Although temporally focused wide-field two-photon microscopy (TFM) can perform depth resolved wide field imaging, it cannot avoid the image degradation due to scattering of excitation and emission photons when imaging in a turbid medium. Further, its axial resolution is inferior to standard point-scanning two-photon microscopy. We implemented a structured light illumination for TFM and have shown that it can effectively reject the out-of-focus scattered emission photons improving image contrast. Further, the depth resolution of the improved system is dictated by the spatial frequency of the structure light with the potential of attaining depth resolution better than point-scanning two-photon microscopy.

The effects of the axial field components of a focused beam under high NA on the second harmonic generation (SHG) in collagen was examined using a vectorial approach. We find that with high NA, the cross-component terms that are most likely to have an effect on SHG will be ExEx, ExEy, ExEz and EzEz as a result of tight focusing. By considering the tensor and the presence of the other electric field components the possibility of different polarization states of the generated second harmonic as a result of the nonlinear susceptibility tensor making it possible to generate radially polarized modes with linearly polarized beams.

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This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.

We examine the effects of tightly focusing a radially polarized beam with uniform, Gaussian, or Bessel-Gauss pupil functions. The resulting FWHM is smallest for the case of a uniform amplitude profile, while the Bessel-Gauss beam results in the largest FWHM. The uniform amplitude profile also results in an axial field component that increases fastest with increasing NA. The ratio of the axial component to the transverse component is also the greatest for the uniform pupil function. On the other hand, the Bessel-Gauss beam benefits the most from the use of an annulus.