Fluorescence imaging through the second near-infrared window (NIR-II,1000–1700 nm) allows in-depth imaging. However, current imaging systems use wide-field illumination and can only provide low-contrast 2D information, without depth resolution. Here, we systematically apply a light-sheet illumination, a time-gated detection, and a deep-learning algorithm to yield high-contrast high-resolution volumetric images. To achieve a large FoV (field of view) and minimize the scattering effect, we generate a light sheet as thin as 100.5 μm with a Rayleigh length of 8 mm to yield an axial resolution of 220 μm. To further suppress the background, we time-gate to only detect long lifetime luminescence achieving a high contrast of up to 0.45 Ι_contrast. To enhance the resolution, we develop an algorithm based on profile protrusions detection and a deep neural network and distinguish vasculature from a low-contrast area of 0.07 Ι_contrast to resolve the 100 μm small vessels. The system can rapidly scan a volume of view of 75 × 55 × 20 mm³ and collect 750 images within 6 mins. By adding a scattering-based modality to acquire the 3D surface profile of the mice skin, we reveal the whole volumetric vasculature network with clear depth resolution within more than 1 mm from the skin. High-contrast large-scale 3D animal imaging helps us expand a new dimension in NIR-II imaging.

In light-sheet fluorescence microscopy, the axial resolution and field of view are mutually constrained. Axially swept light-sheet microscopy (ASLM) can decouple the trade-off, but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest, which sacrifices the photon efficiency. Here, we report a laterally swept light-sheet microscopy (LSLM) scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter. We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved. Moreover, based on the principle of image scanning microscopy, applying the pixel reassignment to the LSLM images, hereby named iLSLM, improves the optical sectioning. Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning. Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.

The pixel size of a charge-coupled device (CCD) camera plays a major role in the image resolution, and the square pixels are attributed to the physical anisotropy of the sampling frequency. We synthesize the high sampling frequency directions from multiple frames acquired with different angles to enhance the resolution by 1.4 × over conventional CCD orthogonal sampling. To directly demonstrate the improvement of frequency-domain diagonal extension (FDDE) microscopy, lens-free microscopy is used, as its resolution is dominantly determined by the pixel size. We demonstrate the resolution enhancement with a mouse skin histological specimen and a clinical blood smear sample. Further, FDDE is extended to lens-based photography with an ISO 12233 resolution target. This method paves a new way for enhancing the image resolution for a variety of imaging techniques in which the resolution is primarily limited by the sampling pixel size, for example, microscopy, photography, and spectroscopy.

A fast off-axis scanning subvoxel light-sheet microscope enables high-throughput image large-volume specimens at cellular resolution.