TIRF scope SOP
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2008
CFP imaging (405 laser)
GFP imaging (473 laser)
Red imaging (ds-red, mRFP, cherry) (561 laser)
green-red dual color (473+561 and special filtercube)
photoactivation and green imaging (405+473 and special filtercube)
1) Switching on
heater
stage Epi lamp Trans lamp
Shutter
(switch on at back)
PC
(switch on at
back (i.e. normal
front))
lasers
camera
Lasers are switched on at the sockets
Sockets are labeled with the wavelength of
the corresponding laser.
Please do only switch on the lasers you need!
Careful: Please do not touch or change the laser arrangements!! (see pictures)
Coupling of laser fibres and epi lamp into
the optical light path of the microscope
Coupling of the lasers into the fibers:
very delicate – do not touch at all
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TIRF scope SOP
2008
2) Meta Morph
Open Meta Morph “Everything” from the desktop
Taskbar with
journals like:
473 on (to find cells
using TIRF)
473 off
Open the “Acquire”
(once open it stays
open)
and the “Acquire
timelapse” (has to be
reopened after each
movie)
windows
different time-lapse
combinations
Choose
illumination
(laser power!)
Show live (live
stream acquisition)
Choose needed
setting (473, 561,
green-epi etc)
3) TIRF setup for one color
a) Check beamsplitter (in front of camera) is in “one color” mode:
dichroic slider (out)
Lever on
bypass view
b) place your sample on the microscope (using cargille oil) and focus using transmitted light.
c) Switch on the corresponding laser in Meta Morph,
flip back the microscope condenser and change the angle
so that the laser is going to the ceiling
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TIRF scope SOP
2008
You should see an airy disk profile at the ceiling. By changing the position of the lens in the
TIRF condenser you can change the shape of the airy disk.
Good
not so good
d) Switch to camera and show life and find a fluorescent cell
Slowly turn the laser into TIRF and observe how the picture changes on the screen. There
should be a clear transition between epi and TIRF. It is better to go further in TIRF to avoid
partial epi illumination.
(if uncertain, go until the picture disappears completely and then just a little bit back)
the position of the laser should be stable once adjusted, but if you see epi illumination occurring,
just turn the laser a bit more into TIRF
!!! if you cant find a good TIRF position, check that the cells are really flat (i.e. attached to the
coverglass) in transmitted light!!!
4) TIRF setup for two colors
a) Sequential imaging:
 Beamsplitter setup like for one color
 Adjust the first laser as described above
 Once the first laser is in TIRF, change illumination and filtercube and adjust the
second laser to TIRF (the airy disk pattern on the ceiling should be OK, because it
was adjusted for the first laser. If both airy disc patterns are very different you have
to find a compromise between both perfect individual situations. But: do not turn
both lasers completely in epi as the fiber outputs will crash against each other – put
the laser in a position where they are just on the ceiling (~45°))
 Use dual color filtercube and single illumination to take pictures
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TIRF scope SOP
2008
b) Simultaneous imaging
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Adjust beam splitter as described on separate sheet
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Then adjust TIRF for both lasers as described above
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Use dual color filtercube and double illumination for imaging
to change intensity of lasers in dual color setup:
o 473: devices  configure illumination
then change Voltage and save (add/replace)
561: cant be changed online, has to be
manually
5) Switching
off change where the laser enters the
fiber
=> turn “screw” in oder to move the double
prism up and down (down=more intensity)
o
6) Please make sure you switch off everything you switched on earlier (page 1)
(PC, camera, shutter, epi lamp, Nikon trans lamp, lasers…)
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