ASC Z-Resolution
From BioDIP
(Difference between revisions)
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− | + | == requirements == | |
− | + | ||
* Leica Z-resolution mirror (coverslip on top of mirror) | * Leica Z-resolution mirror (coverslip on top of mirror) | ||
− | + | == acquisition == | |
* use immersion for the respective objectives | * use immersion for the respective objectives | ||
* use ~488 nm laser for illumination | * use ~488 nm laser for illumination | ||
Line 11: | Line 10: | ||
* acquire line-stack (XZ-scan) over the reflective side | * acquire line-stack (XZ-scan) over the reflective side | ||
** if XZ-scan is not available: acquire XYZ-stack and reslize it afterwards in Fiji | ** if XZ-scan is not available: acquire XYZ-stack and reslize it afterwards in Fiji | ||
− | + | == analysis == | |
* open the image in Fiji | * open the image in Fiji | ||
** make sure the image is calibrated (use Plugins > LOCI > Bioformats Importer, if File > Open ignores the metadata) | ** make sure the image is calibrated (use Plugins > LOCI > Bioformats Importer, if File > Open ignores the metadata) | ||
Line 17: | Line 16: | ||
* Analyze > Plot Profile for a rough estimation of the FWHM | * 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 | * precise analysis: save the list (button List in the profile window) and measure the FWHM in i.e. Excel | ||
− | + | == images == | |
<gallery> | <gallery> | ||
file:Z-resolution leica mirror.jpg|Leica Z-resolution mirror | file:Z-resolution leica mirror.jpg|Leica Z-resolution mirror |
Revision as of 14:18, 30 September 2009
requirements
- Leica Z-resolution mirror (coverslip on top of mirror)
acquisition
- 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