Endogenous fluorescence provides morphological, spectral, and lifetime contrast that can indicate disease states in tissues. Previous studies have demonstrated that two-photon autofluorescence microscopy (2PAM) can be used for noninvasive, three-dimensional imaging of epithelial tissues down to approximately 150 μm beneath the skin surface. We report ex-vivo 2PAM images of epithelial tissue from a human tongue biopsy down to 370 μm below the surface. At greater than 320 μm deep, the fluorescence generated outside the focal volume degrades the image contrast to below one. We demonstrate that these imaging depths can be reached with 160 mW of laser power (2-nJ per pulse) from a conventional 80-MHz repetition rate ultrafast laser oscillator. To better understand the maximum imaging depths that we can achieve in epithelial tissues, we studied image contrast as a function of depth in tissue phantoms with a range of relevant optical properties. The phantom data agree well with the estimated contrast decays from time-resolved Monte Carlo simulations and show maximum imaging depths similar to that found in human biopsy results. This work demonstrates that the low staining inhomogeneity (∼20) and large scattering coefficient (∼10 mm−1) associated with conventional 2PAM limit the maximum imaging depth to 3 to 5 mean free scattering lengths deep in epithelial tissue.
We demonstrate the use of gold nanorods as molecularly targeted contrast agents for two-photon luminescence (TPL)
imaging of cancerous cells 150 μm deep inside a tissue phantom. We synthesized gold nanorods of 50 nm x 15 nm size
with a longitudinal surface plasmon resonance of 760 nm. Gold nanorods were conjugated to antibodies against
epidermal growth factor receptor (EGFR) and labeled to A431 human epithelial skin cancer cells in a collagen matrix
tissue phantom. Using a 1.4 NA oil immersion objective lens, we found that excitation power needed for similar
emission intensity in TPL imaging of labeled cells was up to 64 times less than that needed for two-photon
autofluorescence (TPAF) imaging of unlabeled cells, which would correspond to a more than 4,000 times increase in
emission intensity under equal excitation energy. However, the aberrations due to refractive index mismatch of the
immersion oil and the sample limit imaging depth to 75 μm. Using a 0.95 NA water immersion objective lens, we
observe robust two-photon emission signal from gold nanorods in the tissue phantoms from at depths of up to 150 μm.
Furthermore, the increase in excitation energy required to maintain a constant emission signal intensity as imaging depth
was increased was the same in both labeled and unlabeled phantom, suggesting that at the concentrations used, the
addition of gold nanorods did not appreciably increase the bulk scattering coefficient of the sample. The remarkable TPL
brightness of gold nanorods in comparison to TPAF signal makes them an attractive contrast agent for early detection of