Biology Reference
In-Depth Information
FIGURE 20.3
Microscope setup for simultaneous, dual-color imaging of two fluorescent single molecules of
different species. For the observations of other dyes, the microscope is also equipped
with 532 and 642 nm lasers. The excitation arm consists of the following optical components:
BP, band-pass filter; DM, dichroic mirror; FD, field diaphragm; L1 and L2, working as a
10
beam expander (L1,
f¼
15 mm; L2,
f¼
150 mm) for 488 nm excitation or as a 4
beam
expander (L1,
f¼
20 mm; L2,
f¼
80 mm) for 594 nm excitation; L3, focusing lens
(
350 mm); M, mirror; ND, neutral density filter; r/4, quarter-wave plate; and S, electronic
shutter. The two-color fluorescence emission signal is split by a dichroic mirror (DM3)
and detected by two cameras at the side and bottom ports. BF, barrier filter; I.I., image
intensifier; PL, projection lens (2
f¼
); and TL, tube lens (1
or 2
).
The image in each arm is projected onto the photocathode of the image intensifier
with a two-stage microchannel plate (C8600-03, Hamamatsu Photonics), which is lens
coupled to the camera.WeuseHamamatsuelectronbombardment charge-coupledcam-
eras (C7190-23). The cameras on the two detecting arms (side and bottom, two cameras
of the samemodel number produced in the same batch placed on each arm) are synchro-
nized frame by frame by coupling the sync out of one camera to the trigger of the second
(gen-locked). Observations are performed only for individual fluorescent spots located
within the central region (20
m
m in diameter) of the illuminated area, in which nonuni-
formitiesappear tobesmall. Thecamera imagesare storedonadigital videotape (8PDV-
184ME, Sony) for postexperiment spatial synchronization.
