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Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinearin-vivoimaging

Müllenbroich, M. Caroline; McGhee, Ewan J.; Wright, Amanda J.; Anderson, Kurt I.; Mathieson, Keith


M. Caroline Müllenbroich

Ewan J. McGhee

Kurt I. Anderson

Keith Mathieson


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.


Caroline M├╝llenbroich, M., Mullenbroich, M. C., McGhee, E. J., Wright, A. J., Anderson, K. I., & Mathieson, K. (2014). Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinearin-vivoimaging. Journal of Biomedical Optics, 19(1), doi:10.1117/1.JBO.19.1.016021

Journal Article Type Article
Acceptance Date Dec 13, 2013
Online Publication Date Jan 27, 2014
Publication Date Jan 27, 2014
Deposit Date Apr 17, 2014
Publicly Available Date Apr 17, 2014
Journal Journal of Biomedical Optics
Print ISSN 1083-3668
Electronic ISSN 1560-2281
Publisher Society of Photo-optical Instrumentation Engineers
Peer Reviewed Peer Reviewed
Volume 19
Issue 1
Article Number 016021
Keywords adaptive optics; aberrations; fluorescence; microscopy; photobleaching
Public URL
Publisher URL
Additional Information M. Caroline M├╝llenbroich, Ewan J. McGhee, Amanda J. Wright, Kurt I. Anderson, and Keith Mathieson, "Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinear in-vivo imaging", Journal of Biomedical Optics, Lihong V. Wang, editor-in-chief, 19(1) 016021 (2014).

Copyright 2014 Society of Photo-Optical Instrumentation
Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited.


Mullenbroich_JBO_2014.pdf (11.7 Mb)

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