High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope

Yongxiao Li; Samantha J. Montague; Anne Brüstle; Xuefei He; Cathy Gillespie; Katharina Gaus; Elizabeth E. Gardiner; Woei Ming Lee

doi:10.1002/jbio.201700341

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope

Yongxiao Li
, Samantha J. Montague
, Anne Brüstle
, Xuefei He
, Cathy Gillespie
, Katharina Gaus
, Elizabeth E. Gardiner
, Woei Ming Lee^*

^*Corresponding author for this work

Cardiovascular Sciences

Research output: Contribution to journal › Article › peer-review

5 Citations (Scopus)

Abstract

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system. (Figure presented.).

Original language	English
Article number	e201700341
Journal	Journal of Biophotonics
Volume	11
Issue number	7
DOIs	https://doi.org/10.1002/jbio.201700341
Publication status	Published - Jul 2018

Bibliographical note

Funding Information:
We thank M. Castanares, Y. Wang, I. Cockburn, M. White and V. Daria for experimental assistance. We are grateful for additional funding support from William Heath. We also recognize B. Condon for making the trigger amplifier electronic circuit. We thank C. Alt (MGH), A. Upadhya, Thomas Mcmenamin and T. Kamal for detailed proof reading of the manuscript.

Funding Information:
We thank M. Castanares, Y. Wang, I. Cockburn, M. White and V. Daria for experimental assistance. We are grateful for additional funding support from William Heath. We also recognize B. Condon for making the trigger amplifier electronic circuit. We thank C. Alt (MGH), A. Upadhya, Thomas Mcmenamin and T. Kamal for detailed proof reading of the manuscript. W.M.L. conceived and supervised the project. W.M.L. and Y.L. designed and constructed the optical system. Y.L. developed the control system, wrote the PScan software and processed and analyzed all the experimental data. Y.L., W.M.L. and S.J.M. performed the in vivo experiments with support from E.E.G., C.G., K.G., A.B. and C.G. X.H. assisted with fluorescence beads and GUVs fabrication. All authors participated in discussions and data interpretation. Y.L. and W.M.L. wrote the paper with input from all authors.

Publisher Copyright:
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Keywords

multiphoton microscopy
optical design and fabrication
scanning microscopy

ASJC Scopus subject areas

General Chemistry
General Materials Science
General Biochemistry,Genetics and Molecular Biology
General Engineering
General Physics and Astronomy

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10.1002/jbio.201700341

Cite this

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title = "High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope",

abstract = "In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r \textasciitilde{}10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system. (Figure presented.).",

keywords = "multiphoton microscopy, optical design and fabrication, scanning microscopy",

author = "Yongxiao Li and Montague, \{Samantha J.\} and Anne Br{\"u}stle and Xuefei He and Cathy Gillespie and Katharina Gaus and Gardiner, \{Elizabeth E.\} and Lee, \{Woei Ming\}",

note = "Funding Information: We thank M. Castanares, Y. Wang, I. Cockburn, M. White and V. Daria for experimental assistance. We are grateful for additional funding support from William Heath. We also recognize B. Condon for making the trigger amplifier electronic circuit. We thank C. Alt (MGH), A. Upadhya, Thomas Mcmenamin and T. Kamal for detailed proof reading of the manuscript. Funding Information: We thank M. Castanares, Y. Wang, I. Cockburn, M. White and V. Daria for experimental assistance. We are grateful for additional funding support from William Heath. We also recognize B. Condon for making the trigger amplifier electronic circuit. We thank C. Alt (MGH), A. Upadhya, Thomas Mcmenamin and T. Kamal for detailed proof reading of the manuscript. W.M.L. conceived and supervised the project. W.M.L. and Y.L. designed and constructed the optical system. Y.L. developed the control system, wrote the PScan software and processed and analyzed all the experimental data. Y.L., W.M.L. and S.J.M. performed the in vivo experiments with support from E.E.G., C.G., K.G., A.B. and C.G. X.H. assisted with fluorescence beads and GUVs fabrication. All authors participated in discussions and data interpretation. Y.L. and W.M.L. wrote the paper with input from all authors. Publisher Copyright: {\textcopyright} 2018 WILEY-VCH Verlag GmbH \& Co. KGaA, Weinheim",

year = "2018",

month = jul,

doi = "10.1002/jbio.201700341",

language = "English",

volume = "11",

journal = "Journal of Biophotonics",

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AU - Li, Yongxiao

AU - Montague, Samantha J.

AU - Brüstle, Anne

AU - He, Xuefei

AU - Gillespie, Cathy

AU - Gaus, Katharina

AU - Gardiner, Elizabeth E.

AU - Lee, Woei Ming

N1 - Funding Information: We thank M. Castanares, Y. Wang, I. Cockburn, M. White and V. Daria for experimental assistance. We are grateful for additional funding support from William Heath. We also recognize B. Condon for making the trigger amplifier electronic circuit. We thank C. Alt (MGH), A. Upadhya, Thomas Mcmenamin and T. Kamal for detailed proof reading of the manuscript. Funding Information: We thank M. Castanares, Y. Wang, I. Cockburn, M. White and V. Daria for experimental assistance. We are grateful for additional funding support from William Heath. We also recognize B. Condon for making the trigger amplifier electronic circuit. We thank C. Alt (MGH), A. Upadhya, Thomas Mcmenamin and T. Kamal for detailed proof reading of the manuscript. W.M.L. conceived and supervised the project. W.M.L. and Y.L. designed and constructed the optical system. Y.L. developed the control system, wrote the PScan software and processed and analyzed all the experimental data. Y.L., W.M.L. and S.J.M. performed the in vivo experiments with support from E.E.G., C.G., K.G., A.B. and C.G. X.H. assisted with fluorescence beads and GUVs fabrication. All authors participated in discussions and data interpretation. Y.L. and W.M.L. wrote the paper with input from all authors. Publisher Copyright: © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

PY - 2018/7

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N2 - In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system. (Figure presented.).

AB - In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system. (Figure presented.).

KW - multiphoton microscopy

KW - optical design and fabrication

KW - scanning microscopy

UR - https://www.scopus.com/pages/publications/85044921926

U2 - 10.1002/jbio.201700341

DO - 10.1002/jbio.201700341

M3 - Article

C2 - 29488344

AN - SCOPUS:85044921926

SN - 1864-063X

VL - 11

JO - Journal of Biophotonics

JF - Journal of Biophotonics

IS - 7

M1 - e201700341

ER -

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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