Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, 3-D functional imaging technique. We investigate the potential for in vivo precancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic cofactor nicotinamide adenine dinucleotide (NADH). The dimethylbenzanthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers are imaged using multiphoton FLIM at 780-nm excitation. The cytoplasm of normal hamster cheek pouch epithelial cells has short (0.29±0.03 ns) and long lifetime components (2.03±0.06 ns), attributed to free and protein-bound NADH, respectively. Low-grade precancers (mild to moderate dysplasia) and high-grade precancers (severe dysplasia and carcinoma in situ) are discriminated from normal tissues by their decreased protein-bound NADH lifetime (p<0.05). Inhibition of cellular glycolysis and oxidative phosphorylation in cell monolayers produces an increase and decrease, respectively, in the protein-bound NADH lifetime (p<0.05). Results indicate that the decrease in protein-bound NADH lifetime with dysplasia is due to a shift from oxidative phosphorylation to glycolysis, consistent with the predictions of neoplastic metabolism. We demonstrate that multiphoton FLIM is a powerful tool for the noninvasive characterization and detection of epithelial precancers in vivo.
The benefits of two-photon fluorescence microscopy of biological samples are vast arising from the utilisation of low
energy light. The two-photon absorption cross sections (σ2) of the di-cation free-base and metallated forms of
hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) are obtained to
ascertain their effectiveness as fluorophores for use in two-photon microscopy. The open-aperture Z-scan and the two-photon
induced fluorescence (TPIF) techniques, each capable of providing information regarding the nonlinear
absorption, are employed to determine σ2 of the various porphyrins at an excitation wavelength of 800 nm.
A significant disparity in the determined values of σ2 using the two methods is observed. This is largely attributed to the
common requirement of higher concentrations used in the open aperture Z-scan method compared with TPIF techniques.
Values of σ2 obtained from the Z-scan experiments are in the order of 10 GM, whilst those obtained from the TPIF
experiments are in the order of 200 GM. Insertion of either protons or metal ions into the macrocycle does not enhance
the σ2 of the porphyrins. Successful two-photon induced fluorescence imaging of BOPP free-base loaded G6 glioma cells
is achieved, confirming the usefulness of this porphyrin in two-photon microscopy.