In the presence of oxygen, eukaryotic cells depend on the mitochondrial electron-transport chain (METC) to produce useful chemical energy in the form of adenosine tri-phosphate (ATP). The first complex of this system, nicotinamide adenine dinucleotide (NAD) (an active coenzyme of niacin) functions as an important intermediary in the transfer of two electrons for cellular energy metabolism. Although it exists in an oxidized (NAD+) and a reduced (dihydronicotinamide adenine dinucleotide - NADH) form, the photo-physics is such that NADH is an intrinsically fluorescent molecule, whereas its oxidized product (NAD+) is not. Fluorescence measurements of endogenous tissue fluorophores such as NADH are attractive for the study and diagnosis of pre-cancers and cancers because the fluorescence properties of this molecule are altered by cellular metabolism [1], which is a hallmark of carcinogenesis.

Multiphoton laser scanning microscopy (MPLSM) non-invasively provides three-dimensional images of cellular and sub-cellular fluorescence intensity with resolution similar to the gold standard for cancer diagnosis, histopathology, in vivo without the need for biopsy, physical sectioning or staining [2-4]. Multiphoton excitation occurs when a fluorophore is excited simultaneously by two photons of half the absorption energy of the fluorophore (or by three photons of one-third the absorption energy of the fluorophore, etc.), and probes the same biological fluorophores as single-photon fluorescence [3]. Multiphoton excitation of NADH occurs in the near infrared wavelength region [5].

Fluorescence intensity and emission spectra of NADH are commonly used for the diagnosis of pre-cancers and cancers, but the diagnostic potential of the fluorescence lifetime (the time a fluorophore remains in its excited state before decaying to the ground state) of NADH has not been well explored. Fluorescence lifetime imaging microscopy (FLIM) is a functional imaging technique that measures the time a fluorophore remains in the excited state after excitation. The excited state lifetime can be altered by the fluorophore microenvironment, including factors such as local pH, temperature, oxygen concentration and protein binding [6-8]. FLIM, which relies on the temporally resolved fluorescence signal, is advantageous over fluorescence intensity or spectral measurements for small volume tissue imaging because it is generally independent of fluorophore concentration, the effects of tissue absorption and scattering, and fluctuations in excitation intensity [8].

MPLSM studies of fluorescence intensity of NADH in normal and neoplastic tissue in an animal model of oral cancer have been carried out by our group [9]. Three-dimensional image stacks of the dysplasia-carcinoma sequence in the hamster cheek pouch were collected in viable, un-sectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. This excitation wavelength likely probes the same fluorophores as the most diagnostic excitation wavelength identified in a preliminary optical spectroscopy study carried out by our group (410 nm excitation) [10]. Morphological and fluorescence intensity differences were able to differentiate normal epithelial tissues (n=15) from pre-cancers (mild to moderate dysplasia and severe dysplasia, n=12) and cancers (CIS and SCC, n=26).

Currently, our group is testing whether FLIM of NADH can detect changes in tissue metabolism with pre-cancer development in vivo using multiphoton microscopy techniques (Figure 1) in the same hamster cheek pouch model of oral carcinogenesis. The most important finding from this study is a statistically significant decrease in the relative abundance and lifetime of protein-bound NADH fluorescence lifetime with the development of pre-cancer in vivo [11]. In the near-term, the findings from multiphoton FLIM studies could guide the design and development of practical time-gated fluorescence detection schemes for clinical applications. In long-term, portable technology could be engineered to enable multiphoton FLIM in a clinical setting. This technology could be used for epithelial pre-cancer detection and metabolic monitoring for tumor therapy.

Normal	Severe Dysplasia
1.4ns	2.4ns

Fig 1: Protein-bound NADH lifetimes measured in vivo from normal tissue and severe dysplasia in the dimethylbenz[]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis at a two-photon excitation wavelength of 780 nm.

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References

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