Light sheet fluorescence microscope with single light sheet illumination enables rapid 3D imaging of living cells. In this paper we show the design, fabrication and characterization of a diffractive optical element producing several light sheets along a 45° inclined tube. The element, which is based on a multi-focal diffractive lens and a linear grating, generates five thin light sheets with equal intensities when combined with a refractive cylindrical lens. The generated uniform light sheets can be applied for the scanning of samples in tubes enabling flow-driven 3-dimensional imaging.

One-dimensional Airy beams allow the generation of thin light-sheets without scanning, simplifying the complex optical arrangements of light-sheet microscopes (LSMs) with an extended field of view (FOV). However, their uniaxial acceleration limits the maximum numerical aperture of the detection objective in order to keep both the active and inactive axes within the depth of field. This problem is particularly pronounced in miniaturized LSM implementations, such as those for endomicroscopy or multi-photon neural imaging in freely moving animals using head-mounted miniscopes. We propose a new method to generate a static Airy light-sheet with biaxial acceleration, based on a novel phase profile. This light-sheet has the geometry of a spherical shell whose radius of curvature can be designed to match the field curvature of the micro-objective. We present an analytical model for the analysis of the light-sheet parameters and verify it by numerical simulations in the paraxial regime. We also discuss a micro-optical experimental implementation combining gradient-index optics with a 3D-nanoprinted, fully refractive phase plate. The results confirm that we are able to match detection curvatures with radii in the range of 1.5 to 2 mm.

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.