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Biophys. J. BioFAST: First Published December 13, 2004. doi:10.1529/biophysj.104.053165
© 2004 by the Biophysical Society.

A more recent version of this article appeared on March 1, 2005.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Skeletal muscle NAD(P)H 2-photon fluorescence microscopy in vivo:Topology and optical inner filters

Emily C Rothstein 1*, Stefanie Carroll 1, Christian A Combs 1, Paul D Jobsis 1 and Robert S Balaban 1

1 National Institutes of Health

* To whom correspondence should be addressed. E-mail: emilyr{at}nih.gov.

Submitted on September 20, 2004
Revised on October 29, 2004
Accepted on 18 November 2004


  Abstract
Two-photon excitation fluorescence microscopy (TPEFM) permits the investigation of the topology of intercellular events within living animals. TPEFM was used to monitor the distribution of mitochondrial reduced nicotinamide adenine dinucleotide (NAD(P)H) in murine skeletal muscle in vivo. NAD(P)H fluorescence emission was monitored (~460 nm) using 710-720 nm excitation. High resolution TPEFM images were collected up to a depth of 150 microns from the surface of the tibalis anterior muscle. The NAD(P)H fluorescence images revealed subcellular structures consistent with subsarcolemmal, perivascular, intersarcomeric and paranuclear mitochondria. In vivo fiber typing between IIB and IIA/D fibers was possible using the distribution and content of mitochondria from the NAD(P)H fluorescence signal. The intersarcomeric mitochondria concentrated at the z-line in the IIB fiber types resulting in a periodic pattern with a spacing of one sarcomere (2.34 ± 0.17 microns). The primary inner filter effects were nearly equivalent to water, however, the secondary inner filter effects were highly significant and dynamically affected the observed emission frequency and amplitude of the NAD(P)H fluorescence signal. These data demonstrate the feasibility, and highlight the complexity, of using NAD(P)H TPEFM in skeletal muscle to characterize the topology and metabolic function of mitochondria within the living mouse.

Key Words: mitochondria, muscle fibers, myoglobin, paranuclear, perivascular




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Copyright © 2004 by the Biophysical Society.