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.

FIGURE 1

Mouse TA topology. (A) Light microscopy (1 μm section) of the mouse TA. Muscle fiber morphology is consistent with conventional mouse skeletal fiber types of Type IIB and Type IIA/D (Hamalainen and Pette, 1993; Sartorius et al., 1998). (B) EM of the mouse TA shows the perivascular mitochondrial (PV) pattern in the skeletal muscle around vessels (V). (C) EM in Type IIB reveals the deposition of mitochondria at the intersection of fibers along the Z-line. Scale bars shown represent 30 μm in panel A, and 1 μm in panels B and C.

FIGURE 2

NAD(P)H fluorescence images of isolated mouse skeletal muscle fibers. Single fibers isolated from the mouse (A) EDL muscle and (B) soleus muscle imaged for NAD(P)H fluorescence. Subsarcolemmal mitochondria (SSM) were evident in the isolated soleus muscle, which is consistent with the in vivo observations. The SSM in these isolated fibers could also represent the perivascular mitochondrial pool. Scale bars shown in both panels A and B represent 10 μm.

FIGURE 3

Montage of a z-stack of NAD(P)H fluorescence images from a mouse TA in vivo. NAD(P)H fluorescence was imaged in 1 μm optical sections from the first site where myocytes were visible (0 μm location) to 150 μm deep into the TA. Every 28th image is presented beginning with 11 μm into the stack at the top left-hand corner and with depth running in a left-to-right direction. The depths of the images are given as a reference; 0.636 μm pixel size, 10.464 μs dwell time, 720 nm excitation, 8-bit resolution, and 410–490 nm emission monitored.

FIGURE 4

Illustrative NAD(P)H TPEFM images in mouse TA in vivo. (A) Illustration of Type IIB (dark highly striated) and Type IIA/D (bright fiber; long axis streaking of mitochondria and SSM pools) within the same field of view; 0.636-μm pixel size, 5.208-μs dwell time, 720-nm excitation, 8-bit resolution, and 410–490 nm emission monitored. (B) Vasculature (V) in the mouse TA is seen as voids in NAD(P)H fluorescence. Mitochondria are visualized through the NAD(P)H signal consistent with paranuclear (PN) and perivascular (PV) mitochondria. N represents nuclei; 0.190-μm pixel size, 1.60 μs dwell time, 720 nm excitation, 8-bit resolution, and 435–485 nm emission monitored. Scale bar shown in panel A represents 20 μm and in panel B represents 10 μm.

FIGURE 5

NAD(P)H fluorescence can be used to estimate sarcomere length in vivo. (A) FFT analysis is performed along the long axis of the cell in a NAD(P)H fluorescence image of a Type IIB fiber (image acquired with 720-nm excitation and 380–550 nm emission monitored with a 0.502 μm pixel size). (B) Filtered power spectrum of the long axis FFT. The average sarcomere length was 2.34 ± 0.17 μm (n = 6 fibers). Scale bar shown in panel A represents 10 μm.

FIGURE 6

Near infrared absorbance spectra of water and mouse skeletal and heart muscle from 650 to 850 nm. (A) Near infrared absorbance (cm⁻¹) was determined for mouse skeletal muscle without hemoglobin. There is no substantial difference between oxygenated (gray) and deoxygenated (black) states, but both do appear to be influenced by the absorbance spectrum of water. However, the absorbance spectra (B) of oxygenated and deoxygenated heart tissue are insignificantly affected by water, but show substantial differences between oxygenated (gray) and deoxygenated (black) states. The degree of NIR absorbance of heart tissue may act as significant primary filter during TPEFM, whereas skeletal muscle NIR absorbance would only have a small primary filter effect similar to that of water.

FIGURE 7

Effect of ischemia/reperfusion on NAD(P)H emission intensity from mouse TA in vivo. (A) Time course of normalized total emission intensity from 380 to 560 nm. The ischemia and reperfusion periods are indicated. Labeled experimental time points are listed as R (reference), A (control), B (early ischemia), C and D (mid-ischemia), E (late ischemia), F (early reperfusion), and G (reperfusion) for indexes to spectral data. (B) Fluorescence spectra from selected time points. (C) Difference spectra performed by subtracting the reference spectrum (R) from the index spectrum. (D) Fluorescence emission time courses (435 and 462 nm) illustrating the different temporal behavior of these two emission wavelengths through the protocol.

FIGURE 8

Secondary inner filter effects on NAD(P)H fluorescence in tissue. Two-photon NAD(P)H fluorescence was monitored with increasing tissue depth from the first site where myocytes were visible (0 μm location) up to 80 μm deep into the tissue using 720 nm excitation. NAD(P)H fluorescence (382–628 nm) using the Zeiss META in lambda mode. Data were normalized and showed peak fluorescence near 460 nm.