ASC Point Spread Function

The point spread function (PSF) represents how an image of a point appears in a microscope. Measuring this PSF can reveal important informations:

• angle of illumination axis
• damage/performance of objective

Having a system with a proper PSF is gets especially important for high resolution and deconvolution work. Analyzing the PSF of an objective can be semi-automated with the "PSF macro" by Laurent Gelman (FMI Basel (Switzerland)).

requirements

• green fluorescent bead sample, preferably sub-resolution (170 nm)

acquisition

• use high resolution objectives (= the ones that people use for high resolution work)
• set up system for z-stack fluorescence
  • blue excitation, green detection (i.e. EX 488 nm, EM 500-550 nm)
  • pinhole at 1 airy unit
  • pixel size of 100 nm
  • Z-step size of 200 nm
  • 100 planes, focal plane of the bead in the middle
  • no oversaturated pixels
• scan field should be located in the center of the field of view (no panning, put the bead roughly in center by eye and stage)
• size of the scan field should not be smaller than 20x20 µm, with the bead in the center (simplifies the analysis)

prepare PSF macro

• download the PSF macro zip file and unzip it
• include the LUT:
  • Windows: locate the Fiji.app folder (i.e. C:\Program Files\Fiji.app), create a folder luts inside (if it's not there yet), place the LUTforPSFs.lut there
  • Mac: locate the Fiji.app folder (i.e. Applications\Fiji), right click on it > Show package content, create a folder luts (if it's not there yet), place the LUTforPSFs.lut there
• install the macro:
  • open Fiji
  • Plugins > Macros > Install...
  • the macro appears in the Macros menu
  • procedure has to be repeated for every new Fiji session

analysis

• load the bead stack into Fiji
  • make sure the metadata is still there (i.e. that the image is scaled)
• check the pixel size: Image > Show Info > Voxel size X
• if necessary, crop image to a single bead (but also consider the next point)
• the macro attempts to crop a 15x15 µm area with the bead in center; if the image dimensions are smaller, extend the area with black: Fiji > Image > Adjust > Canvas Size...
• if necessary (multi-channel data), split channels (Fiji > Image > Color > Split Channels)
• zoom into the image, in a way that you can hit the center of the bead w/o problems
• start the macro: Plugins > Macros > gelman-psf-macro-v4
• enter the microscope information and click OK
• right-click into the center of the bead
  • that's the tricky step: the analysis will be wrong if you don't hit the center
  • depending on the system configuration you might need to first right-click into the center, and then left-click on the image window to get the process startet
• wait for the analysis to be done, result will be a stack of 2 images (see examples below)

PSF macro overview: automatic macro actions / user actions

by Laurent Gelman

• A. Selects the plane with the highest pixel intensity, adjusts display settings, opens the information dialog box.
• 1. Enter information in the dialog window which popped up.
• 2. Zoom in the image to clearly localize the center of the bead (you can also navigate between planes if needed).
• 3. Right clicks with the mouse on the center of the bead.
• B. Crops the image to get 15mmx15mm area centered over the pixel clicked by the user.
• C. Makes projections in X and Y of the stack
• D. Stitches together the cropped area and the projections
• E. Estimates and subtracts background
• F. Takes the square root of the image (to minimize photon noise and to mimic a decrease in histogram gain)
• G. Resizes the image to 550x550 pixels, adjusts display, changes LUT and displays the picture with a standardized name: Date_Scopename_Magnification_NA

images

• 

  PSF macro: parameters dialog, open bead stack

• 

  PSF macro result 1: 500 nm bead*, confocal

• 

  PSF macro result 2: 500 nm bead*, confocal

(*) 500 nm bead = still above resolution limit! Use 170 nm beads to get a proper PSF.