Biomedical Engineering Reference
In-Depth Information
the fluorophore, buffer, embedding matrix, and rejection filter are first selected carefully
with due consideration for how each variable would be controlled when simply optimizing
an experiment for detection of a large number of molecules. Optical isolation of a single mol-
ecule is often the next step as an effective method to further improve signal-to-noise ratio.
This approach attempts to assure that only one molecule is in resonance with the excitation
radiation within an illuminated spot, and ideally results in a situation in which the average
number of molecules in the excitation area is less than one (15).
There are two common experimental conditions that are used to reduce the number of
molecules that are interrogated. The first and easiest approach is to use extremely dilute
sample solutions to deposit or deliver target molecules to a microscopic area where inter-
rogation occurs. Individual molecules in an excitation area (or volume) yield separate
bursts of emission corresponding to different time periods when such molecules are
located within the excitation region of the microscope optics. A second approach is to use
a selective spectroscopic excitation or emission protocol to excite or detect only one mole-
cule in mixture with others in the sample area or volume (16). This method requires that
different molecules in the excitation zone must have sufficiently different excitation or
emission properties so that a signal-to-noise advantage can be achieved.
Several other conditions should be considered to ensure that a fluorescence signal actually
originates from one single molecule (16). Molecules that do not readily aggregate are pre-
ferred for single molecule detection. An emitter should have a substantial absorption and
emission dipole so that spectroscopic properties can be assessed when the molecule is
immobilized. Fluorescence emission from single molecules can exhibit on and off behavior
due to “blinking” or photobleaching. Blinking is a photophysical property that offers dis-
tinctive emission levels, and this characteristic is peculiar to certain single molecule emitters
(and quantum dots). Determination of fluorescence intensity using different concentrations
of fluorophore can be used to correlate the intensity anticipated from individual molecules.
2.3
Methods of Detection
Several optical methods have been used for single molecule detection. Point detection
schemes include confocal (18) and near-field scanning optical microscopies (19). Wide-
field detection schemes include epifluorescence illumination with lamps (20) and
defocused laser excitation (3), and total internal reflection (TIR) methods (21,22). Other
techniques include two-photon fluorescence detection (23) and imaging (24), and sur-
face enhanced Raman spectroscopy (SERS) (25,26). Figure 2.1 suggests the physical prin-
ciples that represent the basis for different instrumental methods (e.g., see [27-37]). The
methods that are presented in this article are meant to be representative but not all inclu-
sive, and will focus on near-field microscopy, CM, wide-field epi-illumination
microscopy, and TIR.
2.3.1
Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy (FCS) measures the time-dependent fluctuating flu-
orescence intensity in a defined area or volume of optical interrogation as the number of
fluorescent molecules changes due to diffusion, or due to transition between fluorescent
and nonfluorescent states (15,28,29). The name of this fluorescence method is given by the
mathematical process of signal analysis. The light emitted from the sampling zone is pro-
portional to the number of fluorescent molecules present, and the fluorescence intensity
signal will change as a result of any chemical and photophysical reactions, and
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