In Vivo Intelligent Fluorescence Endo-Microscopy by Varifocal Meta-Device and Deep Learning.

Endo-microscopy is crucial for real-time 3D visualization of internal tissues and subcellular structures. Conventional methods rely on axial movement of optical components for precise focus adjustment, limiting miniaturization and complicating procedures. Meta-device, composed of artificial nanostructures, is an emerging optical flat device that can freely manipulate the phase and amplitude of light. Here, an intelligent fluorescence endo-microscope is developed based on varifocal meta-lens and deep learning (DL). The breakthrough enables in vivo 3D imaging of mouse brains, where varifocal meta-lens focal length adjusts through relative rotation angle. The system offers key advantages such as invariant magnification, a large field-of-view, and optical sectioning at a maximum focal length tuning range of ≈2 mm with 3 µm lateral resolution. Using a DL network, image acquisition time and system complexity are significantly reduced, and in vivo high-resolution brain images of detailed vessels and surrounding perivascular space are clearly observed within 0.1 s (≈50 times faster). The approach will benefit various surgical procedures, such as gastrointestinal biopsies, neural imaging, brain surgery, etc.

Intelligent Phase Contrast Meta-Microscope System.

Phase contrast imaging techniques enable the visualization of disparities in the refractive index among various materials. However, these techniques usually come with a cost: the need for bulky, inflexible, and complicated configurations. Here, we propose and experimentally demonstrate an ultracompact meta-microscope, a novel imaging platform designed to accomplish both optical and digital phase contrast imaging. The optical phase contrast imaging system is composed of a pair of metalenses and an intermediate spiral phase metasurface located at the Fourier plane. The performance of the system in generating edge-enhanced images is validated by imaging a variety of human cells, including lung cell lines BEAS-2B, CLY1, and H1299 and other types. Additionally, we integrate the ResNet deep learning model into the meta-microscope to transform bright-field images into edge-enhanced images with high contrast accuracy. This technology promises to aid in the development of innovative miniature optical systems for biomedical and clinical applications.

Varifocal Metalens for Optical Sectioning Fluorescence Microscopy.

Fluorescence microscopy with optical sectioning capabilities is extensively utilized in biological research to obtain three-dimensional structural images of volumetric samples. Tunable lenses have been applied in microscopy for axial scanning to acquire multiplane images. However, images acquired by conventional tunable lenses suffer from spherical aberration and distortions. Here, we design, fabricate, and implement a dielectric Moiré metalens for fluorescence imaging. The Moiré metalens consists of two complementary phase metasurfaces, with variable focal length, ranging from ∼10 to ∼125 mm at 532 nm by tuning mutual angles. In addition, a telecentric configuration using the Moiré metalens is designed for high-contrast multiplane fluorescence imaging. The performance of our system is evaluated by optically sectioned images obtained from HiLo illumination of fluorescently labeled beads, as well as ex vivo mice intestine tissue samples. The compact design of the varifocal metalens may find important applications in fluorescence microscopy and endoscopy for clinical purposes.

Simultaneous multi-color optical sectioning fluorescence microscopy with wavelength-coded volume holographic gratings.

Optical sectioning fluorescence microscopy provides high contrast images of volumetric samples and has been widely used for many biological applications. However, simultaneously acquiring multi-color fluorescence images require additional optical elements and devices, which are bulky, wavelength specific, and not cost-effective. In this paper, wavelength-coded volume holographic gratings (WC-VHGs) based optical sectioning fluorescence microscopy is proposed to simultaneously offer multi-color fluorescence images with fine out-of-focus background rejection. Due to wavelength degeneracy, multiplexed WC-VHGs are capable of acquiring multi-wavelength fluorescence images in a single shot, and displaying the laterally separated multi-wavelength images onto CCD. In our system optical sectioning capability is achieved through speckle illumination and HiLo imaging method. To demonstrate imaging characteristics of our system, dual-wavelength fluorescence images of both standard fluorescent microspheres and ex vivo mT/mG mice cardiac tissue are presented. Current results may find important applications in hyperspectral imaging for biomedical research.

Multiplane differential phase contrast imaging using asymmetric illumination in volume holographic microscopy.

SIGNIFICANCE: Differential phase contrast (DPC) is a well-known imaging technique for phase imaging. However, simultaneously acquiring multidepth DPC images is a non-trivial task. We propose simultaneous multiplane DPC imaging using volume holographic microscopy (VHM). AIM: To design and implement a new configuration of DPC-VHM for multiplane imaging. APPROACH: The angularly multiplexed volume holographic gratings (AMVHGs) and the wavelength-coded volume holographic gratings (WC-VHGs) are used for this purpose. To obtain asymmetric illumination for DPC images, a dynamic illumination system is designed by modifying the regular Köhler illumination using a thin film transistor panel (TFT-panel). RESULTS: Multidepth DPC images of standard resolution chart and biosamples were used to compare imaging performance with the corresponding bright-field images. An average contrast enhancement of around three times is observed for target resolution chart by DPC-VHM. Imaging performance of our system is studied by modulation transfer function analysis, which suggests that DPC-VHM not only suppresses the DC component but also enhances high-frequency information. CONCLUSIONS: Proposed DPC-VHM can acquire multidepth-resolved DPC images without axial scanning. The illumination part of the system is adjustable so that the system can be adapted to bright-field mode, phase contrast mode, and DPC mode by controlling the pattern on the TFT-panel.

Volume holographic spatial-spectral imaging systems [Invited].

In this paper, we present an overview of the recent developments in applications of volume holographic imaging techniques in microscopy. In these techniques, three-dimensional imaging incorporates multiplexed volume holographic gratings, which are formed in phenanthrenequinone poly(methyl methacrylate) (PQ-PMMA) photopolymer and act as spatial-spectral filters, to obtain multiplane images from a volumetric object without scanning. We introduce recent major roles of volume holography in different imaging modalities, including large-capacity spatial-spectral multiplane microscopy, digital holographic microscopy, and structured Talbot (or speckle) illumination fluorescence imaging. Among various imaging applications of volume holography, simultaneous multiplane fluorescence microscopy for collecting spatial-spectral information is distinct and has great potential for hyperspectral imaging. Depth selective spatial-spectral information from an object is particularly useful for designing a high-resolution microscope in real-time operation. We further discuss volume holography in particle trapping and beam shaping. In addition, we investigate future prospects of volume holography in microscopy as well as endoscopy.

Conventional volume holography for unconventional Airy beam shapes.

Utilizing multiplexed volume holography, a single optical element, enabling to shape structural variants of non-diffracting Airy wavefronts, from one-dimensional Airy mode to unconventional Airy modes such as vortex Airy and quad Airy modes, has been experimentally realized. Here, beam shaped angularly multiplexed volume holographic gratings (AMVHGs) are recorded in PQ: PMMA photopolymer, where five different spatial wavefronts of Airy beams have been sequentially recorded, for simultaneous reconstruction of different Airy modes, by a conventional Gaussian beam. Spatial and spectral mode selective properties of AMVHGs are demonstrated by narrow-band as well as by broadband light source. In addition, through wavelength degeneracy property, the maximum sensitivity wavelength of blue (488 nm) is used for recording in PQ: PMMA, but the AMVHGs are operated at a broad wavelength band of interest, all the way to longer wavelength in near infrared (850 nm). The K-sphere representation is used to explain the spectral properties of AMVHGs.