Biochemical estimation of NADH concentration is a useful method for monitoring cellular metabolism, because the NADH/NAD + reduction-oxidation pair is crucial for electron transfer in the mitochondrial electron chain. In this article, we present a novel method for deriving functional maps of intracellular reduction-oxidation ratio in vivo via measurement of the fluorescence lifetimes and the ratio of free and protein-bound NADH using two-photon fluorescence lifetime imaging (FLIM). Through systematic analysis of FLIM data from the control cells, it was observed that there is a statistically significant decrease in the fluorescence lifetime of both free and protein-bound NADH and the contribution of proteinbound NADH as cells progress from an early to logarithmic to confluent phase. Potassium cyanide (KCN) treatment and serum starvation of cells yielded similar changes. There was a statistically significant decrease in the fluorescence lifetime of protein-bound and free NADH at the early and logarithmic phase of the growth curve and a statistically significant decrease in the contribution of protein-bound NADH relative to that observed in the control cells at all three phases of the growth curve. The imposed perturbations (confluence, serum starvation, and KCN treatment) are all expected to result in an increase in the ratio of NADH/NAD + . Our studies suggest that the fluorescence lifetime of both the free and the protein-bound components of NADH and the ratio of free to protein-bound NADH is related to changes in the NADH/NAD + ratio.

This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows highresolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a twophoton excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting. (Cancer Res 2005; 65(4): 1180-6)

Background and Objective-The first objective of this study was to evaluate the performance of fluorescence spectroscopy for diagnosing pre-cancers in stratified squamous epithelial tissues in vivo using two different probe geometries with (1) overlapping vs. (2) non-overlapping illumination and collection areas on the tissue surface. Probe (1) and probe (2) are preferentially sensitive to the fluorescence originating from the tissue surface and sub-surface tissue depths, respectively. The second objective was to design a novel, angled illumination fiber-optic probe to maximally exploit the depth-dependent fluorescence properties of epithelial tissues.

It was found that the effects of freezing and thawing on the fluorescence properties of tissue are greater than any changes brought about by degradation of tissue over a time frame of 90 minutes after biopsy. Performing ex vivo fluorescence measurements within a reasonable time window has the advantage of more accurately reproducing the clinically relevant in vivo conditions in the case of the hamster cheek pouch tissue. Therefore, in tissue biopsy studies, the tissue sample should ideally be maintained in an unfrozen state prior to measurement.

We explored the use of diffuse reflectance spectroscopy in the ultraviolet-visible (UV-VIS) spectrum for the diagnosis of epithelial precancers and cancers in vivo. A physical model (Monte Carlo inverse model) and an empirical model (principal component analysis, (PCA)) based approach were compared for extracting diagnostic features from diffuse reflectance spectra measured in vivo from the dimethylbenz[alpha]anthracene-treated hamster cheek pouch model of oral carcinogenesis. These diagnostic features were input into a support vector machine algorithm to classify each tissue sample as normal (n=10) or neoplastic (dysplasia to carcinoma, n=10) and cross-validated using a leave one out method. There was a statistically significant decrease in the absorption and reduced scattering coefficient at 460 nm in neoplastic compared to normal tissues, and these two features provided 90% classification accuracy. The first two principal components extracted from PCA provided a classification accuracy of 95%. The first principal component was highly correlated with the wavelength-averaged reduced scattering coefficient. Although both methods show similar classification accuracy, the physical model provides insight into the physiological and structural features that discriminate between normal and neoplastic tissues and does not require a priori, a representative set of spectral data from which to derive the principal components.

These data show a decreased opportunity and a wide variability in the frequency of successfully completed paramedic technical skills and experiences in this EMS system. Limited exposure to critically ill adult and pediatric patients reaffirms that high-risk skills are performed infrequently. A multifaceted approach should be considered for maintaining provider competency.

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