Three-photon fluorescence imaging of melanin with a CW laser and a dual-wedge scanning system

Abstract

Confocal microscopy offers high resolution optical imaging with sectioning capability. This imaging modality is being developed as a practical tool in non-invasive applications in medical diagnostics and evaluation. In particular, it is being used in the early detection of skin cancer to identify pathological cellular components and, potentially, replace conventional biopsies. Previous work in the field has shown that the detection of melanin and its spatial location and distribution is important in the detection of skin cancer. Melanin absorbs UV and visible light strongly and appears black in images. However, melanin can also appear bright in confocal images since it can scatter the laser light efficiently. This dual contrast mechanism can complicate the detection or identification of melanin by confocal imaging. Earlier, our group has shown that the melanin emission can also be used to detect melanin. The visible emission is strong and can be easily observed with a near-infrared CW laser using low power, this is due to the unique step-wise three photon excitation signature of melanin. The following paper shows that the same step-wise three photon fluorescence can also be achieved with an inexpensive continuous-wave laser using a dual prism (AKA dual-wedge) scanning system. The results are in agreement with images obtained with the larger and more expensive femtosecond laser system used earlier. This finding is important since it shows that our low-cost configuration, which is portable and can be miniaturized, can be extended for practical non