Chemistry Reference
In-Depth Information
microfabricated metal barriers to further restrict
the
fluorescence excitation
volume [17].
Imaging cameras and spot detectors with the sensitivity to record single molecule
emission are commercially available. A type of imaging detector that has come into
wide use for single molecule fluorescence microscopy is the Electronic Multiplying
Charge-Coupled Device (EMCCD). Thinned chips, illuminated from the side of the
silicon wafer opposite to the detector junctions (back-illumination), result in increas-
ing the quantum ef
ciency of converting photons into charge carriers to above 90%.
The electron-multiplying shift register increases the gain (output signal/input light)
several hundred-fold while introducing very little noise [18], and thereby effectively
reduces the contribution to noise associated with reading the photo-electron count
from the detector. Cooling the detector chip from
70 C reduces the dark
current. These features have the result that the main source of noise in the image is
the probabilistic nature of detecting limited numbers of photons (shot noise). Several
manufacturers supply EMCCD cameras with 512
30 to
512 pixel arrays reading out up to
30 full frames per second.
Single spot photon counting detectors, such as avalanche photodiodes (APDs),
have much higher time resolution (
1MHz) than imaging cameras and high
quantum yield extending to longer wavelengths. They do not need to be cooled,
simplifying construction and reducing their cost. Background count rates as low as
10 counts per second allow counting of photons arriving signi cantly above this
rate [19]. Limitation to a single position in the microscopic field is a disadvantage
because typical experiments result inmany individual molecules worth recording per
microscopic field. A spot detector also requires very accurate motion of the micro-
scope stage to position an individual molecule conjugate toward the detector [20]. For
molecular motor research, the molecules of interest move during the experiment,
and localizing them in line with a spot detector is only a temporary condition. Thus
cameras, mostly EMCCDs, have dominated the experiments. For other research
areas in which the molecules of interest can diffuse into a limited observation region,
spot detectors are used [16]. Arrays of photodiodes are becoming available that retain
high time resolution with single-photon counting sensitivity.
Snells law for refraction at an interface is n 1 sin
>
q
¼
n 2 sin
q
2 , where n 1 and
q
1 are
1
the refractive index (n g ¼
1.515) and incident angle in the glass, and n 2 and
q 2 are the
refractive index in w ¼
1.33) and refracted angle in the aqueous medium. Angles
are relative to the optical axis. Figure 3.2A shows the directions for a series of incident
and refracted rays at various angles depicted by different colors. Because n 2 <
n 1 ,
q 2 >q 1 , the refracted rays are bent away from the optical axis. At a certain critical angle
of incidence,
sin 1 (n 2 /n 1 ), where sin
1, the refracted ray would be parallel to
the interface (a ray at a slightly higher angle than the blue ray in Figure 3.2A). At
q 1 >q c , there is no real solution for
q c ¼
q 2 ¼
q 2 , resulting in total internal re ection (purple
ray). For cover slip glass and water the critical angle is
sin 1 (1.33/1.515)
61.4 .
The numerical aperture of the objective required to obtain higher illumination angles
is greater than a critical value, NA c ¼
q c ¼
¼
n g sin (sin 1 (n w /n g ))
n w . Thus,
TIRFobjectivesmust have NA o substantially greater than 1.33, the index of refraction
of water. Another point that follows from this relationship is that the coupling fluid
n g sin
q c ¼
¼
 
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