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Molecular order in dynamic systems

All functions of cells are directed. For example, cells move in a specific direction or divide at a certain site and orientation so the two daughter cells are the right size. This direction in living systems emerges from nanoscale arrangement  of molecules, or molecular order. 

Resolving ordered arrangement of molecules in live cells is difficult with light microscopes, even with current fluorescence super-resolution methods. But, the intrinsic polarization of fluorescent emission is a sensitive reporter of molecular order averaged over the spatial resolution of the microscope. Thus, even if it is difficult to resolve molecular order in cells with light microscopes, one can measure the orientation and alignment of biomolecules using fluorescence polarization. Fluorescence polarization is an under-exploited property in multi-dimensional fluorescence microscopy. 

https://sites.google.com/site/shalinmehta/research/dynamicorder/DipoleRadiation.png?attredirects=0


Instantaneous fluorescence PolScope


Fluorescence polarization is a robust reporter of molecular orientation and degree of alignment. Unbiased analysis of molecular order requires viewing the fluorescent specimen through at least three different polarization orientations. Earlier methods acquired these polarization-resolved images sequentially, leading to motion artifacts when imaging fast moving specimens. In addition, earlier methods could not analyze orientation of many single molecules forming a higher-order assembly. In Tomomi Tani's lab, we built an instantaneous fluorescence polarization microscope (instantaneous fluorescence PolScope) to address this measurement challenge.

https://sites.google.com/site/shalinmehta/research/dynamicorder/instantaneousFP+Factin.png

Tracking molecular orientation and flow

I developed computational algorithms that retrieve molecular orientation and flow of fluorescent particles from time and polarization-resolved images taken with instantaneous fluorescence PolScope. This computational microscopy approach opens a new avenue of understanding emergence of microscale ordered assemblies in live cells from nanoscale alignment of molecules.

References
1.
S. B. Mehta, M. McQuilken, P. La Riviere, P. Occhipinti, A. Verma, R. Oldenbourg, A. S. Gladfelter, and T. Tani, "Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells," Proceedings of the National Academy of Sciences PNAS Plus, https://doi.org/10.1073/pnas.1607674113 (2016). Preprint: http://biorxiv.org/content/early/2016/08/12/068767


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