Biology Reference
In-Depth Information
Data
acquisition
EMCCD
Two emission channel
OptoSplit II
Laser
Yokogawa
Spinning-Disk
Objective
Cell specimen
FIGURE 14.5
Schematic representation of spinning disk microscope setup. Pathway of excitation as well as
emission light through the setup is shown schematically. Laser excitation light passes
spinning disk and Yokogawa dichroic mirror (DM) into the objective exciting the specimen.
The emission passes pinhole spinning disk and is reflected by DM and guided to the OptoSplit
II that separates the emission with a filterset of dichroic mirror and emitters into two
emission channels. This results in two spatially similar, but spectrally different images on both
half sides of the EMCCD camera.
14.3.5.3
TIRF microscope
The extension configuration available at our TIRF microscope allows us to alter the
excitation incident angle stepless to switch easily between wide field and TIRF
mode. FRET measurements were conducted in two modes: wide field (incident angle
0
) and TIRF (incident angle 75.48
). Due to the difference in signal intensity,
