Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging
We have developed a nonlinear adaptive optics microscope utilizing a deformable membrane mirror (DMM) and demonstrated its use in compensating for system- and sample-induced aberrations. The optimum shape of the DMM was determined with a random search algorithm optimizing on either two photon fluore...
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Society of Photo-optical Instrumentation Engineers (SPIE)
2014
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| Online Access: | https://eprints.nottingham.ac.uk/2820/ |
| _version_ | 1848790883988668416 |
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| author | Mullenbroich, M. Caroline McGhee, Ewan J. Wright, Amanda J. Anderson, Kurt I. Mathieson, Keith |
| author_facet | Mullenbroich, M. Caroline McGhee, Ewan J. Wright, Amanda J. Anderson, Kurt I. Mathieson, Keith |
| author_sort | Mullenbroich, M. Caroline |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | We have developed a nonlinear adaptive optics microscope utilizing a deformable membrane mirror (DMM) and demonstrated its use in compensating for system- and sample-induced aberrations. The optimum shape of the DMM was determined with a random search algorithm optimizing on either two photon fluorescence or second harmonic signals as merit factors. We present here several strategies to overcome photobleaching issues associated with lengthy optimization routines by adapting the search algorithm and the experimental methodology. Optimizations were performed on extrinsic fluorescent dyes, fluorescent beads loaded into organotypic tissue cultures and the intrinsic second harmonic signal of these cultures. We validate the approach of using these preoptimized mirror shapes to compile a robust look-up table that can be applied for imaging over several days and through a variety of tissues. In this way, the photon exposure to the fluorescent cells under investigation is limited to imaging. Using our look-up table approach, we show signal intensity improvement factors ranging from 1.7 to 4.1 in organotypic tissue cultures and freshly excised mouse tissue. Imaging zebrafish in vivo, we demonstrate signal improvement by a factor of 2. This methodology is easily reproducible and could be applied to many photon starved experiments, for example fluorescent life time imaging, or when photobleaching is a concern. |
| first_indexed | 2025-11-14T18:19:42Z |
| format | Article |
| id | nottingham-2820 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T18:19:42Z |
| publishDate | 2014 |
| publisher | Society of Photo-optical Instrumentation Engineers (SPIE) |
| recordtype | eprints |
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| spelling | nottingham-28202020-05-04T16:41:16Z https://eprints.nottingham.ac.uk/2820/ Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging Mullenbroich, M. Caroline McGhee, Ewan J. Wright, Amanda J. Anderson, Kurt I. Mathieson, Keith We have developed a nonlinear adaptive optics microscope utilizing a deformable membrane mirror (DMM) and demonstrated its use in compensating for system- and sample-induced aberrations. The optimum shape of the DMM was determined with a random search algorithm optimizing on either two photon fluorescence or second harmonic signals as merit factors. We present here several strategies to overcome photobleaching issues associated with lengthy optimization routines by adapting the search algorithm and the experimental methodology. Optimizations were performed on extrinsic fluorescent dyes, fluorescent beads loaded into organotypic tissue cultures and the intrinsic second harmonic signal of these cultures. We validate the approach of using these preoptimized mirror shapes to compile a robust look-up table that can be applied for imaging over several days and through a variety of tissues. In this way, the photon exposure to the fluorescent cells under investigation is limited to imaging. Using our look-up table approach, we show signal intensity improvement factors ranging from 1.7 to 4.1 in organotypic tissue cultures and freshly excised mouse tissue. Imaging zebrafish in vivo, we demonstrate signal improvement by a factor of 2. This methodology is easily reproducible and could be applied to many photon starved experiments, for example fluorescent life time imaging, or when photobleaching is a concern. Society of Photo-optical Instrumentation Engineers (SPIE) 2014-01-27 Article PeerReviewed Mullenbroich, M. Caroline, McGhee, Ewan J., Wright, Amanda J., Anderson, Kurt I. and Mathieson, Keith (2014) Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging. Journal of Biomedical Optics, 19 (1). 016021. ISSN 1560-2281 adaptive optics; aberrations; fluorescence; microscopy; photobleaching http://biomedicaloptics.spiedigitallibrary.org/article.aspx?articleid=1819302 doi:10.1117/1.JBO.19.1.016021 doi:10.1117/1.JBO.19.1.016021 |
| spellingShingle | adaptive optics; aberrations; fluorescence; microscopy; photobleaching Mullenbroich, M. Caroline McGhee, Ewan J. Wright, Amanda J. Anderson, Kurt I. Mathieson, Keith Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging |
| title | Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging |
| title_full | Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging |
| title_fullStr | Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging |
| title_full_unstemmed | Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging |
| title_short | Strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging |
| title_sort | strategies to overcome photobleaching in algorithm based adaptive optics for nonlinear in-vivo imaging |
| topic | adaptive optics; aberrations; fluorescence; microscopy; photobleaching |
| url | https://eprints.nottingham.ac.uk/2820/ https://eprints.nottingham.ac.uk/2820/ https://eprints.nottingham.ac.uk/2820/ |