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Journal of Biomedical Optics • Open Access

Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy
Author(s): Emma Salomonnson; Laura Anne Mihalko; Kathryn E. Luker; Vladislav V. Verkhusha; Gary D. Luker

Paper Abstract

Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the state-of-the-art technology for longitudinal imaging studies in tissues and living animals. Successful analysis of separate cell populations or signaling events by intravital microscopy requires optimal pairing of multiphoton excitation wavelengths with spectrally distinct fluorescent proteins. While prior studies have analyzed two photon absorption properties of isolated fluorescent proteins, there is limited information about two photon excitation and fluorescence emission profiles of fluorescent proteins expressed in living cells and intact tissues. Multiphoton microscopy was used to analyze fluorescence outputs of multiple blue, green, and red fluorescent proteins in cultured cells and orthotopic tumor xenografts of human breast cancer cells. It is shown that commonly used orange and red fluorescent proteins are excited efficiently by 750 to 760 nm laser light in living cells, enabling dual color imaging studies with blue or cyan proteins without changing excitation wavelength. It is also shown that small incremental changes in excitation wavelength significantly affect emission intensities from fluorescent proteins, which can be used to optimize multi-color imaging using a single laser wavelength. These data will direct optimal selection of fluorescent proteins for multispectral two photon microscopy.

Paper Details

Date Published: 11 September 2012
PDF: 9 pages
J. Biomed. Opt. 17(9) 096001 doi: 10.1117/1.JBO.17.9.096001
Published in: Journal of Biomedical Optics Volume 17, Issue 9
Show Author Affiliations
Emma Salomonnson, Univ. of Michigan Medical School (United States)
Laura Anne Mihalko, Univ. of Michigan Medical School (United States)
Kathryn E. Luker, Univ. of Michigan Medical School (United States)
Vladislav V. Verkhusha, Albert Einstein College of Medicine of Yeshiva Univ. (United States)
Gary D. Luker, Univ. of Michigan Medical School (United States)

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