Skeletal Muscle NAD(P)H Two-Photon Fluorescence Microscopy In Vivo: Topology and Optical Inner Filters

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 ( approximately 460 nm) using 710-720 nm excitation. High-resolution TPEFM images were collected up to a depth of 150 microm from the surface of the tibialis 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 microm). 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.

NADH changes during hypoxia, ischemia, and increased work differ between isolated heart preparations

“…The potential risk of ischemic damage during the heart excision and cannulation heart procedure is also a limitation, so procedure times were minimized. Finally, a cautionary note: the wavelengths at which NADH is imaged should be carefully selected to prevent signal contamina-H536 ISOLATED HEART PREPARATION AFFECTS NADH CHANGES tion by absorbance changes of other tissue components, such a myoglobin (26).…”

Label-free microscopy and stress responses reveal the functional organization of Pseudodiaptomus marinus copepod myofibrils

“…On this basis sarcomere microarchitecture has been analyzed by SHG microscopy in various animal models and in human biopsies (Friedrich et al, 2010;Rouede et al, 2013). Two-photon excited fluorescence (TPEF) microscopy of flavoproteins and NAD(P)H has been used to characterize the distribution of mitochondria along myofibrils (Huang et al, 2002;Rothstein et al, 2005). In skeletal muscle fibers organelles containing CH 2 rich molecules such as the T-tubular system and lipids droplets have been visualized by Coherent Anti-Stokes Raman Scattering (CARS) when the symmetric stretch Raman active vibration of the CH 2 group is targeted at 2845 cm À1 (Brackmann et al, 2010;Pfeffer et al, 2011).…”

“…Flavins that switch between the fluorescent oxidized form and the nonfluorescent reduced form allow imaging the deoxygenation and reoxygenation of mitochondria during ischemia reperfusion in mouse hepatocytes (Lu et al, 2014). The distribution of reduced NAD(P)H fluorescence in mouse skeletal muscle reveals the existence of intersarcomeric populations of mitochondria (Rothstein et al, 2005).…”

Symposium Presentations

“…Another very important development has been the use of multi-photon excitation schemes to observe subcellular events in vivo (15,16). This form of microscopy allows us to go deep into tissues while maintaining the resolution we need to examine a single cell with a microscope; this gives us a 1-to 2-m spatial resolution deep (Ͼ300 m) in the intact animal.…”