From bench to bedside with advanced confocal microendoscope

Confocal microscopy is an indispensable tool for three-dimensional (3-D) imaging in both intravital and clinical settings. Because of its optical sectioning property achieved from its optical pinhole, it allows serial image stacking of each focus image plane to re-create volumetric imaging. Conventional single-axis confocal (SAC) microscopes have a tradeoff between resolution, field of view (FOV), and objective lens size, since a high numerical aperture (NA) lens is needed for sufficient resolution, and a long focal length is needed for a large FOV and working distance (WD). A dual-axes confocal (DAC) microscope architecture has been proposed utilizing two overlapping low NA beams, which effectively decouples these tradeoffs [1]. The DAC microscope offers several advantages over the SAC design. First, the higher NA required by the SAC design implies a smaller WD and smaller FOV. In contrast, the DAC design has a long WD, providing room for a postobjective MEMS scanner. Second, in the SAC design, the transverse resolution is substantially better than the axial resolution, while the DAC design provides balanced resolutions in all spatial dimensions. Third, the DAC design has superior optical sectioning because light scattered along the illumination path outside the focal volume couples to the output spatial filter with very low efficiency, enhancing both detection sensitivity and dynamic range. Previously, MEMS-scanner-based DAC microscopes have been demonstrated in a tabletop setup [2], and then in the first miniaturized proto-type system on a V-block with 1300- and 488-nm-wavelength, respectively [3, 4].