Chemistry Reference
In-Depth Information
which acquires
film of a large
field-of-view (FOV) near video rate (30-ms frames), but
may be signi
cantly faster for smaller FOVs (
1 kHz). As mentioned above, such
sensitive CCD cameras allow single-molecule detection to be undertaken on a wide
area. Themain drawback is that the time resolution is currently limited to thems time
range. This does not allow time-correlation information below the millisecond time
scale (see data analysis below) or
fluorescence lifetime information to be obtained.
Gated CCD cameras are available to obtain
fluorescence lifetime information, but a
large proportion of signal is lost using gating techniques [48].
The combination of wide-area detection and photon counting would rem-
ove the timing drawbacks of TIR methods, or a allow detection with high time
resolution from many confocal spots simultaneously. Such a detector is under
development [49, 50].
9.3.5
Data Reduction and Analysis Methods
The most general way to view single-molecule data is as a stream of photon-detection
events. All of the single-molecule data reduction and analysis methods branch off
from this point. Data reduction is the process by which the raw data is transformed
into time traces, histograms, and correlations, and sorted into single-molecule events
that can then be analyzed. Data analysis is the process by which the reduced data is
interpreted to provide quantitative results.
9.3.5.1 Photon Streams and Films
When detected, a photon carries several pieces of information. There is the arrival
time of the photon, its polarization, energy or wavelength, and spatial position at the
image plane. With current detection schemes, not all of this information can be
obtained simultaneously.
For confocal microscopy with APD detection, only the arrival time of the photon is
directly recorded. The spatial position of the photon is only used to exclude out-of-
focus light at the pinhole. In order to obtain other information about wavelength and
polarization, detection channels are de
ned using optical
filters and dichroic and
polarizing beamsplitters that correspond to speci
c polarization and wavelength
ranges. Spatial information about the sample is obtained by scanning the sample, the
beam [51], or even the objective (Microtime 200, Picoquant, Berlin, Germany). The
information obtained from such detection schemes is a list of photons, containing
the arrival time and detection channel. In
fluorescence lifetimemeasurements using
TCSPC, two arrival times are obtained for each photon detected: one measured with
respect to the beginning of the measurement (at a relatively low temporal resolu-
tion of
10 ns), and onemeasured as a time difference with respect to the laser pulse
(typically at higher time resolution, of
4
-
50 ps). Some data acquisition techniques
acquire data as numbers of photon counts over
fixed time widths (photon binning).
The photon timing technique is in general preferred (because of higher information
content), unless photon count rates are so high that the size of the data
files becomes
unmanageable.
