conducting-multiple-imaging-modes-with-one-fluorescence-microscope

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

University of Chicago

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CHNHPbBr perovskites and measure differences between nanocrystal films and micron scale crystals.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest.

T-box riboswitches are -regulatory RNA elements that regulate the expression of proteins involved in amino acid biosynthesis and transport by binding to specific tRNAs and sensing their aminoacylation state. While the T-box modular structural elements that recognize different parts of a tRNA have been identified, the kinetic trajectory describing how these interactions are established temporally remains unclear. Using smFRET, we demonstrate that tRNA binds to the riboswitch in two steps, first anticodon recognition followed by the sensing of the 3' NCCA end, with the second step accompanied by a T-box riboswitch conformational change. Studies on site-specific mutants highlight that specific T-box structural elements drive the two-step binding process in a modular fashion. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches, and suggest such binding mechanism is kinetically beneficial for efficient, co-transcriptional recognition of the cognate tRNA ligand.

Article	Year
A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest. The Review of scientific instruments\| PubMed ID: 27910631	2016
Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand. eLife\| PubMed ID: 30251626	2018

Jiacheng Zhang

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