Optical
System
The optical system
consists of the light source and modulator, the microscope, the emission port
optics, the shutters, the detectors, some custom adapters, and the filters
located in the microscope and emission port.
Light Sources:
Coherent Mira Ti-Sapph Laser, femtosecond
option, tunable from 700-950nm.
Argon
Ion Laser, principle lines at 488 and 514nm, fiber-optically coupled through SM
fiber.
Mercury
lamp (Olympus).
Coherent Optical Second Harmonic Generator
for extending Mira range to include 350-475nm.
Modulator:
The
Conoptics Electro-Optic Modulator (EOM)
attenuates the laser beam depending on the voltage potential applied to the
Pockels Cell crystals the beam passes through. This potential can be hundreds
of volts but can be controlled remotely by a smaller (0 to 2 Volt) signal input
connector provided. The modulator bandwidth is DC to 200kHz but in our system
is limited to 10kHz by the bandwidth of our analog control signal.
Microscope:
Olympus IX-70 inverted microscope
with side input turret option (for laser excitation) and special dichroic
mounts for side excitation; side emission port option for photon counting.
Thorlabs beam expander mounted to side
input turret to fill back aperture of objective.
UPlanApo
60X water immersion objective.
Emission Port Optics:
The
emission port optics consist of a series of Thorlabs lens tubes, rubber axle
boots, stage-mounted pinhole, and other accessories, all connected to the
microscope side emission port via a custom C-mount adapter. We modelled this
after a setup we saw at the Laboratory for
Fluorescence Dynamics at the University of Illinois-Urbana. The emitted
photons are focused onto Avalanche Photodiode Detectors (APDs) that have shutters
mounted to their inputs via a custom adapter. Detector alignment is
accomplished by adjusting the X, Y, and Z stage micrometers on which the
detectors are mounted. Here is a schematic:
Emission
Port Features:
Configurable
for two-color, anisotropy, or cross-correlation measurements.
Fluorophor-specific
filters can be mounted at each detector.
Configurable
3-lens or single lens system.
Achromatic
AR-coated lenses.
Pinhole
on XYZ mount for confocal work.
Easy
detector alignment using XYZ mounting stages.
Shutters:
Uniblitz LS-6 Shutters with C-mount
adapters.
Less
than 1ms closing time.
Note:
"Sync" versions of shutter incorporate a light source which must be
inactivated!
Uniblitz VMM-D4 Shutter Controller accepts
TTL control pulses.
Detectors:
Two Perkin-Elmer
SPCM AQR-15 APDs.
50cps
maximum dark count.
170
micron diameter active area.
Photon
detection efficiency 70% typical at 650nm, >10% from 420nm to 1000nm.
Adapters:
Click the link for mechanical drawings (JPEG images).
Microscope side
port-to-C-mount adapter
APD-to-translation stage adapter
Filter Sets:
A
filter set consists of 1) a microscope dichroic for passing only emission
(higher, except for two-photon excitation experiments) wavelengths and
reflecting lower-wavelength excitation light to the sample, 2) A bandpass
filter for passing only wavelengths within the emission spectrum of the
fluorophor, 3) a beamsplitter if two-channel detection is desired--for example,
a FRET experiment would use a dichroic beamsplitter that reflected the
"donor" emission spectrum but passed the "acceptor"
emission spectrum. Losses are cumulative and include all filters, lenses
(including the objective and its collection angle), and the detector
efficiency.
Shown
below is an example of Cy3 losses for a maximum throughput Cy3/Cy5 FRET pair,
not taking into account lens losses and the numerical aperture (collection
angle) of the objective. The brown spectrum is what is left from the original
Cy3 emission spectrum (in dark blue). The filter set was purchased from Chroma Scientific.
Last updated August
10, 2003