Marsico Hall Microscopes Facility (MHMF.ORG)

Fiji ASL Estimation

Outline:

HBE Airway cultures are imaged on a Leica Confocal in x-z scanning mode using a 40x 1.25 NA immersion objective or higher

• ASL is marked with 10kD or 70kD dextran conjugated with e.g. Alexa 594 or Alexa 647
• Cultures on T-clear frames, or similar, are placed on a glass bottom dish (#1.5 thickness 20 mm diameter)
• Microscope environmental chamber is set to 37C, 5% CO2, 85% relative humidity
• x-z scanning is done with 1024^2 pixels, minimum zoom is 1.66 giving 0.17 um square pixels
• Optionally counterstaining is done with Calcein Green-AM or Calcein Orange-AM
• Use 40x 1.25NA glycerol immersion objective or better
• Note that Glycerol immersion objective has a coverslip correction collar which should be adjusted for maximum clarity (minimum spherical aberration).  There is a different scale for 37C versus 23C.
• X-Z scans are acquired at multiple positions on the cultures, avoiding edges and the center and stored in a .LIF file
• Frame averaging of 4 was used
• .LIF files  are processed with the Bio-formats-importer-2.0.2.ijm macro
  • Individual .TIF files are created for each position
• ASL are measured along the entire length of the airway captures in the image using "DoList2021.ijm" which calls "PLOTPROFILE12.ijm"

Software setup

• "Bio-formats-importer-2.0.2.ijm" can be located anywhere
• "PLOTPROFILE12.ijm" should be stored in the c:\win64apps\fiji\macros or equivalent
• "DoList2021.ijm" can be located anywhere. Drag into main (FIJI Is Just) ImageJ window . Or add to StartupMacros.ijm - it is setup to execute with a "3" key press (make sure any other macro using the "3" is removed or commented out)
• .LIF file(s) can be located anywhere with read & write privileges

Running:

• 1. Run "Bio-formats-importer-2.0.2.ijm" which prompts for location of .LIF files(s).  Images in the .LIF file(s) are exported as .TIF files will be placed into the same directory.
• 2. DoList2021.ijm and will prompt for the location of the .TIF files (unfortunately the prompt box will not display the actual files - just select the directory)
  • An "asl_fwhm_summary.txt" file is created and stores the resulting average measurement for each .TIF image.  Measurements are in pixel units but the "asl_fwhm_summary.txt" file will list the um/pixel calibration for each .TIF file.
  • Note: Data added to asl_fwhm_summary.txt is cumulative.  So rerunning scans will duplicate data already in the file.
  • Each .TIF image will create a directory with the same name as the .TIF minus the .tif extension
  • This directory will contain a text file with the fit parameters for each vertical position and a plot of all the fits.  Note ASL thickness estimate is the fwhm (full width half maximum) of the fit and is 2.35 times the "d" parameter of the fit.

Notes:

• Please report suggestions, errors or problems to Michael.  Screen captures & samples images can be helpful.
• Line fits of ASL height are based on a Gaussian y=a+(b-a)*exp(x-c)*(x-c)/(2*d*d) where d is the wide factor and gives a fwhm as 2.35 * d

Protocol:

• Install FIJI ( https://fiji.sc )      Note FIJI is a super set of ImageJ - Note in the future FIJI will replace ImageJ

Appendix:

An asl_fwhm_summary.txt file typical contents:

Gaussian fit (units are pixels) - Rejected if any side peaks exists +/-1.5*d - with tolerance 36 using Array.findMaxima(); y=a+(b-a)*exp(x-c)*(x-c)/(2*d*d)
Adjust 'closeness'& 'tolerance' in C:\..\Fiji.app\macro\PLOTPROFILE12.IJM (near line 35)
fwhm is ASL height in pixels
Image_name                         fwhm   avg    sd    count avg.r^2 sd.r^2 x&z-microns
C3-Project001.lif - Image013.tif   37.62  16.01  2.87  1024  0.974   0.022  0.1711 0.1711
C3-Project001.lif - Image014.tif   37.61  16.01  2.49  1024  0.980   0.014  0.1711 0.1711
C3-Project001.lif - Image015.tif   39.08  16.63  2.52  1024  0.968   0.015  0.1711 0.1711
C3-Project001.lif - Image016.tif   39.02  16.60  3.61  1024  0.972   0.018  0.1711 0.1711
C3-Project001.lif - Image017.tif   39.03  16.61  2.87  1022  0.968   0.027  0.1711 0.1711
C3-Project001.lif - Image018.tif   40.81  17.37  3.61  1024  0.965   0.029  0.1711 0.1711
C3-Project001.lif - Image019.tif   38.32  16.31  2.76  1024  0.970   0.024  0.1711 0.1711
C3-Project001.lif - Image020.tif   38.35  16.32  2.91  1024  0.983   0.011  0.1711 0.1711
C3-Project001.lif - Image021.tif   36.20  15.40  1.46  1024  0.983   0.007  0.1711 0.1711
C3-Project001.lif - Image022.tif   35.70  15.19  1.61  1024  0.979   0.008  0.1711 0.1711
C3-Project001.lif - Image023.tif   36.78  15.65  2.49  1024  0.976   0.015  0.1711 0.1711
C3-Project001.lif - Image024.tif   40.07  17.05  3.25  1024  0.971   0.018  0.1711 0.1711

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