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dedicated excitation and band pass emission (barrier) filters that are coupled in the slider. This makes it
easy to selectively excite most fluorophores properly and separate them from other fluorphores relatively
well. Unfortunately, fluorophores with a large Stokes shift (like PI or Q-dots, which are excited by UV or
near UV light, but emit in the green, red or far red range) may not work with this system.
A manual shutter slide is located behind the filter slider (Fig. 2, “ND”). There are three
positions in this slider: all the way to the right blocks all light from the arclamp (for brightfield), the
middle position is 50% of the arclamp output, and all the way to the left is 100% of the arclamp output.
You should use as little light for live cell imaging as possible (both sliders pushed in) to forestall
photodamage. Most imaging can be done with only the 50% slider pushed in (well suited for your eyes,
too). You can use full light for dim samples, but be aware that you are probably getting high
autofluorescence and probes will bleach quickly. And remember to always
check your controls with the same illumination intensities!
A brightfield lightsource is available (above the scope) and is
controlled via the green switch (on/off) and knob (intensity up/down) on the
back right of the microscope base (see above). There is a makeshift foil
“shutter” to block brightfield light at the top of the condenser. It is
sometimes easier to control the brightfield light by pulling this in and out
than to turn it on and off with the switch.
The MicroManager Software
All of the configuration of this microscope is manual, the software is only for image
capture (i.e. is only aware of the camera). The software will not change the microscope settings, and it
will not be aware of any changes that you have made to the microscope (or did not make to the
microscope but should have, as the case may be). Once you have your sample in view with the
magnification and color you want, and are ready to take an image, start the MicroManager software.
MicroManager is built on top of ImageJ, the same free, open source software we recommend for image
processing. When MicroManager opens, you will see the main MicroManager System window. To get a
live preview of your data, push in
the slider on the lefthand side of
the microscope, up near the oculars
(it’s labeled) and hit the “Live”
button. If you don’t see an image,
make sure the ocular/camera slider
on the side of the microscope is
pushed in, then increase the
exposure time. The default
exposure time is 0.0 ms, which is
obviously way too little. You can
also use the “auto” button, which
will autoscale the image display to
make even low signals appear on
the screen. The “Full” button
returns the image display to the full
range of whatever bit-depth you
Fig. 3 Brightfield control
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