3.2 Basic Confocal Operation - Sequential
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:
- Setup the confocal normally for simultaneous scanning. i.e. find
sample, focus, choose filters, etc.
- Setup laser powers, PMT gain (voltage) and PMT offset
(black-level) to roughly obtain
an image while simultaneously scanning.
- If doing a z-series find the beginning and ending levels now and choose a
- Disable one or more PMTs, but leave at least one active
- Set the laser power used to excite the de-activated PMTs to zero percent
at the AOTF controls
- Readjust gain and offset if necessary of the reaming active
- From the filter settings box press the "save" button,
enter a new name (e.g. myname-fitc) and hit "OK". The laser power, PMT
gain and offset and filter setting are now saved in this filter settings file (e.g.
myname-fitc). If PMT gain or laser power are changed the setting must be resaved,
since this parameters saved in the file will be loaded and used during the sequential scan.
- Now reactivate the PMT for the other channel(s) in the filter settings
- De-activate the PMTs and zero the power settings for the
channels used in the first scan
- Set the AOTF laser power for the second channel and adjust the gain and
- Save the settings for the second channel by pressing "save"
in the filter settings box, enter another new name (e.g. myname-texas)
- From the filter settings box press the "seq" button and
check "activate sequential"
- Choose the fist desired filter setting (e.g. myname-fitc) and press
- Choose another filter setting (e.g. myname-texas) and press "add"
- Choose additional filters.
- Set these check boxes: add frames, change filters at each section
- Press "series" button to start acquisition
- Save image data using "file -> save_as"
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.