ASC Z-Resolution
From BioDIP
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(Created page with '== Z-Resolution == === requirements === * Leica Z-resolution mirror (coverslip on top of mirror) === acquisition === * use immersion for the respective objectives * use ~488 nm l...') |
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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) | ||
| − | * draw a line over the peak (select line tool from the toolbar, left-click for start point, hold shift, left-click for end point) | + | * draw a vertical line over the peak intensity (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 | * 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 | ||
file:Fiji z-resolution FWHM.jpg|Fiji: XZ-scan, Z-resolution profile | file:Fiji z-resolution FWHM.jpg|Fiji: XZ-scan, Z-resolution profile | ||
</gallery> | </gallery> | ||
| + | [[category:ASC]] | ||
| + | __NOTOC__ | ||
Latest revision as of 09:51, 1 October 2009
[edit] requirements
- Leica Z-resolution mirror (coverslip on top of mirror)
[edit] 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
[edit] 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 vertical line over the peak intensity (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
[edit] images
Leica Z-resolution mirror
Fiji: XZ-scan, Z-resolution profile
