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| − | MPI-CBG LMF
| + | This section suggests protocols for the evaluation of high end light microscopes. The tests should be carried out in a regular manner. Although the protocols are set up for the use with [[confocal]] laser scanning microscopes (CLSM), most of them can be adopted for the use with wide-field systems and other types of light microscopes. |
| − | | + | # [[ASC Fluorescence and Brightfield Imaging|Fluorescence and Brightfield Imaging]] |
| − | Advanced System Check Protocol (draft)
| + | # [[ASC Field Illumination|Field Illumination]] |
| − | | + | # [[ASC Color Overlay|Color Overlay]] |
| − | [[image:Conva10xok.jpg with bar.jpg|thumb]]
| + | # [[ASC Point Spread Function|Point Spread Function]] |
| − | [[image:Conva10xok-1.jpg with bar.jpg|thumb]] | + | # [[ASC Z-Resolution|Z-Resolution]] |
| − | == Fluorescence and Brightfield Imaging ==
| + | The individual protocols are optimized for PDF output via the "Print as PDF"-feature in the sidebar. |
| − | **purpose: test general performance of the system - basic functions work fine? + Koehler illumination, light paths ok?
| + | [[category:Manuals|A]] |
| − | **requirements: Zeiss/Leica Convallaria demo sample
| + | [[category:ASC]] |
| − | **work flow: acquire two images in basic resolution (512x512), blue ex / green em plus transmitted light image, using 10x objective
| + | |
| − | == Field of Illumination ==
| + | |
| − | ** purpose: check for even illumination
| + | |
| − | ** requirements: mirror
| + | |
| − | ** work flow: 80/20 mirror, 488 nm laser, pinhole 1 AU. focus on reflective side of mirror. try to obtain "japanese flag".
| + | |
| − | == Point Spread Function (PSF) ==
| + | |
| − | **purpose:
| + | |
| − | **requirements:
| + | |
| − | **work flow:
| + | |
| − | *overlay UV/V and VIS (confocal systems with separate UV/V laser coupling)
| + | |
| − | **purpose: check the coupling precision of the UV/V laser
| + | |
| − | **requirements: 0.2/0.5µm fluorescent beads sample
| + | |
| − | **work flow: open pinhole(s), use high resolution apochromatic lens (NA 1.2 or above), acquire two images (UV/V plus VIS channel) with good sampling (pixel size ~100nm)
| + | |
| − | | + | |
| − | === PSF Macro ===
| + | |
| − | [[image:2009-09-29 MPI11 63x 1.4 FWHMa 3822nm - FWHMl 456nm.jpg|thumb|PSF macro result: 500 nm bead, confocal]] | + | |
| − | [[image:2009-09-29 MPI11 63x 1.4 FWHMa 3822nm - FWHMl 456nm graphs.jpg|thumb|PSF macro result 2: 500 nm bead, confocal]] | + | |
| − | ==== requirements for bead stacks ====
| + | |
| − | * 100 planes, 200 nm spacing
| + | |
| − | * optimal: single bead in center of image
| + | |
| − | * the macro attempts to crop a 15x15 µm area with the bead in center; if the image dimensions are already smaller, extend the area: Fiji > Image > Adjust > Canvas Size...
| + | |
| − | ==== install LUT ====
| + | |
| − | * Mac: go to applications folder > fiji: ctrl+click on the Fiji(.app) icon > Show package content
| + | |
| − | * PC: go to \Program Files\Fiji.app (or to the place you installed Fiji)
| + | |
| − | * create a folder called "luts", if it's not there yet
| + | |
| − | * drag and drop the *.lut into it
| + | |
| − | * (re)start fiji
| + | |
| − | ==== install macro ====
| + | |
| − | * Fiji > Plugins > Macros > Install...
| + | |
| − | * select macro txt file
| + | |
| − | * macro gets installed to that menu for the current session
| + | |
| − | ==== load bead stack ====
| + | |
| − | * Fiji > File > Open
| + | |
| − | * select bead stack file
| + | |
| − | * if necessary, split channels (Fiji > Image > Color > Split Channels)
| + | |
| − | * if necessary, crop image to a single bead
| + | |
| − | ==== run macro ====
| + | |
| − | [[image:Set parameters.jpg|thumb|PSF macro: parameters dialog, open bead stack]] | + | |
| − | | + | |
| − | ==== description by Laurent ====
| + | |
| − | Thanks a lot for your interest in our ImageJ Macro, which I join as an attachment.
| + | |
| − | | + | |
| − | You need to install it each time you start ImageJ. Go to >Plugins>Macros>Install...
| + | |
| − | | + | |
| − | Select it in the dialog window and click "open".
| + | |
| − | | + | |
| − | To run it, you need before to open a stack. We take stacks of 100 planes, spaced by
| + | |
| − | 0.2mm, for all objectives and all microscopes.
| + | |
| − | Then go to >Plugins>Macros>MIPs for PSFs for all microscopes to run the Macro.
| + | |
| − | | + | |
| − | Automatic Macro actions / User actions:
| + | |
| − | | + | |
| − | 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 saves the
| + | |
| − | picture in a JPEG format with a standardized name: Date_Scope
| + | |
| − | name_Magnification_NA.jpg.
| + | |
| − | | + | |
| − | == Z-Resolution ==
| + | |
| − | | + | |
| − | *leica mirror
| + | |
| − | *XZY Scan
| + | |
| − | *Zoom 16.00
| + | |
| − | *30% 488nm laser
| + | |
| − | *30/70 splitter
| + | |
| − | *pmt 1 > detection slit around 488nm
| + | |
| − | *scan speed 200 Hz
| + | |
| − | *line average 4
| + | |
| − | *512x256
| + | |
| − | *8 bit
| + | |
| − | *min pinhole diameter!
| + | |
| − | | + | |
| − | | + | |
| − | results @ 1/2 maximum of intensity (+ theoretical leica value):
| + | |
| − | | + | |
| − | 100x/1.4 oil > 486 nm (235.8 nm)
| + | |
| − | 63x/1.32 oil > 660 nm (285.8 nm)
| + | |
| − | 40x/1.25 oil > 836 nm (334.4 nm)
| + | |
| − | 10x/0.3 dry > 1021 nm (4767.6 nm)
| + | |
This section suggests protocols for the evaluation of high end light microscopes. The tests should be carried out in a regular manner. Although the protocols are set up for the use with confocal laser scanning microscopes (CLSM), most of them can be adopted for the use with wide-field systems and other types of light microscopes.
The individual protocols are optimized for PDF output via the "Print as PDF"-feature in the sidebar.
