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Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation
Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The me...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Molecular Diversity Preservation International (MDPI)
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3907809/ https://www.ncbi.nlm.nih.gov/pubmed/24378851 http://dx.doi.org/10.3390/ijms15010261 |
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author | Biener, Gabriel Stoneman, Michael R. Acbas, Gheorghe Holz, Jessica D. Orlova, Marianna Komarova, Liudmila Kuchin, Sergei Raicu, Valerică |
author_facet | Biener, Gabriel Stoneman, Michael R. Acbas, Gheorghe Holz, Jessica D. Orlova, Marianna Komarova, Liudmila Kuchin, Sergei Raicu, Valerică |
author_sort | Biener, Gabriel |
collection | PubMed |
description | Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects. |
format | Online Article Text |
id | pubmed-3907809 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Molecular Diversity Preservation International (MDPI) |
record_format | MEDLINE/PubMed |
spelling | pubmed-39078092014-01-31 Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation Biener, Gabriel Stoneman, Michael R. Acbas, Gheorghe Holz, Jessica D. Orlova, Marianna Komarova, Liudmila Kuchin, Sergei Raicu, Valerică Int J Mol Sci Article Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects. Molecular Diversity Preservation International (MDPI) 2013-12-27 /pmc/articles/PMC3907809/ /pubmed/24378851 http://dx.doi.org/10.3390/ijms15010261 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland http://creativecommons.org/licenses/by/3.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Biener, Gabriel Stoneman, Michael R. Acbas, Gheorghe Holz, Jessica D. Orlova, Marianna Komarova, Liudmila Kuchin, Sergei Raicu, Valerică Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation |
title | Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation |
title_full | Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation |
title_fullStr | Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation |
title_full_unstemmed | Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation |
title_short | Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation |
title_sort | development and experimental testing of an optical micro-spectroscopic technique incorporating true line-scan excitation |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3907809/ https://www.ncbi.nlm.nih.gov/pubmed/24378851 http://dx.doi.org/10.3390/ijms15010261 |
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