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− | == Fluorescence and Brightfield Imaging ==
| + | 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. |
− | [[image:Conva10xok.jpg with bar.jpg|thumb|Convallaria, 10x/0.45 objective, 488 nm excitation, BP505-550 detection]]
| + | # [[ASC Fluorescence and Brightfield Imaging|Fluorescence and Brightfield Imaging]] |
− | [[image:Conva10xok-1.jpg with bar.jpg|thumb|Convallaria, 10x/0.45 objective, 488 nm illumination, transmitted light detection]]
| + | # [[ASC Field Illumination|Field Illumination]] |
− | First step of the system check. The purpose is to test the general performance of the system: Do the basic functions work fine? Does the acquisition software behave as it should? Can one set up Koehler illumination? Are the transmitted and the reflected beam-paths ok? Although this test does not represent high-resolution work, it might reveal general problems with the system.
| + | # [[ASC Color Overlay|Color Overlay]] |
− | * requirements: basic sample with fluorescence and contrast in transmitted light (i.e. Zeiss/Leica Convallaria demo sample)
| + | # [[ASC Point Spread Function|Point Spread Function]] |
− | === acquisition ===
| + | # [[ASC Z-Resolution|Z-Resolution]] |
− | * set up the beam-path for fluorescence and transmitted light
| + | The individual protocols are optimized for PDF output via the "Print as PDF"-feature in the sidebar. |
− | * use basic air objective (i.e. 10x/0.4)
| + | [[category:Manuals|A]] |
− | * acquire two images in basic resolution (i.e. 512x512)
| + | [[category:ASC]] |
− | | + | |
− | == Field of Illumination ==
| + | |
− | ** purpose: check for even illumination
| + | |
− | ** requirements: mirror
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− | ** 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:
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− | *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)
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− | | + | |
− | === 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
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− | * 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
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− | * 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...
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− | | + | |
− | Select it in the dialog window and click "open".
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− | | + | |
− | 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.
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− | | + | |
− | Automatic Macro actions / User actions:
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− | | + | |
− | A. Selects the plane with the highest pixel intensity, adjusts display settings,
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− | opens the information dialog box.
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− | | + | |
− | 1. Enter information in the dialog window which popped up.
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− | | + | |
− | 2. Zoom in the image to clearly localize the center of the bead (you can also
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− | navigate between planes if needed).
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− | | + | |
− | 3. Right clicks with the mouse on the center of the bead.
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− | | + | |
− | B. Crops the image to get 15mmx15mm area centered over the pixel clicked by the user.
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− | | + | |
− | C. Makes projections in X and Y of the stack
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− | | + | |
− | D. Stitches together the cropped area and the projections
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− | | + | |
− | E. Estimates and subtracts background
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− | | + | |
− | F. Takes the square root of the image (to minimize photon noise and to mimic a
| + | |
− | decrease in histogram gain)
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− | | + | |
− | 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 ==
| + | |
− | [[image:Z-resolution leica mirror.jpg|thumb|Leica Z-resolution mirror]] | + | |
− | [[image:Fiji z-resolution FWHM.jpg|thumb|Fiji: XZ-scan, Z-resolution profile]] | + | |
− | === acquisition ===
| + | |
− | * requirements: Leica Z-resolution mirror (coverslip on top of mirror)
| + | |
− | * use immersion for the respective objectives
| + | |
− | * use ~488 nm laser for illumination
| + | |
− | * set main beamsplitter to reflection (i.e. 30/70 or 80/20 splitter, AOBS in reflection mode)
| + | |
− | * set detection to the range of the laser wavelength (i.e. LP420, LP470, spectral detection 480-500 nm)
| + | |
− | * close pinhole to minimum
| + | |
− | * set acquisition to good quality (laser power high enough, line average, low scan speed, no saturated pixels)
| + | |
− | * acquire line-stack (XZ-scan) over the reflective side
| + | |
− | ** if XZ-scan is not available: acquire XYZ-stack and reslize it afterwards in Fiji
| + | |
− | === analysis ===
| + | |
− | * open the image in Fiji
| + | |
− | ** make sure the image is calibrated (use Plugins > LOCI > Bioformats Importer, if File > Open ignores the metadata)
| + | |
− | * draw a line over the peak (select line tool from the toolbar, left-click for start point, hold shift, left-click for end point)
| + | |
− | * Analyze > Plot Profile for a rough estimation of the FWHM
| + | |
− | * precise analysis: save the list (button List in the profile window) and measure the FWHM in i.e. Excel
| + | |
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.