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Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy
Published on: December 3, 2012
Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones
Atsushi Matsuda1, Lin Shao, Jerome Boulanger
1Department of Biochemistry and Biophysics, The Keck Center for Advanced Microscopy, University of California San Francisco, San Francisco, California, United States of America.
Plos One
|September 22, 2010
Summary
This study introduces a high-resolution Photoactivated Localization Microscopy (PALM) technique using enhanced algorithms and conventional EGFP. The method achieves super-resolution imaging of chromatin structure, overcoming limitations of existing methods.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Photoactivated localization microscopy (PALM) offers nanoscale resolution but is limited by specialized fluorescent probes and high background noise.
- Existing methods struggle with incorporating specific fluorescent molecules and effectively reducing autofluorescence or out-of-focus signals in 3D imaging.
Purpose of the Study:
- To develop a high-resolution PALM method adaptable for common biological samples, overcoming limitations of probe requirements and background noise.
- To apply this improved PALM technique for visualizing higher-order chromatin structures with nanoscale precision.
Main Methods:
- Utilized enhanced algorithms for processing raw PALM images, incorporating denoising and deconvolution to mitigate biological background noise.
- Employed a conventional enhanced green fluorescent protein (EGFP) as the photoconvertible fluorophore, activated by blue light in the presence of reduced riboflavin.
- Applied the developed PALM method to image Drosophila mitotic chromosomes labeled with H2AvD-EGFP.
Main Results:
- Demonstrated efficient conversion of EGFP emission from green to red using riboflavin, achieving a photon yield comparable to other bright photoconvertible proteins.
- Showcased significant improvement in spatial resolution through image pre-processing, enabling reconstruction from noisy datasets.
- Revealed filamentous chromatin structures approximately 70 nm in size in Drosophila mitotic chromosomes, achieving resolutions comparable to electron microscopy.
Conclusions:
- The developed PALM technique facilitates super-resolution imaging of biological samples using common fluorescent proteins like EGFP and improved image processing.
- This method effectively addresses challenges posed by biological background noise and probe limitations, enabling visualization of fine cellular structures.
- Provides unprecedented nanoscale insights into chromatin organization, specifically visualizing histone variant-specific filaments.
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