FIJI/Image J for Quantification Hands on session
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1 FIJI/Image J for Quantification Hands on session Dr Paul McMillan Biological Optical Microscopy Platform
2 Hands on demonstrations FIJI set up Line Profile Thresholding Area of stain Cell confluence Nuclei counting Cell segmentation Advanced segmentation Intensity over time Euclidean Distance Measurements Kymogram Macro Recording Batch Macro Analysis
3 FIJI set up Edit/Options/Colours Foreground: White Background: Black Selection: Yellow Process/Binary/Options Iterations: 1, Count: 1 Black Background (Tick ) Pad edges when eroding (Tick) EDM output: Overwrite Edit/Options/Memory and threads Set memory 60-70% of memory Plugins/Utilities/Monitor Memory Clear memory as we go Plugins/Utilities/Find commands Control L (PC) or Command L (Mac)
4 Line analysis Open Gams.tif, Gams (green) & Gams (red) Change line tool to segmented line (right click to change) Draw line along filament Restore selection (Control + Shift E) Plot line profile on gams (green) & gams (red) Analyse/plot profile, Control K (PC) or Command K (Mac) Save plot as xls OPTIONAL ITEMS Edit/Selection/Straighten, line width 10 pixels Change line colour (Edit/Options/Colours/Selection) Change width of line (Edit/Options/Line width) Save line as overlay (Image/Overlay/Selection) Control B (PC) or Command B (Mac)
5 Thresholding Open kidney (green).tif Image/adjust/auto threshold Select white objects on black background Show threshold values in log window Select one that s works best Find threshold values in log window Apply selected Threshold Image/Adjust/Threshold Control + Shift T (PC) Command + Shift T (Mac)
6 Area of stain Open kidney (green).tif Images/adjust/threshold Use method & settings identified in auto threshold Analyse/set measurements Area, integrated density, mean gray value, area fraction Limit to threshold Measure Analyse/measure Control M (PC) Command M (Mac) Right click (or Analyse/Set measurements) to change measurement settings
7 Cell confluency Open BPAE (green) Set threshold Image/Adjust/Threshold Control + Shift T (PC) Command + Shift T (Mac) Measure Analyse/Measure Control M (PC) Command M (Mac) Right click (or Analyse/Set measurements) to change measurement settings
8 Nuclei Counting Open BPAE (blue).tif Image/Adjust/Threshold Set lower threshold level = 70 Set higher threshold level = 255 Apply threshold Process Binary/watershed Analyze/analyze particles Size = 100-infinity Circularity = Show = outlines Exclude on edges Tick Display results, Summarise, Exclude on edges
9 Cell segmentation
10 Cell segmentation Open Cell.tif (from Segmentation) & Duplicate Watershed (Save final image as MASK 1.tif) Process/Find Maxima (Noise = 400, exclude on edges, segmented particles) Whole cell stain (Save final image as MASK 2.tif) Threshold duplicated image (min = 388, max 4095) but don t apply yet Process/smooth & apply threshold Cell outlines (Save final image as MASK 3.tif) Process/Image Calculator Mask 1 AND Mask 2 Analyze/Analyze Particles (Size = 250 Inf, exclude on edges, show masks) Invert LUT Process/Binary/Fill Holes Analyse the images Analyze/Set Measurement (Area, Shape, Int Den, Mean, Perimeter, Ferets, Display label. REDIRECT to Cell.tif Analyse Particles (size = 250-infinity, Show = outlines, display, clear, summarize, exclude on edges) Try as above, but also select ROI manager
11 Advanced segmentation Cytoplasmic masks (Cells minus nucleus) Open Nuclei.tif Threshold & create binary (Save as Mask N) Process/Binary/Watershed Process/Image calculator/ Mask 3 SUBSTRACT MASK N Perinuclear mask Open Mask N & Duplicate Process/Binary/Dilate (on Mask N-1.tif) & Process/Binary/Watershed Process/Binary/Erode (on Mask N.tif) Process/Image calculator/mask N-1 SUBTRACT Mask N
12 Intensity over time Open Calcium flux.tif Draw ROI on bottom right cell Analyze/Set Measurement Mean Gray value Display label Image/Stacks/Plot Z axis Profile Repeat on background Analyze/Tools/ROI manager Add multiple ROIs Show all Select More, Multi Measure, Measure all 50 slides, one row per slice
13 Euclidean distance measurement Open Nuclei.tif, apply threshold & create binary image Process/Binary/Options Configure EDM to 16 bit Edit/Invert Process/Binary/Distance Map Apply 16 colours LUT Analyze/Set Measurements Mean gray value, limit to threshold, display label Threshold to select background (use 1-29 threshold) Analyze/Measure Average distance = Pixels (read out is always in pixels) Calculate distance in microns on calibrated images Image/Properties, Control + Shift P (PC), Command + Shift P (Mac) Covert using pixel dimensions
14 Kymogram Open tracking.tif from Kymogram Image/Colour/Split channels Draw line form ROI tools (Hold shift to keep it straight) Select channel two and restore selection (Ctrl + Shift E) Select channel 1, Analyze/Multi Kymporaph/Multi Kymograph Set line width to 29 (must be an odd number) Image/Rename, rename channel 1 Repeat Kymograph (as above) for Channel 2 Merge Channels (Channel 1 = green, Channel 2 = Red)
15 Macro Recording Macro Recording: Plugins/Macros/Record Repeat a simple analysis used earlier (e.g. Nuclei Counting) Remove any mistakes or unwanted steps (e.g. Select Window for image opening) Rename macro & press Create Close all open images and open an image to test (e.g. BPAE (blue.tif for the nuclei counting) Hit Run and it should perform the steps automatically File/Save (or Save as) to define where to save it to Always save with.ijm file extension for FIJI to read the macro Macro Running: Plugins/Macros/Run to use the Macro again
16 Batch Macro Analysis Process/Batch/Macro Copy macro details into the dialogue box Define input folder (where the images to be analysed are) Define output folder (if you are saving output images) File name contains Filter images analysed based on file name (e.g. only images with ch01) Use test to make sure the code works Press process for all images to be analysed
17 Hands on demonstrations FIJI set up Line Profile Thresholding Area of stain Cell confluence Nuclei counting Cell segmentation Advanced segmentation Intensity over time Euclidean Distance Measurements Kymogram Macro recording Batch Macro analysis
18 Copyright The University of Melbourne 2011
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Revision 1.2 March 2017 Contents 1... 1 1.1 Calibration Workflow... 2 1.2 Create Master Calibration Frames... 3 1.2.1 Create Master Dark & Bias... 3 1.2.2 Create Master Flat... 5 1.3 Calibration... 8
2.0 4 Easy Ways to Delete Background to Transparent with GIMP. 2.1 Using GIMP to Delete Background to Transparent
1.0 Introduction As JPG files don't support transparency, when you open a JPG image in GIMP with the purpose of making the background transparent. The first thing you must to do is Add Alpha Channel. It