Project Summary:
protein-protein
used
combine
becoming
and
designing
for
adapted
used
this
imaging
detector
utilized
different
channel
lightsheet
pipeline
handling
fluorescence
Fluorescence anisotropy (or polarization) is routinely used for investigating
interactions at high resolution in cellular environments. This imaging approach is
in onjunction with TIRF, confocal or epi-illumination and lately there have been efforts to
i t with lightsheet. Among the available fluorescence imaging modalities, lightsheet is
a method of choice for 3D imaging of live samples because of its fast-imaging speed
gentleness. We ropose to leverage our experience in building lightsheet systems and
polarization optics to advance the field of fluorescence polarization i maging. Our aim
this proposal i s to combine these two modalities in a manner such that it can easily be
and used for biological applications and quantitative analysis. Our previous research has
a specifically designed Wollaston prism for imaging single color fluorescence anisotropy. In
application, we propose to build a dual-color optical splitter for fluorescence polarization
which will employ the Wollaston prism for polarization separation and a single area
such as sCMOS camera (Aim 1a). Fabricated nanostructured pinhole arrays will be
to characterize the optical splitter for field dependent distortions, transmission losses in
channels (due to various optical components), and to validate the registration of multi-
for polarization computation. We will also combine this splitter with the home-built
systems for 3D live imaging of cells and embryos (Aim 1b). A tailored image analysis
along with a user-friendly graphical user interface (GUI) will be developed for data
and quantitative image analysis (Aim 2). Our proposed approach would allow us t o map
anisotropy for investigating protein-protein interactions in 3D in live samples.
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