Optical Sectioning Research Articles

Protective effects of neuropeptide Y treatment on retina in a mouse model of experimental glaucoma

Purpose: Neuropeptide Y (NPY) is an endogenous 36‐amino acid linear peptide with tyrosine residues on both ends of the molecule. Cumulative evidence suggests that NPY and its receptors in the CNS may be a potential neuroprotective targets in various degenerative conditions. This study aimed to investigate the protective effects of NPY in the retina in an experimentally induced glaucoma animal model.Methods: Weekly intracameral microbead injections were performed to induce sustainably increased intraocular pressure (IOP) and NPY was administered intravitreally for 2 months. Wild‐type C57BL/6J mice (n = 40) were categorized into four major groups i) control, ii) control + NPY, iii) high IOP model, & iv) high IOP + NPY. Inner retinal function was evaluated by using positive scotopic threshold response (pSTR) amplitudes. Eye and optic nerve sections were then immunostained with pNFH (phosphorylated neurofilament heavy chain), GFAP (glial fibrillary acidic protein) and Iba‐1(Ionized calcium‐binding adaptor molecule 1) antibodies to understand the protective effects of NPY treatment.Results: IOP elevation was observed in the microbead model (control, 10.5 ± 0.75; microbead, 27.35 ± 1.92 mmHg). Functional data revealed a significant decline in the pSTR amplitudes in high IOP conditions (p < 0.0001, n = 10) compared to the control group, and a significant increase in pSTR was observed in the high IOP + NPY group (p < 0.001, n = 10). Optic nerves stained with pNFH revealed a significant protective effect of NPY treatment against high IOP‐induced axonal damage (p < 0.01, n = 4). Increased GFAP expression observed in high IOP condition was significantly decreased with NPY treatment both in the retina (p < 0.001, n = 4) and optic nerves (p < 0.01, n = 4). Similarly, microglial activation evaluated by Iba‐1 expression was also significantly decreased by NPY treatment both in the retina (p < 0.01, n = 4) and optic nerves (p < 0.01, n = 4).Conclusions: NPY treatment significantly reduced inner retinal functional loss and optic nerve damage against high IOP‐induced glaucomatous injury. NPY also reduced the microglial activation and reactive gliosis in experimental glaucoma. Our ongoing investigation will reveal specific NPY receptors activation and biochemical mechanism underlying these protective effects in the retina.

Microfluidic device for the high-throughput and selective encapsulation of single target cells.

Droplet-based microfluidic technologies for encapsulating single cells have rapidly evolved into powerful tools for single-cell analysis. In conventional passive single-cell encapsulation techniques, because cells arrive randomly at the droplet generation section, to encapsulate only a single cell with high precision, the average number of cells per droplet has to be decreased by reducing the average frequency at which cells arrive relative to the droplet generation rate. Therefore, the encapsulation efficiency for a given droplet generation rate is very low. Additionally, cell sorting operations are required prior to the encapsulation of target cells for specific cell type analysis. To address these challenges, we developed a cell encapsulation technology with a cell sorting function using a microfluidic chip. The microfluidic chip is equipped with an optical detection section to detect the optical information of cells and a sorting section to encapsulate cells into droplets by controlling a piezo element, enabling active encapsulation of only the single target cells. For a particle population including both targeted and non-targeted particles arriving at an average frequency of up to 6000 particles per s, with an average number of particles per droplet of 0.45, our device maintained a high purity above 97.9% for the single-target-particle droplets and achieved an outstanding throughput, encapsulating up to 2900 single target particles per s. The proposed encapsulation technology surpasses the encapsulation efficiency of conventional techniques, provides high efficiency and flexibility for single-cell research, and shows excellent potential for various applications in single-cell analysis.

A new instrumentation for simultaneous terahertz and mid-infrared spectroscopy in corrosive gaseous mixtures.

The correct interpretation of infrared (IR) observations of planetary atmospheres requires an accurate knowledge of temperature and partial and global pressures. Precise laboratory measurements of absorption intensities and line profiles, in the 200-350K temperature range, are, therefore, critical. However, for gases only existing in complex chemical equilibria, such as nitrous or hypobromous acids, it is not possible to rely on absolute pressure measurements to measure absolute integrated optical absorption cross sections or IR line intensities. To overcome this difficulty, a novel dual-beam terahertz (THz)/mid-IR experimental setup has been developed, relying on the simultaneous use of two instruments. The setup involves a newly constructed temperature-controlled (200-350K) cross-shaped absorption cell made of inert materials. The cell is traversed by the mid-IR beam from a high-resolution Fourier transform spectrometer using along a White-cell optical configuration providing absorption path lengths from 2.8 to 42m and by a THz radiation beam (82.5GHz to 1.1THz), probing simultaneously the same gaseous sample. The THz channel records pure rotational lines of molecules for which the dipole moment was previously measured with high precision using Stark spectroscopy. This allows for a determination of the partial pressure in the gaseous mixture and enables absolute line intensities to be retrieved for the mid-IR range. This new instrument opens a new possibility for the retrieval of spectroscopic parameters for unstable molecules of atmospheric interest. The design and performance of the equipment are presented and illustrated by an example of simultaneous THz and mid-IR measurement on nitrous acid (HONO) equilibrium.

Intelligent Beam Optimization for Light-Sheet Fluorescence Microscopy through Deep Learning.

Light-sheet fluorescence microscopy (LSFM) provides the benefit of optical sectioning coupled with rapid acquisition times, enabling high-resolution 3-dimensional imaging of large tissue-cleared samples. Inherent to LSFM, the quality of the imaging heavily relies on the characteristics of the illumination beam, which only illuminates a thin section of the sample. Therefore, substantial efforts are dedicated to identifying slender, nondiffracting beam profiles that yield uniform and high-contrast images. An ongoing debate concerns the identification of optimal illumination beams for different samples: Gaussian, Bessel, Airy patterns, and/or others. However, comparisons among different beam profiles are challenging as their optimization objectives are often different. Given that our large imaging datasets (approximately 0.5 TB of images per sample) are already analyzed using deep learning models, we envisioned a different approach to the problem by designing an illumination beam tailored to boost the performance of the deep learning model. We hypothesized that integrating the physical LSFM illumination model (after passing it through a variable phase mask) into the training of a cell detection network would achieve this goal. Here, we report that joint optimization continuously updates the phase mask and results in improved image quality for better cell detection. The efficacy of our method is demonstrated through both simulations and experiments that reveal substantial enhancements in imaging quality compared to the traditional Gaussian light sheet. We discuss how designing microscopy systems through a computational approach provides novel insights for advancing optical design that relies on deep learning models for the analysis of imaging datasets.

4D-STEM Optical Sectioning of Dopants in Diamond

4D-STEM Optical Sectioning of Dopants in Diamond

Recent Advances in Super-Resolution and Optical Sectioning of Digital-Micromirror Device-Based Structured-Illumination Microscopy (Invited)

Recent Advances in Super-Resolution and Optical Sectioning of Digital-Micromirror Device-Based Structured-Illumination Microscopy (Invited)

Ratiometric near-infrared fluorescent probe for nitroreductase activity enables 3D imaging of hypoxic cells within intact tumor spheroids.

Fluorescent molecular probes that report nitroreductase activity have promise as imaging tools to elucidate the biology of hypoxic cells and report the past hypoxic history of biomedical tissue. This study describes the synthesis and validation of a "first-in-class" ratiometric, hydrophilic near-infrared fluorescent molecular probe for imaging hypoxia-induced nitroreductase activity in 2D cell culture monolayers and 3D multicellular tumor spheroids. The probe's molecular structure is charge-balanced and the change in ratiometric signal is based on Förster Resonance Energy Transfer (FRET) from a deep-red, pentamethine cyanine donor dye (Cy5, emits ∼660 nm) to a linked near-infrared, heptamethine cyanine acceptor dye (Cy7, emits ∼780 nm). Enzymatic reduction of a 4-nitrobenzyl group on the Cy7 component induces a large increase in Cy7/Cy5 fluorescence ratio. The deep penetration of near-infrared light enables 3D optical sectioning of intact tumor spheroids, and visualization of individual hypoxic cells (i.e., cells with raised Cy7/Cy5 ratio) as a new way to study tumor spheroids. Beyond preclinical imaging, the near-infrared fluorescent molecular probe has high potential for ratiometric imaging of hypoxic tissue in living subjects.

A REVIEW ON BENZANTHRONE LUMINOPHORES FOR RAPID AND HIGH-RESOLUTION IMAGING OF PARASITES BY CONFOCAL LASER SCANNING MICROSCOPY

Nowadays, is growing interest in investigation of parasites and their anatomic structure. Most common tools are linked with usage of luminophores and luminescent microscopy techniques. In recent years, a variety of fluorescent probes have been developed and widely used to realize the visualization of certain structures [1]. The benzanthrone compounds captivated a lot of interest as fluorescent probes for biomedical technologies because of remarkable spectral properties and negligible fluorescence in the aqueous phase [2]. Notably, the spectral characteristics of benzanthrone dyes meet the criteria for an ideal bio-imaging agent, featuring a high extinction coefficient, bright fluorescence, photo-, thermo- and chemical stability, and reduced background signal [3]. Fluorescent molecular dyes, currently used to study cell membranes, make lipid structures visible through optical techniques [4] and act as potential fluorescent probes for biomolecules [5]. There are studies in the literature where the benzanthron phosphors have confirmed their application for visualization of biological objects [6-8]. Despite high activity in this field, still it`s huge request for specific fluorescent probes for biological objects. The confocal laser scanning microscope (CLSM) is a powerful tool for providing high-resolution optical sections. CLSM can be used to analyse images of morphological structures and to place organ or organ systems of interest in their anatomical context. Luminescence imaging techniques are increasingly utilized for exploring the structure and properties of biological objects. Laser-induced fluorescence stands out as a sensitive method, capable of detecting even a single-molecule under specific conditions [9], which makes it a powerful bioanalytical tool for life sciences [10 12]. Benzanthrone derivates are used for CLSM imaging as fluorescence probe for various biological objects because benzanthrones render the specimen fluorescent to examine the stained samples by optical sectioning [13,14]. This review provides inputs in utilisation of different benzanthrone luminophores for examination of the parasites.

A spatial carrier dynamic quantitative differential phase imaging method

Differential interference contrast (DIC) microscopy is an important method for quantitative phase imaging (QPI) due to its high compatibility regarding to partial coherent light illumination and excellent optical sectioning ability. However, limited by the on-axis interferometric configuration of Nomarski DIC microscopic imaging system, it is difficult to achieve single-shot phase retrieval. Here, we propose a simple and efficient method for dynamic quantitative differential phase imaging (QDPI) by introducing a spatial carrier device into DIC microscope. A Wollaston prism (WP) is placed on the imaging plane of DIC microscope and then projected by a 4f imaging system, so two orthogonally polarized imaging beams with a certain shearing amount will interfere on the conjugate plane of the 4f imaging system and form a spatial carrier DIC image, thus the QDPI can be conveniently realized. Importantly, the system not only possesses high temporal phase stability provided by the common-path geometries and low spatial phase noise due to partially coherent light illumination, but also the advantages of simple optical implementation, no additional phase distortion, high light energy utilization efficiency, and high signal-to-noise ratio. The experimental result demonstrates that using the proposed method, the phase measurement sensitivity reaches 0.003 rad, the corresponding applicability is verified by a polystyrene microsphere crown and biological cells.

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms

Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400–650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.

Peripapillary intrachoroidal cavitation at the crossroads of peripapillary myopic changes.

To analyze the prevalence of peripapillary intra-choroidal cavitation (PICC) in eyes with gamma peripapillary atrophy (γPPA), in eyes with peripapillary staphyloma (PPS) and in those combining γPPA and PPS and to analyze border tissue discontinuity in PICC. This prospective cross-sectional non interventional study included highly myopic eyes. Non-highly myopic eyes were used as control. Radial and linear scans centered on the optic nerve head were performed using spectral-domain optical coherence tomography. Variables were analyzed along the twelve hourly optical coherence tomography sections in both eyes of each subject. A total of 667 eyes of 334 subjects were included: 229 (34.3%) highly myopic eyes and 438 (65.7%) non highly myopic eyes. The mean age of the highly myopic group was 48.99±17.81y. PICC was found in a total of 40 eyes and in 13.2% (29/220) of highly myopic eyes. PICC was found in 10.4% (40/386) of eyes with γPPA, in 20.5% (40/195) of eyes with PPS and in 22.7% (40/176) of those combining γPPA and PPS. All the eyes with PICC showed the co-existence of γPPA and PPS whereas none of the eyes presenting only one of these entities exhibited PICC. A border tissue discontinuity in the γPPA area was found in all eyes with PICC. We confirm the presence of a border tissue discontinuity in the γPPA area of all eyes with PICC. These findings suggest the involvement of mechanical factors in the pathogenesis of PICC which may contribute to PICC-related visual field defects.

Volume holographic illuminator for Airy light-sheet microscopy.

Airy light sheets combined with the deconvolution approach can provide multiple benefits, including large field of view (FOV), thin optical sectioning, and high axial resolution. The efficient design of an Airy light-sheet fluorescence microscope requires a compact illumination system. Here, we show that an Airy light sheet can be conveniently implemented in microscopy using a volume holographic grating (VHG). To verify the FOV and the axial resolution of the proposed VHG-based Airy light-sheet fluorescence microscope, ex-vivo fluorescently labeled Caenorhabditis elegans (C. elegans) embryos were imaged, and the Richardson-Lucy deconvolution method was used to improve the image contrast. Optimized parameters for deconvolution were compared with different methods. The experimental results show that the FOV and the axial resolution were 196 µm and 3 µm, respectively. The proposed method of using a compact VHG to replace the common spatial light modulator provides a direct solution to construct a compact light-sheet fluorescence microscope.

Three-dimensional imaging in reflection phase microscopy with minimal axial scanning.

Reflection phase microscopy is a valuable tool for acquiring three-dimensional (3D) images of objects due to its capability of optical sectioning. The conventional method of constructing a 3D map is capturing 2D images at each depth with a mechanical scanning finer than the optical sectioning. This not only compromises sample stability but also slows down the acquisition process, imposing limitations on its practical applications. In this study, we utilized a reflection phase microscope to acquire 2D images at depth locations significantly spaced apart, far beyond the range of optical sectioning. By employing a numerical propagation, we successfully filled the information gap between the acquisition layers, and then constructed complete 3D maps of objects with substantially reduced number of axial scans. Our experimental results also demonstrated the effectiveness of this approach in enhancing imaging speed while maintaining the accuracy of the reconstructed 3D structures. This technique has the potential to improve the applicability of reflection phase microscopy in diverse fields such as bioimaging and material science.

Multidimensional Widefield Infrared-Encoded Spontaneous Emission Microscopy: Distinguishing Chromophores by Ultrashort Infrared Pulses.

Photoluminescence (PL) imaging has broad applications in visualizing biological activities, detecting chemical species, and characterizing materials. However, the chemical information encoded in the PL images is often limited by the overlapping emission spectra of chromophores. Here, we report a PL microscopy based on the nonlinear interactions between mid-infrared and visible excitations on matters, which we termed MultiDimensional Widefield Infrared-encoded Spontaneous Emission (MD-WISE) microscopy. MD-WISE microscopy can distinguish chromophores that possess nearly identical emission spectra via conditions in a multidimensional space formed by three independent variables: the temporal delay between the infrared and the visible pulses (t), the wavelength of visible pulses (λvis), and the frequencies of the infrared pulses (ωIR). This method is enabled by two mechanisms: (1) modulating the optical absorption cross sections of molecular dyes by exciting specific vibrational functional groups and (2) reducing the PL quantum yield of semiconductor nanocrystals, which was achieved through strong field ionization of excitons. Importantly, MD-WISE microscopy operates under widefield imaging conditions with a field of view of tens of microns, other than the confocal configuration adopted by most nonlinear optical microscopies, which require focusing the optical beams tightly. By demonstrating the capacity of registering multidimensional information into PL images, MD-WISE microscopy has the potential of expanding the number of species and processes that can be simultaneously tracked in high-speed widefield imaging applications.

Three-dimensional refractive index microscopy based on the multi-layer propagation model with obliquity factor correction

Multi-layer propagation model-based intensity diffraction tomography (IDT) is a three-dimensional (3D) microscopy imaging technique to reveal the structural information of optically thick or multiple scattering samples. However, the performance of existing multi-layer propagation models deteriorates with increasing illumination angle, which can prevent good 3D refractive index (RI) imaging. To overcome this limitation, we incorporate the obliquity factor (OF) into the multi-layer propagation model. Numerical simulation validates that the forward propagation and the RI reconstruction both can be considerably improved. Then, the 3D RI reconstruction performance is quantitatively and qualitatively validated by optical experiments on polystyrene microspheres and peony pollen grains, respectively. In addition, excellent optical sectioning and high spatial resolution are experimentally demonstrated on various unlabeled fixed and living samples, including an Arabidopsis root, HeLa and HCT-116 cells. Time-lapse 3D imaging of living samples is performed for the first time for multi-layer propagation model-based IDT, demonstrating its potential application to some biological and medical studies.

Super‐Resolution Two‐Photon Fluorescence Tomography Through the Phase‐Shifted Optical Beatings of Bessel Beams for High‐Resolution Deeper Tissue 3D Imaging

AbstractA novel z‐scanning‐free epi‐detected super‐resolution two‐photon fluorescence tomography (TPFT) technique enabling super‐resolution deeper tissue 3D imaging is reported. To accomplish this, a unique method is conceived by generating the phase‐shifted optical beatings of Bessel beams (PS‐OB3) with a spatial light modulator (SLM) to break the diffraction limit for enhancing both the lateral and axial resolutions as well as improving the penetration depth in TPFT for super‐resolution deeper tissue imaging. By electronically varying the optical beating frequency and the phase shifts of the beating patterns through SLM, the depth‐resolved TPF signals about the volumetric tissue are encoded in the spatial frequency domain and hence, a series of depth‐resolved TPF images can be retrieved by implementing inverse fast Fourier transform without a need of mechanical depth‐scanning. PS‐OB3 TPFT provides ≈1.3‐ and 2‐fold improvements in lateral and axial resolutions in comparison with conventional point‐scan TPF imaging. It is also illustrated that the epi‐detected PS‐OB3 TPFT imaging with inherent scattering‐resilient properties of the Bessel beams employed gives over 2‐fold improvement in imaging depth in porcine brain tissue compared to conventional point‐scan Gaussian beam TPF imaging. The z‐scanning‐free optical sectioning ability of PS‐OB3 method developed in TPFT is universal, which can be readily extended to practically any other nonlinear optical imaging modalities for super‐resolution deeper 3D imaging in biological and biomedical tissues.

Rapid 3D isotropic imaging of whole organ with double-ring light-sheet microscopy and self-learning side-lobe elimination.

Bessel-like plane illumination forms a new type of light-sheet microscopy with ultra-long optical sectioning distance that enables rapid 3D imaging of fine cellular structures across an entire large tissue. However, the side-lobe excitation of conventional Bessel light sheets severely impairs the quality of the reconstructed 3D image. Here, we propose a self-supervised deep learning (DL) approach that can completely eliminate the residual side lobes for a double-ring-modulated non-diffraction light-sheet microscope, thereby substantially improving the axial resolution of the 3D image. This lightweight DL model utilizes the own point spread function (PSF) of the microscope as prior information without the need for external high-resolution microscopy data. After a quick training process based on a small number of datasets, the grown-up model can restore sidelobe-free 3D images with near isotropic resolution for diverse samples. Using an advanced double-ring light-sheet microscope in conjunction with this efficient restoration approach, we demonstrate 5-minute rapid imaging of an entire mouse brain with a size of ∼12 mm × 8 mm × 6 mm and achieve uniform isotropic resolution of ∼4 µm (1.6-µm voxel) capable of discerning the single neurons and vessels across the whole brain.

Improving optical sectioning with spinning disk structured illumination microscopy.

A new fluorescence microscopy technique for optical sectioning was investigated. This technique combined Spinning Disk microscopy (SD) with Structured Illumination Microscopy (SIM), resulting in more background removal than either method. Spinning Disk Structured Illumination Microscopy (SD-SIM) resulted in higher signal-to-background ratios. The method detected and quantified a dendritic spine neck that was impossible to detect with either SIM or SD alone.

Tissue optical clearing methods for microscopy: A review of their application in neuroscience

Recent advances in microscopy have enabled cellular-resolution imaging of thick tissue samples or even whole organs. The natural opacity of organs and tissues acts as a barrier to light penetration and must be removed to visualise structures of interest on a three-dimensional scale. Tissue optical clearing methods achieve sample transparency while also preserving fluorescently labelled epitopes. This innovative approach to sample preparation effectively enhances traditional histological sections and, with the aid of light sheet microscopy, enables optical sectioning and three-dimensional reconstruction of entire organs, even whole brains. Light sheet microscopy of optically cleared brain samples is a valuable method in neuroscience that is used in neuro-oncology, traumatic brain injury, ischemic brain injury, and neurodegenerative disease research.In this review, we describe tissue optical clearing methods used to achieve optical transparency in brain samples. This quickly developing field has a significant potential for producing cutting-edge uses in neuroscience research.

Management of the axial modulation of the illumination pattern in structured illumination microscopy using an extended illumination source.

We have designed and implemented an approach for three-dimensional (3D) structured illumination (SI) microscopy (SIM) based on a quasi-monochromatic extended source illuminating a Wollaston prism to improve robustness, light efficiency and flexibility over our previous design. We show through analytical and experimental verification of the presented theoretical framework for our proposed tunable structured illumination microscopy (TSIM) system, that a simple and accurate determination of the axial modulation of the SI pattern is achieved, enabling a realistic characterization of the system's effective optical transfer function (OTF). System performance as a function of the extended source size is investigated with simulations. Results from a comparative performance analysis of the proposed TSIM system and traditional SIM systems show some advantages over the traditional two-wave and three-wave interference SIM systems. We show that by controlling the source size and thereby the axial modulation of the 3D SI pattern, the TSIM scheme offers increased OTF compact support and improved optical sectioning capability, quantified by the integrated intensity, under certain conditions, which may be desirable when imaging optically thick samples. The additional tunability of the 3D SI pattern, provides a unique opportunity for OTF engineering in our TSIM system.