Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy

Emma Salomonnson, Laura Anne Mihalko, Vladislav Verkhusha, Kathryn E. Luker, Gary D. Luker

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article number096001
JournalJournal of Biomedical Optics
Volume17
Issue number9
DOIs
StatePublished - Sep 2012

Fingerprint

Spectrum analysis
spectrum analysis
Microscopic examination
microscopy
proteins
Proteins
Cells
cells
Wavelength
Photons
Imaging techniques
Fluorescence
wavelengths
excitation
Tissue
Color
photons
Lasers
color
Green Fluorescent Proteins

Keywords

  • fluorescent protein
  • intravital microscopy
  • live cell imaging
  • two photon excitation

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biomaterials
  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics

Cite this

Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy. / Salomonnson, Emma; Mihalko, Laura Anne; Verkhusha, Vladislav; Luker, Kathryn E.; Luker, Gary D.

In: Journal of Biomedical Optics, Vol. 17, No. 9, 096001, 09.2012.

Research output: Contribution to journalArticle

Salomonnson, Emma ; Mihalko, Laura Anne ; Verkhusha, Vladislav ; Luker, Kathryn E. ; Luker, Gary D. / Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy. In: Journal of Biomedical Optics. 2012 ; Vol. 17, No. 9.
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