract Background...}}|-| style="border:none;padding:0.0194in;"| [[File:raw_rolling_ball_back_corr.jpg]]| style="border:none;padding:0.0194in;"| [[File:rolling_ball_back_corr.jpg]]| style="border:none;padding:0.0194in;"| [[File:processed_rolling_ball_back_corr.jpg]] |} Once the background has been evened, final adjustments can be made with the ''Brightness/Contrast'' control.
1{| style="border-spacing:0;"| style="border:none;padding:0. flatten the stack (''Image/Stacks/Zproject0194in;" | [[File:bright_contr_roll_ball.jpg]]| style="Projection typeborder: Maximimumnone;padding:0.0194in;"''),| [[File:histogram_roll_ball.jpg]]| style="border:none;padding:0.0194in;"| [[File:bright_contr_control_roll_ball.jpg]]|}
2. Adjust the&nbsp;brightness and contrast&nbsp;if required.=== ROI background correction ===
3The rolling-ball algorithm takes a lot of time. Ensure To speed up the new process with an image is selected that has a more even background, select a region of interest from the background and subtract the mean value of this area for each slice from each slice. Use the selection tools to select an area of background and click run the "More button" in menu command {{bc | Process | Subtract Background}}. This macro will subtract the mean of the ROI manager then select "Label"from the image plus an additional value equal to the standard deviation of the ROI multiplied by the scaling factor you enter. The default for this is 3.
&nbsp;This macro, because it also works with stacks, can be used on time-courses with varying backgrounds.
&nbsp{| style="border-spacing:0;"| style="border:none;padding:0in;"| <center>Before correction</center>| style="border:none;padding:0in;"| <center>Background intensity over time</center>| style="border:none;padding:0in;"| <center>After ''ROI_BG_Correction''</center>|-| style="border:none;padding:0in;"| [[File:roi_back_corr_before.gif]]| style="border:none;padding:0in;"| [[File:roi_back_corr_during.gif]]| style="border:none;padding:0in;"| [[File:roi_back_corr_after.gif]]|}
== 4.6 Obtaining timestamp data ===== 4.6.1 Zeiss LSM =Flat-field correction ==Once imported via the Zeiss LSM panel, the timestamp data can be extracted with the panel’s ‘Apply t-stamp’ button. This will then ask you if you want the timestamp to be added to the image, or displayed in a text file for saving/pasting to excel etc.
=== 4.6.2 Noran Proper correction ===In most instances the x-axis data, i.e. time of each frame, can be calculated from acquisition rates and frame number (e.g. frame 301 acquired with the acquisition rate set to 1 frame per 0.5 seconds was acquired at 25 minutes). However, the acquisition rate is non-linear, in the above experiment frame 301 was actually acquired at 25 minutes 12 seconds. Each frame has stored with it a “timestamp”, the precise time (in nanoseconds!) that it was acquired. This information can be extracted from an opened movie.
The movie must be opened as Use this technique on brightfield images. You can correct uneven illumination or dirt/dust on lenses by acquiring a stack and "flat-field" reference image ''with the timestamps can be extracted with same intensity illumination as the command: “experiment''Import/Noran timestamp (msec)''”&nbsp;&nbsp;while the Noran Movie is open and selected.&nbsp;The timestamp data appears in flat field image should be as close as possible to a field of view of the “''Results''” windowcover slip without any cells/debris. To copy dataThis is often not possible with the experimental cover slip, click so a fresh cover slip may be used with approximately the right mouse button on same amount of buffer as the windows, select “''Select All''”, then right-click again and select&nbsp;''Copy''. The timestamp data (accompanied by the movie filename) can then be pasted into Excelexperiment.
The Noran SGI plugins are not bundled with the ImageJ package. To receive them, please contact{| style="border-spacing:0;"| colspan="3" | [[mailtoFile:tonyc@uhnresearchflat_field_correction.ca tonyc@uhnresearch.cagif]]|-|| <nowikicenter>&nbsp;or their author, Greg Joss '''RAW'''</center>|| <center>'''Flat-field'''</center>|| <gjoss@bio.mq.edu.aucenter>, Dept of Biology, Macquarie University, Sydney, Australia.'''Processed'''</nowikicenter>|}
=== 4# Open both the experimental image and the flat-field image.6.3 Biorad ===This can be accessed via # Click the menu command “''Image/Show Info…Select all''”. Scroll down button on the flat-field image and it should give measure the time at which each slice was acquiredaverage intensity. This can be selectedvalue, the k1 value, copied will appear in to Excel and the time number obtained by searching and replacing (Excel menu command “results window.# Use the ''Edit/ReplaceImage Calculator plus''plugin ({{bc | Analyze | Tools | Calculator plus}}) .# i1 = experimental image; i2 = flat-field image; k1 = mean flat-field intensity; k2 = 0. Select the text, leaving only the time data. The “elapsed” time can then be calculated by subtracting row 1 from all subsequent rows"''Divide"'' operation.
== 4.7 Pseudo-linescan ==“Linescanning” is a mode of acquisition common to many confocal microscopes where a single pixel wide line is acquired over a period of time instead of This can also be done using the norma1 2-D, x-y image. Usually this allows faster acquisition. The single pixel wide images over {{bc | Process | Image Calculator}}function with the time course are stacked from left to right to generate a 2-D image (i,e,&nbsp;''x''32-bit Result''t'')[[Image:intens5option checked.jpg]]Then adjust the brightness and contrast and convert the image to 8-bit.
A “pseudo-linescan” is the generation of a linescan-type x-t plot from a 3-D (''x, y, t'')&nbsp;timecourse and can be useful in displaying 3-D data (x, y, t) in 2 dimensions[[File:calculator_plus_flat_field.jpg]]
A line of interest must be drawn followed by the command: “''Image/Stacks/Reslice”''&nbsp;or keyboard “/”. It will prompt for the line width. Enter the width of line you wish to be averaged. A pseudo=== Pseudo-linescan “stack” will be generated, each slice representing the pseudo-linescan of a single-pixel wide line along the line of interest. To average the pseudo-linescan “stack”, select “''Image/Stack/Z-Project…''” and select the&nbsp;''Average''&nbsp;command. A polyline can be used although this will only allow a single pixel slice to be made.correction ===
This example shows the elementary calcium events preceding a calcium wave. HeLa cell loaded with the calcium{| style="border-sensitive fluorophore, Fluo-3 and imaged whilst responding to application of histamine.&nbspspacing:0;'''A'''. Frame taken from time-course at the peak of the calcium-release response. ('''B''') The line of pixels along X-Y was taken and stacked side by side from right-to left to generate a "pseudo-line scan| colspan="2" style="border:none;padding:0in;"| [[File:pseudoCorrectionImage. This allows visualisation of the progression of the wave from its initiation site.gif]]
Sometimes it is not possible to obtain a flat-field reference image. It is still possible to correct for illumination intensity, though not small defects like dust, by making a "pseudo-flat field" image by performing a large-kernel filter on the image to be corrected. For those working with DIC images, this is particularly useful because they generally have an intrinsic, and distracting, gradient in illumination.
&nbsp;ImageJ assumes stacks to This can be&nbsp;''z''-series rather than&nbsp;''t''-series so many functions related to accomplished simply by subtracting the thirdGaussian-dimension blurred image version of an the image stack are called “''z-”''&nbsp;something – e.g. “z-axis profile” is intensity over time plot.
==   ==== 4.8 FRAP Analysis ==The FRAP profiler plugin will analyse the This can also be used with stacks for brightfield time-courses that vary in intensity of a beached ROI over with time and normalise this against the intensity of the whole cell. It will then find the minimum intensity in the bleach ROI and fit the recovery&nbsp; from Doing this pointwith stacks can be time consuming.
To use|-| style="border:none;padding:0in;"| [[File:pseudoCorrRawCorrected.gif]]| style="border:none;padding:0in;"| The first RAW image (top) is pseudo-flat field corrected. Here the pseudo-flat field corrects for the uneven illumination, but does not correct for the dust specks. Look at this compared to the result of a proper flat-field correction above.
1. Open the&nbsp;ROI manager.|}
2. Draw around the bleached ROI and Add to the ROI manager.=== FFT background correction ===
3. Draw around the whole cell and add to ROI manager. The normalisation corrects You can correct for the bleaching dues to image acquisition uneven illumination and assumes the whole cell is horizontal "scan lines" in transmitted light images acquired using confocal microscopes by using the field of viewnative FFT bandpass function ({{bc | Process | FFT | Bandpass Filter...).}}
'''You can experiment with the settings to optimize the filtering and also choose to filter structures down to a certain number of pixels. The default value is 40 pixels. You can filter small structures up to a certain value. The plugin assumes default value is 3 pixels. The user can choose from a drop down menu whether to suppress stripes with None, Horizontal, or Vertical. The tolerance of direction can be chosen. The default is 5%. Finally, the larger user can choose whether to allow autoscale after filtering, saturation of the last two ROIs in image when autoscaling, whether or not to display the ROI manager is the whole cell ROI filter, and the smaller the bleached ROIwhether or not to process an entire stack.'''
4[[File:newFftBandFilter. Run the FRAP profiler plugin (''Plugins>Stacks - T-functions>FRAP_Profiler'')jpg]]
5. The plugin will return the intensity vs time plot; the normalised intensity vs time plot of the bleached area and the curve fit.== Masking unwanted regions ==
The normalisation corrects for the bleaching dues to image acquisition and assumes the whole cell is in the field of view.=== Simple masking ===
'''normalised intensity at time t = It&nbsp;<nowiki>= (Ib</nowiki>t&nbsp;÷ Ibmax)&nbsp; ÷ (Ict&nbsp;÷ Icmax)'''Use one of the ROI tools to draw around the area of interest and then select: {{bc | Edit | Clear outside}}. This will change the area outside the selected region to the background value.
'''Ibt<nowiki>= intensity of bleached ROI at time t.</nowiki>'''== Complex masking ===
More sophisticated masking can be done by ''thresholding'Ict<nowiki>= intensity of whole cell at time t' the image and subtracting the new binary image from the original image.</nowiki>'''
# Duplicate the image, or, if it’s a stack, generate an ''average projection'' of a few frames.
# Threshold this image with the menu command {{bc | Image | Adjust | Threshold}}.
# Hit the Auto button and adjust the sliders until all the cells are highlighted red.
# Click ''Apply''. Check the following box: ''black foreground, white background''. You should now have a white and black image with your cells black and background white. If you have white cells and black background, invert the image with {{bc | Edit | Invert}}.
# This can be smoothed with the command {{bc | Process | Smooth}} and the black area enlarged slightly with {{bc | Process | Binary | Dilate}} to give a better mask.
# Using the regular Image calculator {{bc | Process | Image calculator}} subtract this black and white "mask" image from your original image or stack.
[[Category:Cookbook]]
[[Category:Tutorials]]
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