In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

Abstract

Metabolic imaging of the relative amounts of reduced NADH and FAD and the microenvironment of these metabolic electron carriers can be used to noninvasively monitor changes in metabolism, which is one of the hallmarks of carcinogenesis. This study combines cellular redox ratio, NADH and FAD lifetime, and subcellular morphology imaging in three dimensions to identify intrinsic sources of metabolic and structural contrast in vivo at the earliest stages of cancer development. There was a significant (P < 0.05) increase in the nuclear to cytoplasmic ratio (NCR) with depth within the epithelium in normal tissues; however, there was no significant change in NCR with depth in precancerous tissues. The redox ratio significantly decreased in the less differentiated basal epithelial cells compared with the more mature cells in the superficial layer of the normal stratified squamous epithelium, indicating an increase in metabolic activity in cells with increased NCR. However, the redox ratio was not significantly different between the superficial and basal cells in precancerous tissues. A significant decrease was observed in the contribution and lifetime of protein-bound NADH (averaged over the entire epithelium) in both low- and high-grade epithelial precancers compared with normal epithelial tissues. In addition, a significant increase in the protein-bound FAD lifetime and a decrease in the contribution of protein-bound FAD are observed in high-grade precancers only. Increased intracellular variability in the redox ratio, NADH, and FAD fluorescence lifetimes were observed in precancerous cells compared with normal cells.

Fig. 1.

In vivo three-dimensional multiphoton images of the redox ratio (fluorescence intensity of FAD/NADH) (a–c), the mean NADH lifetime (α₁ × τ₁ + α₂ × τ₂) (d–f), and the mean FAD lifetime (g–i) from tissues diagnosed as normal (a, d, and g), low-grade precancer (b, e, and h), and high-grade precancer (c, f, and i). The numbers in the corner of each image indicate the depth below the tissue surface in μm, and each image is 100 × 100 μm. There are a different color bars for a–c (in calibrated units), d–f, and g–i. The redox images were created by merging the calibrated blue NADH image (800-nm excitation with a 490-nm short-pass filter) with the calibrated red FAD image (890-nm excitation) into an RGB image for the purposes of this figure only.

Fig. 2.

Percentage of change in the nuclear-to-cytoplasmic ratio (NCR, nuclear diameter/cell diameter) and redox ratio between the top and bottom plane of the cellular epithelium within an animal, calculated from the multiphoton redox images. The mean and 95% confidence interval were calculated for animals diagnosed as normal (n = 36 bottom/top cell pairs), low-grade precancer (n = 42 bottom/top cell pairs), and high-grade precancer (n = 33 bottom/top cell pairs). Paired Wilcoxon tests revealed a significant increase in NCR and decrease in redox ratio for basal cells compared with superficial cells in normal tissue (*, P < 0.05) but no change with depth in precancerous tissues (P > 0.05).

Fig. 3.

Mean and 95% confidence interval of the volume-averaged NADH (a) and FAD (b) lifetime variables. All values were obtained from in vivo multiphoton FLIM images, and variables were averaged for all image planes within the epithelium of an animal for normal (n = 6), low-grade precancerous (n = 8), and high-grade precancerous animals (n = 7). Significant differences (P < 0.05) exist between normal vs. low-grade precancer and normal vs. high-grade precancer (*). There were no significant differences between low- and high-grade precancers (P > 0.05).

Fig. 4.

Mean and 95% confidence interval of the coefficient of variation (cell standard deviation/cell mean) of the NADH (a) and FAD (b) lifetime variables. All values were obtained from in vivo multiphoton FLIM images, and variables were averaged for all cells within an animal for normal (n = 6), low-grade precancerous (n = 8), and high-grade precancerous animals (n = 7). Significant differences (P < 0.05) exist between normal vs. low-grade precancer and normal vs. high-grade precancer (*). There were no significant differences between low- and high-grade precancers (P > 0.05). c.u., calibrated units.