A schematic and photograph of the benchtop VHIS are shown in Figs. 2(a) and 2(b), respectively, depicting the system’s key components. The interchangeable illumination consists of either a 690 nm broadband LED (H2A1-H690, Roithner Lasertechnik GmbH, Vienna, Austria) for reflectance imaging or a 635 nm laser diode (BWF, B&W Tek, Incorporated, Newark, Delaware) for fluorescence imaging. In either scheme, illumination light passes through a pellicle beam splitter (BP150, Thorlabs Incorporated, Newtown, New Jersey) and then enters the , 0.55 numerical aperture (NA) objective lens with a 3.64-mm focal length (ULWDMSPlan50, Olympus America Incorporated, Center Valley, Pennsylvania). A Köhler illumination configuration is used to provide uniform illumination at the sample. Optical relays are used to project the pupil to the hologram plane which prevents beam shifting and signal loss. Reflected or fluorescent light originating from the sample is captured by the objective lens, redirected by the pellicle beam splitter, and filtered by the 665 nm long-pass filter (ET665LP, Chroma Technology Corporation, Bellows Falls, Vermont), which blocks the 635-nm excitation illumination in fluorescence mode, and reaches the multiplexed volume hologram. Here, the hologram diffracts and disperses the light along the -axis of the image. The multiplexed gratings in the hologram are designed to select light from the tissue surface and 50 μm below the surface. The light from these two channels is focused by a camera lens (AC254-075-A, Thorlabs Incorporated, Newtown, New Jersey) and projected side-by-side onto a CCD camera (ORCA-R2, Hamamatsu Corporation, Middlesex, New Jersey). VHIS images were acquired using HCImage Live software (Hamamatsu Corporation, Middlesex, New Jersey). The position of the tissue specimen is adjusted using a three-axis micropositioning stage (460A Series, Newport Corporation, Irvine, California) with a digital micrometer head (DM713, Thorlabs Incorporated, Newtown, New Jersey) for -axis control. As assembled, the VHIS provides lateral resolution of approximately 4 μm and an axial resolution of approximately . Future adaptation into a clinical endoscopic VHIS for in vivo imaging will be achieved by replacing the objective lens with a high NA endoscope and incorporating the hologram and all other optical components into a sealed handle for use by the oncological surgeon.