**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.