ASC Z-Resolution
From BioDIP
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