Biomedical Engineering Reference
In-Depth Information
useful for investigating the kinetic behavior (diffusion and binding) of proteins in
living cells and have been widely used by biologists to study a range of proteins
(especially membrane proteins) since the emergence of GFP [
112
-
114
]. In a FRAP
experiment, the fluorescent molecules in a selected region of interest of a cell
are bleached using high-intensity excitation light (bleaching phase), followed by
monitoring the diffusion of new fluorescent molecules into the bleached area over
a period of time with low-intensity excitation light (recovery phase); fitting the
recorded data into a defined kinetic model can provide quantitative estimates for
the mobile fraction of the fluorescent molecules and the rate of mobility.
Fluorescence correlation spectroscopy (FCS)
(also called fluorescence fluc-
tuation spectroscopy), originally developed by Magde, Elson, and Webb [
115
],
has become a major imaging tool for studying molecular diffusion, binding,
and interactions both
in vivo
and
in vitro
[
116
-
120
]. FCS measurements are
usually carried out at selected points of a sample on a confocal or 2P scanning
microscope equipped with a high numerical aperture objective lens and photon-
counting PMT or APD detectors. At each point, the intensity fluctuations due to
number of fluorescent molecules moving into or out of the excitation volume are
recorded and then analyzed to estimate the diffusion rate and concentration of the
fluorescent molecules. Compared to FRAP, FCS can reveal much faster dynamics
such as blinking [
121
]. Fluorescence cross-correlation spectroscopy (FCCS) is an
extended version of FCS and allows studying the dynamic interactions between two
differently colored species [
117
,
118
,
120
].
Image correlation spectroscopy (ICS)
(also called image correlation microscopy)
and associated techniques, including image cross-correlation spectroscopy (ICCS)
[
122
-
125
], raster image correlation spectroscopy (RICS), and cross-correlation
RICS [
121
,
126
,
127
], were derived from FCS and FCCS and have also been used
to measure diffusion coefficients, flow rates, and concentrations of fluorescently
labeled proteins in cells. Image correlation techniques can be implemented on
a regular confocal or TPE scanning microscope. Using both ICS- and FCS-
related techniques, quantitative characterization of the diffusion and concentration
parameters based on a defined diffusion model requires calibration of the size of the
excitation volume for fitting the correlation function calculated from the measured
data into the model.
Superresolution microscopy
refers to a collection of several new fluorescence
microscopy techniques, which provide sub-diffraction-limited resolutions. Due to
the diffraction limit elucidated by Ernst Abbe in the late 1800s, the fundamental
resolution limit of light microscopy is approximately 200 nm. Recently, several
techniques including STED microscopy, PALM, and STORM have been demon-
strated to circumvent this physical barrier and to achieve lateral resolutions of
less than 50 nm [
128
-
130
]. STED microscopy [
131
-
133
] uses two lasers: one for
excitation of the fluorophores to induce their fluorescent state and the other for
de-excitation of the fluorophores by means of stimulated emission; this achieves
the sub-diffraction-limited resolution by engineering the point spread functions
of the two laser beams so that only the fluorophores within a sub-diffraction-
limited volume get excited. Both PALM [
134
-
138
]andSTORM[
139
-
141
] utilize
Search WWH ::
Custom Search