Biomedical Engineering Reference
In-Depth Information
different fluorescence
properties related to open and closed conformations of
biopolymers.
Two-photon molecular excitation is performed by very high local intensity provided
by tight focusing in a laser scanning microscopy (LSM) (Denk et al., 1990; Heinze et al.,
2000). This technique is combined with the temporal concentration of femtosecond
pulsed lasers that produce a stream of pulses with a pulse duration of about 100 fs at a
repetition rate of about 80 MHz. An average incident laser power which can saturate the
fluorescence output has been estimated as about 50 mV (about
Advantages of the two-photon laser spectroscopy are as follows: high resolution,
tolerance of infrared light by biological objects, different selection rule and vibronic
coupling. The latter feature allows the accomplishment of simultaneous two- photon and
one-photon excitation.
A dual-color cross-correlation fluorescence spectroscopy (DCCFS) appears to be a
variant of SMM that is the most suitable for direct monitoring of enzymatic reactions
(Winkler et al., 1999; Heinze et al., 2000). In DCCFS experiments, a sample, containing
two fluorophores with different emissions in each molecule, is irradiated with two lasers
(or with one laser) to perform simultaneous excitation of the fluorophores. The DCCFS
in combination with the confocal laser microscopy allows the separation of microcopic
volume with two different fluorophores from volume with only one of them and,
therefore, the monitoring of dissociation of the dual-labeled molecules or association of
two single-labeled molecules. The confocal fluorescence coincidence analysis has been
employed for a rapid homogeneous assay for restriction endonuclease EcoRI (Winkler et
al., 1999). This methodology has been improved by the application of two-photon
excited dual-color cross-correlation spectroscopy on the level of single diffusing
molecules (Heinze et al., 2000). A double-strand of DNA was labeled with Rhodamin
green and Texas red. The kinetics of the enzymatic cleavage of the labeled DNA by
restriction endonuclease was monitored by this new technique.
Two-photon laser fluorescence
In confocal spectroscopy, the exciting laser beam is focused to a diffraction- limited
sport by illumination of a high numerical aperture objective. A pinhole in the image
plane serves as a field diagram and discriminates against out-of-focus fluorescence. The
optically defined detection volume is usually of the order of
liters. The high
resolution of technique in the single
particle regime allows the investigation of
molecular objects at nanomolar concentrations. Fluorescence correlation spectroscopy
is an effective tool for measurement of local concentrations, investigation of partile
diffusion, intramolecular dynamics, association and dissociation rates and enzymatic
activity (Denk et al., 1990; Winkler et al., 1990; Schwille et al., 2000; Elson and Rigler,
2000; Heinze et al., 2000).
Two-photon excitation of a fluorescent within the cross section of the day molecule
about is an induced probe for time about by laser light in the visible or
near UV spectral range (Denk et. al., 1990). Such an excitation requires instaneous
photon flux densities of the order of
-
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