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III
Detailed Sequence of Microscopy
5 TIRF microscopy
30
5
TIRF microscopy
When the laser beam is focused on the objective’s pupil surface, the objective-emitted laser
beam should form parallel rays.
As the laser beam is focused on the edge of the objective’s pupil surface, the optical path of the
laser beam tilts. If the amount of tilt is a critical angle or greater relative to the specimen, the
illumination state becomes total reflection.
In contrast, if the laser beam is not parallel, non-parallel rays will pass through the cover glass
even when the optical path of the laser beam is tilted at a critical angle or greater relative to the
specimen, degrading image quality or damaging the specimen. It is essential for users to adjust
the focal point of the laser beam each time the objective is changed.
In TIRF microscopy, images are captured by TV camera and observed on the monitor.
(1) Close the Epi-fl attachment shutter.
Move the shutter lever of the cassatte
holder to the “C” position to close the
Epi-fl attachment shutter. The illumination
light for Epi-fl observations is blocked.
(2) Open the eyepiece shutter.
Switch the eyepiece shutter knob to
“Confocal” .
The shutter for the laser beam’s optical
path opens.
Then, observations from the eyepiece
becomes unavailable.
(3) Change the optical path.
1. Select the TIRF microscopy. (Pull out the mirror switch lever on the TIRF2
attachment.)
2.
Switch the microscope’s optical path to a port to which the TV camera is attached.
(Use the optical path switch knob, LIGHT PATH key etc.)
(4) Select the excitation method.
Rotate the excitation method changeover ring to insert the filter cube suitable for the
specimen.
(5) Open the laser shutter.
Press the shutter button on the shutter control unit to open a desired optical path.
Set the dial to "Bino" for normal
observation. (Eyepiece shutter open)
Set the dial to "Confocal" for TIRF
observation. (Eyepiece shutter close)
Summary of Contents for TIRF2
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