3.2  Basic Confocal Operation - Sequential Scanning

Brief Overview: 

Sequential multiple channel fluorescence scanning is used to avoid or reduce bleed through (cross talk).  Cross talk can have a number of sources.  For examples filters my not cut off sufficient light emitted away from the peak emission wavelength, especially with bright fluorescence, allowing that light to impinge on adjacent fluorophore channels.  Also fluorescent coupling transfer (& perhaps FRET) can cause adjacent fluorophores to be excited by the emitted fluorescence light.  

Sequential scanning is more complex to set up on the LeicaTCS-NT than simultaneous scanning, but avoids the severe problem of cross talk that can invalidate meaningful interpretation of image data obtained from multiply labeled samples.  On some confocal systems (e.g. Biorad, Zeiss) the default scanning mode is sequential.  

A Guide to Setting Up Sequential Scanning - Initial Setup:

Notes.  If  PMT gain, AOTF laser power, PMT offset, filter wheels are changed the named filter setting must be re-saved in the sequential box, otherwise the settings will revert back to the old settings when the sequential scan is started when "series" button is pressed.

These notes are rather terse.  Some effort will be dedicated to clarifying them, but not much due to the new Leica scanning software that will be available in the future.


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