OPERATION
Carl Zeiss
Adjusting the TIRF Effect on the TIRF Slider man.
Laser TIRF 3
80
423683-7144-001
02/2014
•
Now activate epi-fluorescence illumination by pressing the
RL
button on the right-hand side of the
microscope stand.
•
Set the beam path of the microscope in such a way that the light from the specimen is deflected to
the camera.
•
If you use the Slider Laser TIRF man., turn the TIRF angle adjusting screw (Fig. 4-8/
2
) carefully to mid-
position. If you use the TIRF slider mot., set the TIRF angle to 0° using the software. In this position,
the specimen is illuminated as in epi-fluorescence.
On the monitor, the camera image of the object should be visible in epi-fluorescence with laser
illumination.
•
It is possible to see deep into the volume of the sample by moving the focus motor up and down
slightly. Only fluorescing movable beads can now observed.
•
Change the TIRF angle slowly until you can no longer see the camera image. A sharp contrast
interface, which marks the transition to total reflection, will be visible.
•
Change the TIRF angle slowly in the opposite direction until you can see an image (carrying on
changing the TIRF angle will bring about the change from TIRF to epi-fluorescence).
•
Epi-fluorescence may be selected by pressing the
RL
button.
•
It is very easy to test whether the TIRF angle has been reached, by shifting the focus upwards and
downwards slightly in a running live image.
−
Once the total reflection zone has been reached, there is no focal plane outside the glass/medium
barrier in which the fluorescing particles are sharply in focus.
−
This TIRF test can also be used as a quality criterion for the actual sample.
−
If there are still Epi elements in the image in calibrated system status (see Section 4.8), although a
TIRF angle has nominally been set, it can be assumed that the cell / sample is suboptimal.
Summary of Contents for TIRF 3
Page 1: ...Laser TIRF 3 Imaging System Operating Manual...
Page 110: ......
