TY - JOUR
T1 - Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy
AU - Salomonnson, Emma
AU - Mihalko, Laura Anne
AU - Verkhusha, Vladislav V.
AU - Luker, Kathryn E.
AU - Luker, Gary D.
N1 - Funding Information:
This work was supported by United States National Institutes of Health National Cancer Institute Grants R01CA136553, R01CA136829, R01CA142750, and P50CA093990. The authors thank James Lopez for helpful discussions.
PY - 2012/9
Y1 - 2012/9
N2 - Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the stateof- 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.
AB - Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the stateof- 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.
KW - fluorescent protein
KW - intravital microscopy
KW - live cell imaging
KW - two photon excitation
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U2 - 10.1117/1.JBO.17.9.096001
DO - 10.1117/1.JBO.17.9.096001
M3 - Article
C2 - 22975677
AN - SCOPUS:84870618399
SN - 1083-3668
VL - 17
JO - Journal of Biomedical Optics
JF - Journal of Biomedical Optics
IS - 9
M1 - 096001
ER -