Advanced system check

MPI-CBG LMF

Advanced System Check

Protocol (draft)

• fluorescence and bright field imaging (all systems)
  • purpose: test general performance of the system - basic functions work fine? + Koehler illumination, light paths ok?
  • requirements: Zeiss/Leica Convallaria demo sample
  • work flow: acquire two images in basic resolution (512x512), blue ex / green em plus transmitted light image, using 10x objective
• field of illumination
  • purpose: check for even illumination
  • requirements: mirror
  • work flow: 80/20 mirror, 488 nm laser, pinhole 1 AU. focus on reflective side of mirror. try to obtain "japanese flag".
• psf
  • purpose:
  • requirements:
  • work flow:
• 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)

PSF Macro

install LUT

• Mac: go to applications folder > fiji: ctrl+click on the Fiji(.app) icon > Show package content
• create "luts" folder 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
• 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