**Page is still
under development (must add narrative for
optical tweezers).**
We invented an optical approach, laser-tracking microrheometry
(LTM), to quantify cellular mechanics during pathogen
infection. With subnanometer and near-microsecond
resolution, we can track the Brownian “dancing” of
microscopic particles, and appropriate analysis
of their motions reveals their mechanical microenvironment.
In polymeric test materials, LTM is very accurate
(<15%) and very fast (~1s) in measuring mechanical
properties. LTM provides unique abilities for noninvasive
mapping of subcellular mechanical properties.
Despite its revolutionary promise, LTM has three limitations
and, hence, three areas of improvement:
1) Add complementary optical
tweezers. For statistical
confidence, the duration of data acquisition
during LTM must be 3-10 times longer
than the slowest mechanical response
of interest. To achieve ~25% certainty
of mechanical modulus at 1s, 3s of data
must be acquired. By averaging longer
windows of time, there is the implicit
assumption that underlying processes
have stationary kinetic probabilities.
Although this is not a problem for reconstituted
polymers, dynamic cells clearly violate
this assumption if observation periods
are extended too long. Instead, cells
must be directly deformed and observed
at the relevant time scales. A new generation
of optical tweezers must be built to
complement LTM so that both LTM
and deformations by optical forces can
occur simultaneously.
2) Add
three-dimensional tracking. Our
current version of LTM uses only two-dimensional
tracking, hence limiting us to very flat
cells. Because most cells are inherently
three-dimensional, many cellular phenomena,
including secretion and internal particle
trafficking, cannot be measured by our
equipment. The optical design for three-dimensional
tracking is straightforward, but developing
real-time calibration strategies in living
cells and maintaining compatibility with
optical tweezers are the project challenges.
3) Develop
fluorescence-based LTM. For
its high spatial resolution, LTM is currently
limited to flat, optically "clean" cells.
Because of the presence of out-of-focus
scatterers, measuring the mechanics of
thick cells, which include most secretory
cells, and dense subcellular regions,
including inside the nucleus, are not
feasible. Fluorescence offers a method
to reject out-of-focus light scatterers. Using
laser-based fluorescence excitation, fluorescence
correlation spectroscopy (FCS) is
commercially available and can measure
diffusion, size and affinities of biomolecules.
We propose to modify FCS techniques and
apply the analysis of LTM so that local
mechanical properties can be determined
in new areas of living cells.
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