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Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate

Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse have been requi...

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Autores principales: Lisicovas, Viktoras, Mariserla, Bala Murali Krishna, Sahoo, Chakradhar, Harding, Reuben T., Man, Michael K. L., Wong, E Laine, Madéo, Julien, Dani, Keshav M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789829/
https://www.ncbi.nlm.nih.gov/pubmed/31357714
http://dx.doi.org/10.3390/mps2030065
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author Lisicovas, Viktoras
Mariserla, Bala Murali Krishna
Sahoo, Chakradhar
Harding, Reuben T.
Man, Michael K. L.
Wong, E Laine
Madéo, Julien
Dani, Keshav M.
author_facet Lisicovas, Viktoras
Mariserla, Bala Murali Krishna
Sahoo, Chakradhar
Harding, Reuben T.
Man, Michael K. L.
Wong, E Laine
Madéo, Julien
Dani, Keshav M.
author_sort Lisicovas, Viktoras
collection PubMed
description Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse have been required in order to provide the high peak power densities for effective two-photon excitation over the large area. However, this configuration suffers from reduced signal intensity due to the low repetition rate, saturation effects due to increased excitation fluences, as well as faster photobleaching of the fluorescence probe. In contrast, with the recent advent of high repetition rate, high pulse energy laser systems could potentially provide the advantages of high repetition rate systems that are seen in traditional two-photon microscopes, while minimizing the negatives of high fluences in WF-TeFo setups to date. Here, we use a 100 microjoule/high repetition rate (50–100 kHz) laser system to investigate the performance of a WF-TeFo two-photon microscope. While using micro-beads as a sample, we demonstrate a proportionate increase in signal intensity with repetition rate, at no added cost in photobleaching. By decreasing pulse intensity, via a corresponding increase in repetition rate to maintain fluorescence signal intensity, we find that the photobleaching rate is reduced by ~98.4%. We then image live C. elegans at a high repetition rate for 25 min. as a proof-of-principle. Lastly, we identify the steady state temperature increase as the limiting process in further increasing the repetition rate, and we estimate that repetition rate in the range between 0.5 and 5 MHz is ideal for live imaging with a simple theoretical model. With new generation low-cost fiber laser systems offering high pulse energy/high repetition rates in what is essentially a turn-key solution, we anticipate increased adoption of this microscopy technique by the neuroscience community.
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spelling pubmed-67898292019-10-16 Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate Lisicovas, Viktoras Mariserla, Bala Murali Krishna Sahoo, Chakradhar Harding, Reuben T. Man, Michael K. L. Wong, E Laine Madéo, Julien Dani, Keshav M. Methods Protoc Article Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse have been required in order to provide the high peak power densities for effective two-photon excitation over the large area. However, this configuration suffers from reduced signal intensity due to the low repetition rate, saturation effects due to increased excitation fluences, as well as faster photobleaching of the fluorescence probe. In contrast, with the recent advent of high repetition rate, high pulse energy laser systems could potentially provide the advantages of high repetition rate systems that are seen in traditional two-photon microscopes, while minimizing the negatives of high fluences in WF-TeFo setups to date. Here, we use a 100 microjoule/high repetition rate (50–100 kHz) laser system to investigate the performance of a WF-TeFo two-photon microscope. While using micro-beads as a sample, we demonstrate a proportionate increase in signal intensity with repetition rate, at no added cost in photobleaching. By decreasing pulse intensity, via a corresponding increase in repetition rate to maintain fluorescence signal intensity, we find that the photobleaching rate is reduced by ~98.4%. We then image live C. elegans at a high repetition rate for 25 min. as a proof-of-principle. Lastly, we identify the steady state temperature increase as the limiting process in further increasing the repetition rate, and we estimate that repetition rate in the range between 0.5 and 5 MHz is ideal for live imaging with a simple theoretical model. With new generation low-cost fiber laser systems offering high pulse energy/high repetition rates in what is essentially a turn-key solution, we anticipate increased adoption of this microscopy technique by the neuroscience community. MDPI 2019-07-28 /pmc/articles/PMC6789829/ /pubmed/31357714 http://dx.doi.org/10.3390/mps2030065 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lisicovas, Viktoras
Mariserla, Bala Murali Krishna
Sahoo, Chakradhar
Harding, Reuben T.
Man, Michael K. L.
Wong, E Laine
Madéo, Julien
Dani, Keshav M.
Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate
title Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate
title_full Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate
title_fullStr Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate
title_full_unstemmed Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate
title_short Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate
title_sort improving signal and photobleaching characteristics of temporal focusing microscopy with the increase in pulse repetition rate
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789829/
https://www.ncbi.nlm.nih.gov/pubmed/31357714
http://dx.doi.org/10.3390/mps2030065
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