7.
Confocal Aperture, 4.10
– Controls the diameter of the pinhole.
8.
Kalman Averaging, 4.11 –
Allows averaging signals from multiple scans to reduce noise.
9.
Information, 4.12 –
Opens a window displaying the current pixel size.
Focus X2 and Focus X4 settings switch to 2
µ
s dwell time.
These scan settings reduce resolution in
Y axis by 2-fold and 4-fold, respectively. Use these settings with the AutoHV switch selected.
2.3 Settings for the fluorescence detectors.
Each fluorophore selected from the Dye List will activate one of the photomultiplier tube (PMT)
channels and set its appropriate filters. Three settings control the sensitivity and signal to noise ratio for
each detector. Refer to “Optimizing the Image” for details on these settings.
1.
HV, 5.1
– The primary adjustment for the PMT. HV controls the detector sensitivity and
amplification.
a. try to stay below 750, higher settings will introduce detector noise in the image
b. higher HV may used with averaged images to allow reduced laser intensity
2.
Gain, 5.2
– This multiplies the analog electrical signal created by the PMT by a constant, before
it is digitized. While this can provide ‘brighter’ images, the noise and background intensities are
increased as well.
a. leave set to “1” unless working with very low HV settings.
3.
Offset, 5.3
– The Offset will raise or lower all pixel intensities to control the “Black Level” of
the image. It should be set so that the darkest values in the image are above zero.
a. leave set to default values of approximately 5 to 7
b. set the display to gray scale, pixels with intensity = 0 will be pseudocolored blue
c. set the Offset so that no blue pixels ( intensity = 0) are present
2.4 Additional detector controls.
1.
Laser power level, 5.4
- changing from this window may be more convenient than scrolling over
to the laser control window.
2.
Active detector check mark, 5.7
– removing this check mark will turn off the channel and its
associated laser line.
3.
Group number, 5.5
- allows channels to be grouped for sequential channel acquisition.
a. channels that do not share bleedthrough, e.g. Alexa488 and Cy5, they can be combined
into the same group.
b. channels in the same group are scanned simultaneously to reduce the amount of time
required for sequential acquisition
Olympus Fluoview-1000 User’s Guide
V.M. Bloedel Hearing Research Center, Core for Communication Research
Center on Human Development and Disability, Digital Microscopy Center
May 11, 2011
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