Multiphoton redox ratio imaging for metabolic monitoring in vivo.

Metabolic monitoring at the cellular level in live tissues is important for understanding cell function, disease processes, and potential therapies. Multiphoton imaging of the relative amounts of NADH and FAD (the primary electron donor and acceptor, respectively, in the electron transport chain) provides a noninvasive method for monitoring cellular metabolic activity with high resolution in three dimensions in vivo. NADH and FAD are endogenous tissue fluorophores, and thus this method does not require exogenous stains or tissue excision. We describe the principles and protocols of multiphoton redox ratio imaging in vivo.

[1]  N. Ramanujam Fluorescence spectroscopy of neoplastic and non-neoplastic tissues. , 2000, Neoplasia.

[2]  Melissa C Skala,et al.  Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues. , 2005, Cancer research.

[3]  K. Fujita [Two-photon laser scanning fluorescence microscopy]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[4]  B. Chance,et al.  Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. , 1979, The Journal of biological chemistry.

[5]  Kevin W Eliceiri,et al.  Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities. , 2003, The Review of scientific instruments.

[6]  B. Chance,et al.  Metabolic heterogeneities in rapidly metabolizing tissues , 1989 .

[7]  A. Zvyagin Multiphoton endoscopy , 2007 .

[8]  N. Ramanujam,et al.  In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia , 2007, Proceedings of the National Academy of Sciences.

[9]  N Ramanujam,et al.  Low Temperature Fluorescence Imaging of Freeze-trapped Human Cervical Tissues. , 2001, Optics express.

[10]  John White,et al.  Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability , 1999, Nature Biotechnology.

[11]  R. Lotan,et al.  Autofluorescence Microscopy of Fresh Cervical-Tissue Sections Reveals Alterations in Tissue Biochemistry with Dysplasia¶ , 2001, Photochemistry and photobiology.

[12]  Fritjof Helmchen,et al.  Miniaturization of Fluorescence Microscopes Using Fibre Optics , 2002, Experimental physiology.

[13]  C. Baudelet,et al.  Effect of anesthesia on the signal intensity in tumors using BOLD-MRI: comparison with flow measurements by Laser Doppler flowmetry and oxygen measurements by luminescence-based probes. , 2004, Magnetic resonance imaging.

[14]  Watt W Webb,et al.  Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. , 2002, Biophysical journal.

[15]  M. Dewhirst,et al.  Tumor oxygenation: a matter of supply and demand. , 1996, Anticancer research.

[16]  A. Mehta,et al.  Multiphoton endoscopy: optical design and application to in vivo imaging of mammalian hippocampal neurons , 2003, Conference on Lasers and Electro-Optics, 2003. CLEO '03..

[17]  Min Gu,et al.  Two-photon fluorescence endoscopy with a micro-optic scanning head. , 2003, Optics letters.