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Dipole-like backscatter stimulated Raman scattering for in vivo imaging



Stimulated Raman scattering (SRS) scanning microscopy has the potential to enable label-free in vivo imaging for research and clinical medicine. Volume SRS from focus occurs in the forward scattered direction. Therefore, multiple scattering events are required to direct the light out of the tissue, reducing imaging depth and resolution. Here, a method called Stokes interference SRS (SISRS) is introduced that operates by the addition to the standard pump and stimulated emission probe beams a third beam called the donut beam. The donut is close in wavelength to the probe beam and, after passage through a π phase plate, forms an annular beam in the focal plane with bright nodes above and below focus. The donut beats with the probe beam, and when they destructively interfere with each other, the microscope's 3-D stimulated emission focal spot is reduced to subwavelength dimensions. A subwavelength focal volume emits a dipole pattern of SRS with forward and backscatter lobes, enabling high-resolution single-backscatter imaging from deep within tissues. The reduction of the focal volume also increases the resolution of the scanning image creating imaging beyond the diffraction limit. SISRS imaging may provide in vivo label-free Raman images comparable with that achieved in stained in vitro tissues in all planes of section. Copyright © 2014 John Wiley & Sons, Ltd.