Biology Reference
In-Depth Information
laser
laser
laser
laser
A
P
z
x
--
y
stage
Sample
Oil
Ba
Condenser
aperture
diaphragm
Tube
C
Entrance
Focal
I
tube
adjustment
s
adjustment
Split phot
o
detector
Detector lateral adjustment
SCHEME 15.2
Schematic of the 4-color SMD-TIRF microscope with alternating-laser excitation (ALEX). The 561 nm
frequency-doubled, diode-pumped, solid-state (DPSS) laser and the 405, 488, and 642 nm diode lasers
are controlled by TTL signals generated by an Arduino microcontroller. The lasers are combined into a
single polarization-maintaining optical fiber using the kineFLEX/iFLEX-Adder system from Qioptiq. The
kineFLEX fiber is connected to the Zeiss TIRF3 slider. A quad band-pass filter (BP1) with quad band-pass
dichroic mirror (BS1) direct the laser beam to the Zeiss
/1.45NA oil immersion TIRF
objective. In the emission light path, a dual-notch filter (NP1) and single-notch filter (NP2) block residual
laser light. The CRISP autofocus unit is equipped with a customized 785 nm LED. The dichroic mirror
(BS2) directs the infrared beam of the CRISP unit back towards the objective, where the glass-water
interface of the sample reflects the beam. The split photo detector of the CRIPS unit monitors the position of
the reflected beam to control the Piezo Z drive in a feedback loop that locks the sample in focus. The
quad band-pass filter (BP2) eliminates any residual light from the LED and Lasers. The QuadView image
splitter separates the emission light path in four different wavelengthwindows that aredefinedby thedichroic
mirrors (BS3-BS5) and band-pass filters (BP3-BP6). The correction lenses (L1 and L2) reduce
chromatic aberration of the long wavelength emission channels. An electron-multiplying charge-coupled
device (EM CCD) camera simultaneously acquires the four subimages generated by the QuadView optic.
The EM CCD exposure is synchronized with the TTL-controlled lasers by the Arduino microcontroller.
A motorized x-y stage enables automated single-molecule experiments on multiwell microtiter plates.
a
-Plan-Fluar 100
