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Multimodal wide-field two-photon excitation imaging: characterization of the technique for in vivo applications.

Authors
  • Hwang, Jae Youn
  • Wachsmann-Hogiu, Sebastian
  • Ramanujan, V Krishnan
  • Nowatzyk, Andreas G
  • Koronyo, Yosef
  • Medina-Kauwe, Lali K
  • Gross, Zeev
  • Gray, Harry B
  • Farkas, Daniel L
Type
Published Article
Journal
Biomedical Optics Express
Publisher
The Optical Society
Publication Date
Jan 01, 2011
Volume
2
Issue
2
Pages
356–364
Identifiers
DOI: 10.1364/BOE.2.000356
PMID: 21339880
Source
Medline
Keywords
License
Unknown

Abstract

We report fast, non-scanning, wide-field two-photon fluorescence excitation with spectral and lifetime detection for in vivo biomedical applications. We determined the optical characteristics of the technique, developed a Gaussian flat-field correction method to reduce artifacts resulting from non-uniform excitation such that contrast is enhanced, and showed that it can be used for ex vivo and in vivo cellular-level imaging. Two applications were demonstrated: (i) ex vivo measurements of beta-amyloid plaques in retinas of transgenic mice, and (ii) in vivo imaging of sulfonated gallium(III) corroles injected into tumors. We demonstrate that wide-field two photon fluorescence excitation with flat-field correction provides more penetration depth as well as better contrast and axial resolution than the corresponding one-photon wide field excitation for the same dye. Importantly, when this technique is used together with spectral and fluorescence lifetime detection modules, it offers improved discrimination between fluorescence from molecules of interest and autofluorescence, with higher sensitivity and specificity for in vivo applications.

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