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Title: Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.

Abstract

Over the past decade optical approaches were introduced that effectively break the diffraction barrier. Of particular note were introductions of Stimulated Emission/Depletion (STED) microscopy, Photo-Activated Localization Microscopy (PALM), and the closely related Stochastic Optical Reconstruction Microscopy (STORM). STORM represents an attractive method for researchers, as it does not require highly specialized optical setups, can be implemented using commercially available dyes, and is more easily amenable to multicolor imaging. We implemented a simultaneous dual-color, direct-STORM imaging system through the use of an objective-based TIRF microscope and filter-based image splitter. This system allows for excitation and detection of two fluorophors simultaneously, via projection of each fluorophor's signal onto separate regions of a detector. We imaged the sub-resolution organization of the TLR4 receptor, a key mediator of innate immune response, after challenge with lipopolysaccharide (LPS), a bacteria-specific antigen. While distinct forms of LPS have evolved among various bacteria, only some LPS variations (such as that derived from E. coli) typically result in significant cellular immune response. Others (such as from the plague bacteria Y. pestis) do not, despite affinity to TLR4. We will show that challenge with LPS antigens produces a statistically significant increase in TLR4 receptor clusters on the cell membrane, presumablymore » due to recruitment of receptors to lipid rafts. These changes, however, are only detectable below the diffraction limit and are not evident using conventional imaging methods. Furthermore, we will compare the spatiotemporal behavior of TLR4 receptors in response to different LPS chemotypes in order to elucidate possible routes by which pathogens such as Y. pestis are able to circumvent the innate immune system. Finally, we will exploit the dual-color STORM capabilities to simultaneously image LPS and TLR4 receptors in the cellular membrane at resolutions at or below 40nm.« less

Authors:
; ;
Publication Date:
Research Org.:
Sandia National Laboratories
Sponsoring Org.:
USDOE
OSTI Identifier:
1030322
Report Number(s):
SAND2010-6756C
TRN: US201124%%115
DOE Contract Number:  
AC04-94AL85000
Resource Type:
Conference
Resource Relation:
Conference: Proposed for presentation at the Biophysical Society 55th Annual Meeting held March 5-9, 2011 in Baltimore, MD.
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; AFFINITY; ANTIGENS; BACTERIA; CELL MEMBRANES; DETECTION; DIFFRACTION; DYES; EXCITATION; LIPIDS; LIPOPOLYSACCHARIDES; MEMBRANES; MICROSCOPES; MICROSCOPY; PATHOGENS; PROTEINS

Citation Formats

Carson, Bryan D., Aaron, Jesse S., and Timlin, Jerilyn Ann. Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.. United States: N. p., 2010. Web.
Carson, Bryan D., Aaron, Jesse S., & Timlin, Jerilyn Ann. Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.. United States.
Carson, Bryan D., Aaron, Jesse S., and Timlin, Jerilyn Ann. Fri . "Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.". United States.
@article{osti_1030322,
title = {Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.},
author = {Carson, Bryan D. and Aaron, Jesse S. and Timlin, Jerilyn Ann},
abstractNote = {Over the past decade optical approaches were introduced that effectively break the diffraction barrier. Of particular note were introductions of Stimulated Emission/Depletion (STED) microscopy, Photo-Activated Localization Microscopy (PALM), and the closely related Stochastic Optical Reconstruction Microscopy (STORM). STORM represents an attractive method for researchers, as it does not require highly specialized optical setups, can be implemented using commercially available dyes, and is more easily amenable to multicolor imaging. We implemented a simultaneous dual-color, direct-STORM imaging system through the use of an objective-based TIRF microscope and filter-based image splitter. This system allows for excitation and detection of two fluorophors simultaneously, via projection of each fluorophor's signal onto separate regions of a detector. We imaged the sub-resolution organization of the TLR4 receptor, a key mediator of innate immune response, after challenge with lipopolysaccharide (LPS), a bacteria-specific antigen. While distinct forms of LPS have evolved among various bacteria, only some LPS variations (such as that derived from E. coli) typically result in significant cellular immune response. Others (such as from the plague bacteria Y. pestis) do not, despite affinity to TLR4. We will show that challenge with LPS antigens produces a statistically significant increase in TLR4 receptor clusters on the cell membrane, presumably due to recruitment of receptors to lipid rafts. These changes, however, are only detectable below the diffraction limit and are not evident using conventional imaging methods. Furthermore, we will compare the spatiotemporal behavior of TLR4 receptors in response to different LPS chemotypes in order to elucidate possible routes by which pathogens such as Y. pestis are able to circumvent the innate immune system. Finally, we will exploit the dual-color STORM capabilities to simultaneously image LPS and TLR4 receptors in the cellular membrane at resolutions at or below 40nm.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2010},
month = {10}
}

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