Iron Man User Guide v. 1.4
9
6)
Adjusting PMTs
These guidelines assume a fixed sample. For live samples, you should try to avoid Auto Expose or
Continuous modes as these will expose your sample to large amounts of excitation light prior to image
acquisition. Instead use Live mode or optimize in one region of slide
and move to another for data acquisition.
This step depends on your experimental design and question. To
ensure that your images are quantitative, you must avoid over- or
understaurating pixels, as you will not be able to properly measure
intensity in this case. It is also important to remember that your
brightest sample may be a particular Z plane, later slide, alternate
timepoint, and these may be oversaturated when you get to them
later.
-
Select the first channel to optimize (check on/off in Imaging
Setup area). I usually prefer to optimize one channel/track
at a time, but if you plan to use multiple lasers on a single
track, make sure these will all be on during optimization.
-
Ensure that your sample is on the brightest Z plane – go to
live mode
and adjust using focus knob.
-
Set lasers to appropriate starting values
- for the Argon
and Diode lasers, 3-5% is usually sufficient, and for the
HeNe lasers, 25-35% is usually a good starting point.
-
Set the pinhole size to 1AU
. Unless you have a deliberate
reason to change this, 1AU is good for most experiments.
(Bigger pinhole = more light = less “confocal”). Don’t go
smaller. Bigger is useful for if you need to image a really
thick sample and do not want to image many 1AU confocal
slices.
-
Click
Auto Expose
. The software will now attempt to set
the gain and offset for that channel.
-
Click
Continuous
, and select the
Range indicator Look Up
table
beneath the image. Oversaturated pixels will appear
red and undersaturated pixels will appear blue. You can
manually adjust the gain and offset to achieve a good
balance between over- and undersaturation for your
experiment. You should adjust the
offset
first. This will
adjust the background. Be aware that if you change this too much you
will also remove signal form your sample. A little goes a LONG way, go
down to add undersaturated pixels, go up if you have too many
undersaturated pixels. After adjusting the offset, adjust the
gain
(master)
. This will adjust the foreground and background signals. If you
are at the high end of gain, bump up laser power; if you are at low end,
reduce laser power.
-
Repeat these steps for all channels in your experiment.
