| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biophys J, October 2000, p. 1692-1694, Vol. 79, No. 4
Department of Chemistry, University of Washington, Seattle, Washington 98195 USA
Molecular Brownian motions in dense
macromolecular suspensions and glassy liquids may involve extensively
correlated particle movements and substantial temporal and spatial
heterogeneity (Cicerone et al., 1996 The translational dynamics of the same mitochondrial reticulum under
different physiological conditions are investigated by both DVFM and
FICS. S(k, t) is computed directly
from the DVFM images for comparison with FICS, and the two methods are found to agree quantitatively. The observed dynamics are shown to arise
from a superposition of thermally excited motions and presumably driven
motions that require ATP synthesis, and both kinds of motions are
characterized in considerable detail. These results shed significant
new light on the complex motions of the mitochondrial reticulum and
their dependence upon metabolic state. However, the full potential of
the FICS method is not yet fully realized and the best may be yet to come.
In subsequent work, the FICS method is extended to obtain the
trajectories of the separate real and imaginary parts of
A(k, t) (A. Marcus, personal
communication), which allows a determination of the complex
S(k, t), instead of just its absolute
magnitude. Although S(k, t) is purely
real for ergodic thermal motions, it becomes complex in the presence of
directional driven motions, including uniform translation. Knowledge of
the complex S(k, t) should allow one
to ascertain the presence of directional driven motions and to
characterize them in regard to direction and velocity. Thus, FICS
should become a powerful tool for investigating protoplasmic streaming
and the intracellular trafficking of particular labeled particles. It
should also be possible to examine the in- and out-of-phase responses
of particle positions to sinusoidal perturbations, such as alternating
electric or shear fields. In addition, any long-lived spatial
heterogeneity of the molecular dynamics should be manifested by a
difference in the decays of the autocorrelation functions of the real
and imaginary parts of A(k, t), and
can be detected in that way. The spatial scale of those heterogeneities could assessed by comparing such differences for a range of
k values. Another promising new application of FICS is the
study of ligand binding kinetics (A. Marcus, personal communication). Whenever binding events are accompanied by a change in quantum yield of
a fluorophore, they contribute along with particle translation to the
decay of S(k, t). By combining
measurements at two or more different values of k, the
relevant chemical relaxation time can be unambiguously assessed. An
important difference between the FICS method and conventional
fluorescence correlation spectroscopy (FCS) with spot illumination, is
that the signal-to-noise ratio increases with increasing N
in FICS, whereas it decreases with increasing N (for zero
fluorescence background) in FCS. Thus, in FICS it should be possible to
work with much greater signals, and thereby access much
more rapid chemical reactions. The FICS method can also be extended to
study the rotational dynamics of fluorophores in a manner analogous to
depolarized dynamic light scattering.
FICS is one of the most promising new techniques for studying
molecular motions in complex systems, including living systems, that
has appeared in recent years. Moreover, the method can be implemented
using Raman scattering, two-photon fluorescence, and four-wave mixing
in addition to normal fluorescence (A. Marcus, personal communication).
ARTICLE
; Ediger et al., 1996; Marcus et
al., 1999; Weeks et al., 2000). Living systems may exhibit even more
complex Brownian dynamics due to the dispersion in their molecular
sizes and shapes. In addition they may also exhibit complex non-thermal
motions that are driven ultimately by ATP hydrolysis. Although the
translations of individual molecular motors along their conjugate
filaments have been extensively studied, little is known about other
non-thermal motions, such as how cytoskeletal motions within the cell
affect the movements of other molecules, complexes, and organelles. The use of fluorescent labels to track the motions of particular species is
now common, and in principle digital video fluorescence microscopy (DVFM) is the tool of choice for simultaneously tracking the positions of all labeled particles in the field of view in order to study their
complex motions. However, DVFM suffers from certain limitations: 1)
photobleaching of the fluorescent labels restricts the time over which
any given particle can be tracked; 2) the temporal resolution (time
between images) may be inadequate to examine certain rapid motions; and
3) extensive computation is generally required to reduce the data for
many particle trajectories down to the relevant statistical quantities
that characterize their motions. The new technique of Fourier imaging
correlation spectroscopy (FICS) introduced by Margineantu et al. in
this issue of Biophysical Journal provides an alternative
approach that substantially alleviates these problems. FICS can be
regarded as a fluorescence-based analogue of dynamic light scattering
(DLS) that takes full advantage of both the greater sensitivity and
labeling specificity that is afforded by fluorophores. A traveling
fringe excitation method (Hattori et al., 1996) is extended in order to
vary the wavelength of the fringe (or wave vector k) and to
provide potentially much greater time resolution, and is applied to
analyze microscopically imaged samples. The sample, which contains
fluorophores at positions rj, j = 1,... N, is excited by an optical grating of constant wavelength that translates continuously across the sample at a constant
velocity, so the resulting fluorescence intensity oscillates in time
with a period that is much shorter than the time scale of any molecular
motion of interest. The fluorescence signal varies as a consequence of
the oscillatory excitation and the motion of the fluorophores, which
alters the amplitudes, A(k, t) = 
j
exp[ik · rj], of the different Fourier
components of the fluorophore spatial distribution. The fluorescence is
detected by a lock-in amplifier, whose average output over several
cycles of oscillation is proportional to A(k,
t) for the particular optically excited Fourier component.
Subsequent autocorrelation of A(k,
t) 2 yields the relevant statistical
information regarding the fluorophore dynamics in a manner directly
analogous to the intensity autocorrelation function of dynamic light
scattering. Provided the real and imaginary parts of
A(k, t) are nearly gaussian random
variables, which is the case whenever the fluorescence comes from
numerous independently moving or fluctuating domains, the
autocorrelation function of A(k,
t) 2 provides the square of the
dynamic structure factor, S(k, t) = (1/N)
<jm
exp[ik.(rj(0) 
rm(t))]>. This two-time
correlation function has been theoretically investigated for many
different models of particle motions and fluctuations, and has provided
numerous insights into the dynamics of complex systems studied by DLS. By varying the grating spacing, the dynamics of different Fourier components can be examined one at a time in serial fashion. This is
crucial for understanding the diffusive dynamics of strongly interacting or confined species, as well as certain non-thermal motions.
| |
FOOTNOTES |
|---|
Received for publication 11 August 2000 and in final form 21 August 2000.
Address reprint requests to J. Michael Schurr, Department of Chemistry, P.O. Box 351700, University of Washington, Seattle, WA 98195. Tel.: 206-543-6681; Fax: 206-685-8665; E-mail: schurr{at}chem.washington.edu.
| |
REFERENCES |
|---|
Biophys J, October 2000, p. 1692-1694, Vol. 79, No. 4
© 2000 by the Biophysical Society 0006-3495/00/10/1692/03 $2.00
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
