Abstract

Methods to enable enhanced tissue depth in stimulated emission imaging are discussed. To take advantage of the increased scattering length and reduced absorption in the near infrared, 2–4 photons are used for both molecular excitation and stimulated emission in stimulated fluorescence imaging. The method is called multiphoton-stimulated emission microscopy (MP-STEM). In a confocal microscope, the axial focal spot in 3-photon MP-STEM may be reduced to about ½ the wavelength of the stimulated emission beam, potentially enabling the detection of ballistic backscatter at the tissue surface. Also discussed is a microscope geometry that combines Bessel beam excitation with an orthogonal structured illumination stimulated emission beam. This geometry is called Bessel beam-stimulated emission microscopy (BB-STEM). It is shown that the axial focal spot diameter of the stimulated emission beam may be reduced to less than ½ of the stimulated emission beam wavelength for 1–3 photon stimulated emission processes, enabling dipole backscatter ballistic photons to be detected in both stimulated fluorescence and stimulated Raman processes. Dipole ballistic backscatter detection reduces emitter concentrations detection thresholds by the reduction of the detected stimulated emission beam background noise. Use of multiple time delays between the excitation and emission processes enables the use of these approaches to measure the fluorescent lifetime of species with multiple lifetimes. Also discussed are the challenges of these methods, including the required laser power, focal spot blooming, and array detectors.

© 2016 Optical Society of America

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