Scanning A L Ive I Mage; Creating A N Ew B Eam P Ath S Etting; Simultaneous V S S Equential S Canning M Odes - Leica SP5 confocal User Manual

Scanning a l ive i mage
1. To see a continuously scanned live image of the sample press the Live button at
the bottom of the screen. You can change any of the capture parameters with or
without Live mode enabled.
Creating a n ew b eam p ath s etting
1. Activate the laser lines required by moving the vertical slide bars to >0%, the
laser line will be added to the spectral diagram
2. Activate as many detectors as required using the tick box below each detector
3. Select the emission spectrum label for each channel, choose the
fluorophore/protein you're using if possible. This will add the emission spectrum
of the dye to the diagram.
4. By moving the spectral sliders you can adjust the detectors detection bandwidth.
Click and drag a slider to move it. Hover the cursor over the edge of a slider to
allow the width to be adjusted. Try to match the width of the slider to the
emission spectrum of the dye. Avoid overlapping the spectral sliders with the
excitation laser lines (5nm min separation).
5. Select the Channel colour box for each detector if you'd like to channel the
image colour (Lookup table)
6. Press a detectors PMT text to access that detectors Gain and Offset values
7. Use the Save button to store the adjusted beam path setting. This new setting
will appear below the User Settings heading at the bottom of the drop down list.
Simultaneous v s S equential s canning m odes
1. The process of setting up the beam path setting above describes the setup for
simultaneous scanning. This means that all active laser lines and detectors
scan and acquire simultaneously. This has the advantage of speed but the
disadvantage is that fluorescence cross-talk (signal from one fluorophore
recorded in more than one detector) is more likely.
2. In Sequential scanning mode only one detector/laser is active at any one time,
making cross-talk much less likely but at the expense of speed.
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