10
Acquisition Settings
1. Select the XY window in the left hand margin
2.
Format
changes the size of the image in pixels. For
better xy sampling choose 1024x1024. For speed
choose 512x512.
3.
Speed
determines how long the laser spot dwells over
any particular part of the sample. The higher the
frequency the lower the dwell time and thus the lower
the exposure. Optimal values can range between 400-
100Hz.
4. Tick the
Pinhole
box which will expand the Pinhole window. Press the 1 Airy
button. This selects the best compromise between light throughout, resolution
and noise. It is also responsible for determining the optical section thickness
with higher values increasing the thickness of the optical section.
5. Use the
Zoom Factor
slider to increase the image magnification. This can
increase resolution because the pixel size decreases (microns per pixel).
6.
Line/Frame Averaging
can be used to reduce the image noise level by
scanning each channel a number of times and averaging the resulting signal.
Only use if you have a noisy image. Image capture time is increased as is the
likelihood of phoobleaching.
7.
Rotation
allows the whole scanned field of view to be rotated, if you need to
rotate your image while scanning.
Image Optimisation Procedure
1. Start with a format of 512x512 and a speed of 400Hz. Set the laser power
between 5-10% for a each channel. Do not apply zoom or averaging at this
stage.
2.
Press the Live button hopefully you will see an image of each channel displayed
on the right hand monitor. Each channel has its own window by default and you
can toggle them off/on using the panel on the far right of the image monitor. The
lowest button pictured here toggles the merged view on/off.
3.
Check the detector gain settings for each channel by clicking on the PMT text at
the top of each detector window. Gain is used to amplify the signal thus the
image brightness will increase.
4.
However, high gain levels will increase the noise level in
the images therefore achieving optimum exposure is
always a compromise between laser power, gain and scan
speed.
Summary of Contents for SP5 confocal
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Page 16: ...16 FRAP...
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Page 25: ...25 FRET Acceptor Photobleaching...
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