@@ -140,6 +140,17 @@ OME Tiff is recommended single-channel FRAP experiments. It stores FRAP regions
## Image Acquisition
### Choosing the right illumination
In the illumination dropdown menu, you can choose the illumination that matches your hardware settings - meaning the selected SD & camera (W1 or X1, sCMOS or EM-CCD). Choose the laser line you need to excite your fluorophore (DAPI, GFP, mCherry & Cy5 - meaning 405, 488, 561 and 647 excitation lasers). For each illumination (in case of multi-color imaging) go to 'Show live' adapt the following parameters to achieve the desired image quality (in that order):
- the camera gain (only available for the EMCCD, a good starting value is 300)
- the illumination time (if you have a mobile sample, you might save time by reducing the illumination time)
- the laser power (be aware that higher laser powers and illumination times can result in irreversible photobleaching, which impairs e.g. brightness analysis).
When acquiring the brightfield channel, you only have to adapt the Dialamp.
The hardware autofocus permits focus stabilization under non-bleaching conditions. It uses backreflection of grid pattern generated with a weak LED (850 nm) to measure the distance between the objective and the cover glass and to keep it constant. An aqueous medium (with a refractive index close to that of water) is required. It cannot be used with glycerol-based or hardening mounting media.
