Last updated on Mar 2022
4.4 Laser Configuration Settings
Under
PMT Setting tab
, click on
Dye and Detector
. A list of
commercially available dyes along with its excitation and emission
values are available for selection.
1. Choose the appropriate dye.
2. Select
Add
.
3. It will immediately be reflected under phase 1 where each dye
will be allocated to its respective channel. A transmitted light
channel will also be shown by default.
4. Click
OK
.
•
Under Laser ND filter: Select 10%.
※
If you need more than 10% laser power you can switch
off the ND filter, but most imaging will not need that high
power of a laser.
•
Set your laser power for all your channels at 1%.
•
Emission range could be altered to include a broader range of
signal otherwise you could use the default settings.
•
For detectors:
➢
HSD
: keep HV within 350-500. More than 500 will
produce intense background noise.
➢
SD
:
keep HV within 600-800. More than 800 will
contribute to intense background noise.
Under PMT settings tab:
•
Mode
: Select
VBF
•
Average
: average a given number of scans to get a
higher signal to noise ratio.
➢
In
Line
mode
the averaging will be done on a line-
by-line basis.
➢
In
Frame mode
the averaging will be done frame-
by-frame. Drawback: slower scanning time, more
bleaching, double exposure effect if your sample
moves. Averaging of 2 times will help you to get
better quality images if you can afford to do so.
No averaging or Line averaging is recommended if you are doing live-cell imaging.
•
Sequential scan:
Select
Line
to prevent crosstalk among the fluorophores. If you select
None
, simultaneously imaging is done where cross talk is prominent.
•
Confocal Aperture
: press
Auto
button to make sure pinhole size is set to optimal
confocal imaging at Airy Disk ×1. This gives the most optimum size for the thinnest
optical slice while not sacrificing imaging quality. Increasing the size of the Airy Disk from