Fluorescence microscopy with optical sectioning based on varifocal metalens

Imaging with single-pixel detectors offers a valuable alternative to the conventional focal plane array strategy, especially for wavelengths where silicon-based sensor arrays exhibit lower efficiency. However, the absence of optical sectioning remains a challenge in single-pixel microscopy. In this paper, we introduce a single-pixel microscope with optical sectioning capabilities by integrating single-pixel imaging (SPI) techniques with structured illumination microscopy (SIM) methods. A spatial light modulator positioned at the microscope's input port encodes a series of structured light patterns, which the microscope focuses onto a specific plane of the 3D sample. Simultaneously, a highly sensitive bucket detector captures the light reflected by the object. Optical sectioning is achieved through a high-frequency grating positioned at the microscope's output port, which is conjugated with the spatial light modulator. Utilizing SPI reconstruction techniques and SIM algorithms, our computational microscope produces high-quality 2D images without blurred out-of-focus regions. We validate the performance of the single-pixel microscope (SPM) by measuring the axial response function and acquiring images of various 3D samples in reflected bright-field configuration. Furthermore, we demonstrate the suitability of the optical setup for single-pixel fluorescence microscopy with optical sectioning.

Acquisition time and optical sectioning capability are critical factors in fluorescence imaging. Confocal microscopy is a vital optical imaging method to inherently observe volumetric tissues with fine optical sectioning capability; however, point‐by‐point scanning is time‐consuming. Metasurfaces, a type of flat optics utilizing nano‐scale structures, provide diverse functionalities and extensive flexibility in controlling light wavefronts. Here, meta‐microlens‐array (meta‐MLA) for multifocal confocal fluorescence microscopy to enhance acquisition speed is introduced, reduce photo‐bleaching, and improve energy efficiency while remaining compatible with existing commercial scanning configurations. Point spread function (PSF) in the meta‐MLA confocal lateral and axial directions has been evaluated. Fast optically sectioned images of various samples, including pollen grains and biological tissue phantoms, are performed. Image quality is further enhanced by the Richardson–Lucy (RL) deconvolution method with total variation (TV). The trade‐off between spatial resolution and acquisition speed is overcome using deep neural network models, comparing performance metrics with a conventional confocal microscope. The combination of meta‐MLA confocal and deep learning with superior image quality and fast acquisition will likely extend the clinical applications of miniaturized optical imaging.

Optical sectioning structured illumination microscopy (OS‐SIM) is a fast, minimally invasive 3D imaging technique that has found widespread application in the biosciences. It is based on sample illumination with several illumination fringe patterns featuring distinct mutual phase shifts, from which an axially sectioned image is reconstructed. Its optical sectioning capability is commonly attributed to the attenuation of the fringe modulation of light collected from planes displaced from the focal plane. However, in addition to this effect, which is governed solely by the detection optics, optical sectioning can be further enhanced by confining the fringe modulation axially via partially coherent illumination (PCI). To establish guidelines for optimal illumination field shaping, both theoretically and experimentally are investigated, the optical sectioning strength of OS‐SIM upon variation of the two key parameters, modulation period and angular spectrum of the incident illumination. By using PCI with OS‐SIM, nearly fivefold and 1.4‐fold enhanced axial resolution are achieved for scattering (non‐fluorescent) and fluorescent samples, respectively. This work elucidates the optical sectioning mechanism of OS‐SIM and provides a perspective for further optimization.

Optical sectioning structured illumination microscopy (OS-SIM) provides optical sectioning capability in wide-field microscopy. The required illumination patterns have traditionally been generated using spatial light modulators (SLM), laser interference patterns, or digital micromirror devices (DMDs) which are too complex to implement in miniscope systems. MicroLEDs have emerged as an alternative light source for patterned illumination due to their extreme brightness capability and small emitter sizes. This paper presents a directly addressable striped microLED microdisplay with 100 rows on a flexible cable (70 cm long) for use as an OS-SIM light source in a benchtop setup. The overall design of the microdisplay is described in detail with luminance-current-voltage characterization. OS-SIM implementation with a benchtop setup shows the optical sectioning capability of the system by imaging within a 500 µm thick fixed brain slice from a transgenic mouse where oligodendrocytes are labeled with a green fluorescent protein (GFP). Results show improved contrast in reconstructed optically sectioned images of 86.92% (OS-SIM) compared with 44.31% (pseudo-widefield). MicroLED based OS-SIM therefore offers a new capability for deep tissue widefield imaging.

Dynamic focus control in high-speed optical coherence tomography based on a microelectromechanical mirror

Improved extended depth of focus full field spectral domain Optical Coherence Tomography

Objective To evaluate the accuracy of automated fluorescence microscopic imaging and computer-aided diagnosis system (AFMICADS) in the auxiliary diagnosis of superficial cutaneous fungal infections. Methods Totally, 106 outpatients and inpatients with suspected superficial fungal infections were enrolled from clinical departments of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and September 2018. A total of 126 specimens were collected, including 83 skin scales and 43 nail parings. Each specimen was divided into 3 groups to be examined by conventional fungal microscopy, culture with modified Sabouraud dextrose agar and fluorescence microscopy (artificial fluorescence microscopy and AFMICADS-based fluorescence microscopy) respectively. A positive result was defined as that conventional fungal microscopy and/or fungal culture was positive. Consistency rate, sensitivity and specificity of the 3 microscopic methods were calculated. Statistical analysis was carried out with SPSS 10.0 software by using McNemar test and Kappa test for analyzing difference in the positive rate, as well as consistency, between the 3 microscopic methods and the positive standard, and by using efficiency test for comparing the consistency rate among the 3 microscopic methods. Results Of 126 specimens, 124 (98.4%) were positive for artificial fluorescence microscopy, and 123 (97.6%) for AFMICADS-based fluorescence microscopy. Both positive rates of the above 2 microscopic methods were significantly higher than the positive rate of the positive standard (77.8%, both P 0.05) . Conclusion The accuracy of AFMICADS-based fluorescence microscopy in the diagnosis of superficial cutaneous fungal infections is similar to that of artificial fluorescence microscopy. Key words: Fungi; Microscopy, fluorescence; Diagnosis, computer-assisted; Fluorescence stain; Machine vision; Automated fluorescence microscopic imaging

Imaging a volumetric sample in real time is required to directly observe biological anatomy and mechanisms. There are a variety of microscopic imaging systems, developed to achieve optical sectioning, such as confocal microscopy. However, the drawbacks of existing systems are long scanning time or strong laser power. To overcome such problems and to obtain high-speed optical sectioning images, we demonstrate HiLo structured illumination microscopy by the use of digital micro-mirror device (DMD) and the focal tunable lens (FTL). The proposed system is configured such that hybrid uniform and non-uniform pattern are projected onto DMD to reconstruct optical sectioning images. In addition, FTL is utilized to digitally change observation plane with constant magnification and resolution; thus, mechanical movement is eliminated. Furthermore, in vivo three-dimensional (3D) images of biological samples, including a live Canenorhabditis elegans, are experimentally demonstrated.

Selective Plane Illumination Microscopy (SPIM) is attractive for its ability to acquire 3D images with high 3D spatial resolution, good optical sectioning capability and high imaging speed. However, tradeoffs have to be made when a large field of view (FOV) is required, results in lower axial resolution or worse optical sectioning capability. Here, we present a novel method for 3D imaging by SPIM that is capable to maintain its high 3D spatial resolution and good optical sectioning capability within a large FOV. Instead of trying to generate a large and uniformly thick excitation light sheet, the method tiles a relative small light sheet quickly to multiple positions within the image plane by defocusing the excitation beam used to create the light sheet, and takes one additional image at each position, so that a large FOV can be imaged by repeating this process and stitching all images together. By implementing this method, light sheets with thin thickness and good excitation light confinement can be used for SPIM imaging with slightly compromised imaging speed. The method was investigated through both numerical simulation and experiments, and the imaging performance was demonstrated by imaging fluorescent particles embedded in agarose gel and live C. elegans embryos.

Light-sheet fluorescence microscopy (LSFM) facilitates high temporal-spatial resolution, low photobleaching and phototoxicity for long-term volumetric imaging. However, when a high axial resolution or optical sectioning capability is required, the field of view (FOV) is limited. Here, we propose to generate a large FOV of light-sheet by scanning multiple focus-shifted Gaussian beam arrays (MGBA) while keeping the high axial resolution. The positions of the beam waists of the multiple Gaussian beam arrays are shifted in both axial and lateral directions in an optimized arranged pattern, and then scanned along the direction perpendicular to the propagation axis to form an extended FOV of light-sheet. Complementary beam subtraction method is also adopted to further improve axial resolution. Compared with the single Gaussian light-sheet method, the proposed method extends the FOV from 12 μm to 200 μm while sustaining the axial resolution of 0.73 μm. Both numerical simulation and experiment on samples are performed to verify the effectiveness of the method.

A combined 1300 nm Optical Coherence Microscopy (OCM) and 488 nm confocal reflectance/ fluorescence microscopy system was designed to perform high-resolution, high-specificity imaging of collagen-embedded spheroids. Spheroids of Hyaluronic Acid (HA) synthase-overexpressing breast epithelial cells alone, or co-cultured with adipose stromal cells were imaged. The volumes, acquired either after fixing and staining or longitudinally with labeling, enabled the visualization of the spheroid morphology, luminal structures, cellular organization, and collagen matrix remodeling. The morphology and internal lumen structures of spheroids, as large as 500 μm in diameter, could be obtained from the OCM volumes, even in the presence of dense collagen matrix surrounding the spheroids. The confocal stacks provided superior specificity to discriminate cells from the compacted collagen along the spheroid’s periphery, up to a depth of ~120 μm. The combined use of OCM and confocal imaging on these spheroid models has added to our understanding of how HA may contribute to tumor initiation and invasion.

Fluorescence microscope, image analysis and patch-clamp recording system.

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

We present a volumetric imaging method for biological tissue that is free of mechanically scanning components. The optical sectioning in the system is obtained by structured illumination microscopy (SIM) with the depth of focus being varied by the use of an electronic tunable-focus lens (ETL). The performance of the axial scanning mechanism was evaluated and characterized in conjunction with SIM to ensure volumetric images could be recorded and reconstructed without significant losses in optical section thickness and lateral resolution over the full desired scan range. It was demonstrated that sub-cellular image resolutions were obtainable in both microsphere films and in ex vivo oral mucosa, spanning multiple cell layers, without significant losses in image quality. The mechanism proposed here has the ability to be integrated into any wide-field microscopy system to convert it into a three-dimensional imaging platform without the need for axial scanning of the sample or imaging optics. The ability to axially scan independent of mechanical movement also provides the opportunity for the development of endoscopic systems which can create volumetric images of tissue in vivo.

A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells