ASC Point Spread Function
From BioDIP
(Difference between revisions)
(→analysis) |
|||
Line 16: | Line 16: | ||
* 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) | * 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) | * 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 [[media:Gelman-psf-macro-v4.zip|PSF macro zip file]] and unzip it | * download the [[media:Gelman-psf-macro-v4.zip|PSF macro zip file]] and unzip it | ||
* include the LUT: | * include the LUT: | ||
Line 26: | Line 26: | ||
** the macro appears in the Macros menu | ** the macro appears in the Macros menu | ||
** procedure has to be repeated for every new Fiji session | ** procedure has to be repeated for every new Fiji session | ||
+ | == analysis == | ||
+ | * load the bead stack into Fiji | ||
+ | * 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) | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | F. Takes the square root of the image (to minimize photon noise and to mimic a | + | == PSF macro: automatic macro actions / user actions == |
− | decrease in histogram gain) | + | ''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 == | == images == | ||
<gallery> | <gallery> |
Revision as of 13:45, 1 October 2009
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
- 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)
PSF macro: 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