7
Importantly, there are two filter sliders to attenuate light (ND = neutral density filters) one
on the filter changer just in front of the arc lamp housing, and one in front of the filter changer (Fig. 4).
The filter changer (back) slider imposes an ND2 (100x less light, or 1% illumination), and the scope
(front) slider produces 25% illumination. 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 25% 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!
{optional details} For those who want to know the details, the
following chart reports the contents of the 10 filter slots in each wheel:
The Whl-A 340/380 positions are used to conduct calcium imaging
studies with the indicator fura-2. The 440 exciter and Whl-B 485/535
emission filters are used for real-time FRET probes such as cameleon
sensors. Both of these methods will require the proper dichroic mirror
(filter cube) to be placed into the microscope (not routinely installed at
this moment). We are swapping them to the #3 filter cube position since
few high-speed studies require combinations of fura-2 plus FRET plus
DIC. ColorRED is (600nm LP), colorGRN (500-575 BP), and
colorBLU (500nm SP) filters are intended for (additive) RGB color
capture, but can be used in custom filter designs as well.
Need special filters? Ask the staff about them… we have more filters than turret positions, so CFP, YFP,
Chameleon FRET, FURA, TexRed and Cy7 cubes are available but not installed.
How-To Do (non-ratio) Fluorescence Image Capture
Much of the configuration of the microscope is manual, but the software will queue you
with pop-up windows to change what needs to be changed by hand. Turn on the computer and logon; start
MetaMorph. MetaMorph (see Figure below) is configured to always start with the 20x objective in place
and cubes set to DIC. The software will pop up a window reminding you to change the objective to the
20x to start. The TASKBAR that pops up contains most of the buttons to select an objective, rotate in
filter cubes, select light paths, operate the camera, etc. If the taskbar does not open, you can find it under
Journal Taskbars Load Taskbar. Choose the “main” taskbar. When you choose a function on the
taskbar, a small window will pop up to
tell/remind you if you need to change
something manually.
1) You should always use the main menu
(top bar) to open the ACQUIRE acquire
menu. This menu gives you access to
camera controls. Other ACQUIRE menu
options include acquisition of multiple
wavelengths, Z series, timelapse, etc. Many
of those menus close after you use them, so
you’ll have to go back and re-open them for
more data. If you are collecting multiple
Position Whl A (F1)
(excitation)
Whl B (F2)
(emission)
0 Closed Open
1 Open 450/65
2 340 485/40
3 380+nd1.0 535/30
4 440/20 polarizer
5 480/40 colorRED
6 560/40 colorGRN
7 640/20 colorBLU
8 Open 630/60
9 Open 682/22
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