Two -Photon Standing Wave Fluorescence Photobleaching With Application to the Study of Chromatin Motion
Davis, Sara Kay
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Permalink
https://hdl.handle.net/2142/84149
Description
Title
Two -Photon Standing Wave Fluorescence Photobleaching With Application to the Study of Chromatin Motion
Author(s)
Davis, Sara Kay
Issue Date
2004
Doctoral Committee Chair(s)
Christopher J. Bardeen
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Language
eng
Abstract
A new method of measuring molecular diffusion rates in microscopic sample volumes is described. This method utilizes the standing wave interference created by colliding two counterpropagating laser beams at the focus of two opposing microscope objectives, resulting in a periodic light distribution in a volume on the order of a femtoliter and spatial resolution on the order of 100 nm. This two-photon counterpropagating fluorescence recovery after patterned photobleaching (2P-c-FRAPP) method is applied to the study of diffusive motion of chromatin in live cells and isolated nuclei. The chromatin is labeled using the DNA minor groove binding dye Hoechst 33342 or GFP labeled histone H2B. The diffusion is highly constrained and cannot be observed in a standard, single beam fluorescence recovery after photobleaching experiment. To determine the chemical origin of the diffusion, we study motion in isolated nuclei and vary the strength of the histone-DNA interactions by changing the ionic strength and using chemical and photocross-linking experiments. At higher NaCl concentrations, we see increased chromatin diffusion as the histone-DNA interaction is weakened due to ionic screening, while photocross-linking the core histories to the DNA results in a complete absence of diffusive motion. These trends are consistent with the 100 nm scale motion being correlated with the interactions of histone proteins with the DNA. If chromatin diffusion is connected to the nucleosomal dynamics on much smaller lengthscales, this may provide a way to assay biochemical activity in vivo based on larger scale macromolecular dynamics observed via fluorescence microscopy.
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