Beam Path Configuration For Multi Photon Excitation; Acquisition Conditions For Rics - Zeiss LSM 880 Operating Manual

ZEISS
Line and Frame Mode of Multitracking
• Settings can be used for Line or Frame wise multi-tracking acquisition.
• In Line mode the lines are scanned in turns for all tracks with the corresponding laser lines turned on
exclusively. Preferred for living samples with moving objects. Acquisition time can be reduced using
bidirectional scan mode.
• In Frame mode, the whole frames are scanned in turns for all tracks with the corresponding laser lines
turned on exclusively. This mode can be advantageous for dyes that tend to bleach and need time to
recover.
• There are parameters that can be changed quickly in a line wise acquisition: Digital Gain, Digital
Offset, Laser Line Attenuation,
• Any other changes of track settings of the selected tracks, e. g. different emission filters, dichroic
filters or Detector Gain (Master Gain) settings, need a bit more time to be changed and therefore can
be changed only when Frame mode is used.
• There is a fast Frame mode, which requires identical settings of these parameters.
3.3

Beam Path Configuration for Multi Photon Excitation

• The beam path configuration for multi photon excitation follows the same rules as described for a
single and multi-track configuration (see above)
• Use a MBS 690+ or MBS 760+ as main dichroic beam splitter to deflect the IR excitation light
(690 or 760-1050 nm) onto the specimen.
• On the detection side, use an additional band pass filter in front of the NDD to prevent a
bleed through of IR excitation light to the detector.
3.4

Acquisition Conditions for RICS

For RICS the pixel size should be at least 5 times smaller than the PSF of the objective, so appropriate
zooms have to be employed.
For the C-Apo 40x objective with a beam radial extension of approximately 0.25 um zoom factors of
10-20 will guarantee a sufficient size ratio. In addition, scanning speed should not be too high or slow.
As a rule of thumb for solution studies speeds 7-8, for measurements in cytosol and nucleus of 5-6 and
for membrane studies of 3-4 will be appropriate.
Expression levels should not be too high. A maximum of 1 µM concentration can be tolerated.
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CHAPTER 4 - ANNEX
Beam path Configuration Guide for ...
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LSM 880
10/2014 V_01