Optical Sectioning Research Articles

Volumetric Imaging From Raman Perspective: Review and Prospect

AbstractVolumetric imaging, which supports quantitative and comprehensive assessment of a 3D sample from an entire volume, has attracted tremendous attention in biomedical research. Fluorescence imaging techniques, such as optical sectioning and light sheet microscopy, enable to reconstruct the 3D distribution of chemicals within a sample. However, current methods rely on exogenous labels, from which considerable perturbation may be introduced in living systems. Raman imaging offers a feasible solution to visualize components in biological samples in a label‐free manner. Besides, the integration of Raman microscopy with 3D approaches will benefit the research of biomedical samples on novel devices, which is dominated by the strongly enhanced spatial resolution, imaging speed, and overall field of view as well as complemented more details of samples. In this overview, recent achievements in 3D visualization of biological samples from the Raman perspective, are explored including scanning mechanism, light sheet, tomography strategy, compressive sensing, holography, and tissue clearing. Importantly, these platforms are compatible with biomedical research, thus allowing the imaging of chemical constituents and the distribution of samples in a whole volume. As a unique volumetric imaging tool for biological discovery, these methods may provide a strategy to accelerate new discoveries across diverse fields of research.

Opa1 and MT-Nd6 mutations induce early mitochondrial changes in the retina and prelaminar optic nerve of hereditary optic neuropathy mouse models

Abstract Hereditary optic neuropathies (HON), including dominant optic atrophy and Leber's hereditary optic neuropathy, are genetic disorders characterized by retinal ganglion cell (RGC) degeneration leading to vision loss, mainly associated with mitochondrial dysfunction. In this study, we analyzed mitochondrial distribution and ultrastructure in the retina and longitudinal optic nerve sections of presymptomatic HON mouse models with Opa1 and Nd6 deficiency to identify early mitochondrial changes. Our results show significant mitochondrial fragmentation and increased mitophagy in Opa1+/- mice, indicating early mitochondrial changes prior to neuronal loss. Conversely Nd6P25L mice exhibited mitochondrial hypertrophy, suggesting an adaptive response to compensate for altered energy metabolism. These pre-symptomatic mitochondrial changes were mainly observed in the unmyelinated portion of the RGC axons, where the transmission of the visual information requires high energy expenditure, constituting the specific point of vulnerability in HON. These findings highlight early focal mitochondrial changes prior to neuronal loss in HON and provide insight into presymptomatic therapeutic approaches.

Bi-Plane Multicolor Scanning Illumination Microscopy with Multispot Excitation and a Distorted Diffraction Grating

Multifocus microscopy has previously been demonstrated to provide volumetric information from a single shot. However, the practical application of this method is challenging due to its weak optical sectioning and limited spatial resolution. Here, we report on the combination of a distorted diffraction grating and multifocal scanning illumination microscopy to improve spatial resolution and contrast. DG is introduced in the emission path of the multifocal scanning illumination microscopy, which splits the fluorescence signal from different sample layers into different diffraction orders. After postprocessing, super-resolution wide-field images of different sample layers can be reconstructed from single 2D scanning.

Stereo slit-scanning tomography of the anterior segment of the human eye

We demonstrate a new tomographic imaging device for 2D cross-sectional and 3D volumetric images of the anterior eye segment. The optical system combines optical sectioning with a stereoscopic camera setup to determine the true spatial location of the ocular structures. Moreover, we propose a calibration procedure to estimate the triangulation function that links the stereo camera pixel indices with the eye-referenced 3D spatial coordinates. The proposed instrument will image the entire anterior chamber and contiguous structures, including the corneal limbus, sclera and eyelids. Furthermore, the system can obtain corneal topography. Finally, we studied the repeatability of the instrument and its agreement with a commercial Scheimpflug imaging tomographer in a group of healthy subjects.

From Optical Fiber Communications to Bioimaging: Wavelength Division Multiplexing Technology for Simplified in vivo Large-depth NIR-IIb Fluorescence Confocal Microscopy.

Near-infrared II (NIR-II, 900-1880nm) fluorescence confocal microscopy offers high spatial resolution and extensive in vivo imaging capabilities. However, conventional confocal microscopy requires precise pinhole positioning, posing challenges due to the small size of the pinhole and invisible NIR-II fluorescence. To simplify this, a fiber optical wavelength division multiplexer (WDM) replaces dichroic mirrors and traditional pinholes for excitation and fluorescence beams, allowing NIR-IIb (1500-1700 nm) fluorescence and excitation light to be coupled into the same optical fiber. This streamlined system seamlessly integrates key components-excitation light, detector, and scanning microscopy-via optical fibers. Compared to traditional NIR-II confocal systems, the fiber optical WDM configuration offers simplicity and ease of adjustment. Notably, this simplified system successfully achieves optical sectioning imaging of mouse cerebral blood vessels up to 1000µm in depth. It can discern tiny blood vessels (diameter: 4.57µm) at 800µm depth with a signal-to-background ratio (SBR) of 5.34. Additionally, it clearly visualizes liver vessels, which are typically challenging to image, down to a depth of 300µm.

A Structured Illumination Microscope with the Combination of Optical Sectioning and Lateral Resolution Enhancement for Precise 3D Industrial Metrology

A Structured Illumination Microscope with the Combination of Optical Sectioning and Lateral Resolution Enhancement for Precise 3D Industrial Metrology

High-speed in vivo calcium recording using structured illumination with self-supervised denoising

High-speed widefield fluorescence imaging of neural activity in vivo is fundamentally limited by fluctuations in recorded signal due to background contamination and stochastic noise. In this study, we show background and shot noise-reduced imaging of the ultrafast genetically encoded Ca2+ indicator GCaMP8f in CA1 pyramidal neurons using periodic structured illumination (SI) with computational image reconstruction. We implement what we believe to be a novel reconstruction method for data acquired using periodic structured illumination, termed pseudo-HiLo (pHiLo), that combines a pseudo-widefield (pWF) reconstruction with individual SI frames to perform a HiLo reconstruction. We compare this new technique to interleaved optical sectioning structured illumination microscopy (OS-SIM) and pWF reconstruction. We quantify the performance of each reconstruction by evaluating contrast, transient peak-to-noise ratio (PNR), pairwise correlation coefficients between ΔF/F time courses extracted from individual in-focus cells, and correlation coefficients between each cell with surrounding cell-free background pixels. We additionally incorporate a self-supervised deep learning method for real-time noise suppression (DeepCAD-RT) into our data preprocessing pipeline. At 500 Hz frame rates, we demonstrate a 75% increase in PNR using the denoised pHiLo reconstruction compared to pWF. Utilizing DeepCAD-RT, we show significant PNR improvements using both structured illumination (SI) reconstruction methods with OS-SIM showing a 59% increase in PNR after denoising. Both pHiLo and OS-SIM reconstructions result in a ≈65% decrease in the mean correlation coefficient of the ΔF/F time courses between ROIs in comparison with pWF, indicating the potential to remove background fluorescent transients from out-of-focus cells.

Three-dimensional isotropic imaging of live suspension cells enabled by droplet microvortices

Fast, nondestructive three-dimensional (3D) imaging of live suspension cells remains challenging without substrate treatment or fixation, precluding scalable single-cell morphometry with minimal alterations. While optical sectioning techniques achieve 3D live cell imaging, lateral versus depth resolution differences further complicate analysis. We present a scalable microfluidic method capable of 3D fluorescent isotropic imaging of live, nonadherent cells suspended inside picoliter droplets with high-speed single-cell volumetric readout (800 to 1,200 slices in 5 to 8 s) and near-diffraction limit resolution (~216 nm). The platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intradroplet microvortices, which rotate single cells on their axis to enable optical projection tomography (OPT)-based imaging. This allows gentle (~1 mPa shear stress) observation of cells encapsulated inside nontoxic isotonic buffer droplets, facilitating scalable OPT acquisition by simultaneous spinning of hundreds of cells. We demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells-small cells prone to movement in their suspended phenotype. Our fully suspended, orientation-independent cell morphometry, driven by isotropic imaging and spherical harmonic analysis, enabled the study of primary T cells across various immunological activation states. This approach unveiled six distinct nuclear content distributions, contrasting with conventional 2D images that typically portray spheroid and bean-like nuclear shapes associated with lymphocytes. Our arrayed-droplet OPT technology is capable of isotropic, single live-cell 3D imaging, with the potential to perform large-scale morphometry of immune cell effector function states while providing compatibility with microfluidic droplet operations.

Living histopathology - interrogation of ocular tissues by light: a celebration of the slit-lamp and a repertoire of clinical techniques.

The evolution of the slit-lamp microscope has enabled ophthalmologists to examine the transparent tissues of the eye with histological detail. This paper considers the history and optics of the slit-lamp. Optical sectioning and retro-illumination are discussed; particularly, effective placement of the reflected light beam. A variety of less conventional slit-lamp examination techniques is described. These include remote dark-field retro-illumination, examination through refractive surfaces (particularly, meniscus retro-illumination to demonstrate tear cells and non-contact corneal endothelial specular microscopy), location of vitreous abnormalities by parallax, expanding radial cords of vitreous cells in lymphoma, mirror examination of the superior fornix and corneal epithelial folds in ocular hypotension. It concludes with brief discussions about haemoglobin video imaging, semi-quantification of aqueous outflow volume by aqueous column cross-section area, and autofocus for video-microscopy.

Temperature-insensitive and cost-effective distributed NP-Doped optical fiber sensors

This paper presents the development of a cost-effective distributed optical fiber sensor for temperature-insensitive assessment of mechanical disturbances along an optical fiber cable. The proposed sensor system uses a nanoparticle (NP)-doped optical fiber with enhanced Rayleigh backscattering to provide higher sensitivity and spatial resolution using the transmission and reflection analysis (TRA) approach, where the transmitted and backscattered optical powers are analyzed as a function of the mechanical disturbance. In addition, Fiber Bragg Gratings (FBGs) are used as wavelength filters to provide the wavelength division multiplexing of the proposed device, which enable the use of 3 different NP-doped optical fiber sections for simultaneous detection of multiple curvature conditions in a cost-effective distributed sensing approach. The sensor characterization tests are performed by means of applying curvature angles from 360° to 1080° at different positions along NP-doped fibers (namely 25 mm, 100 mm and 175 mm) at 4 different temperatures of 25 °C, 30 °C, 40 °C and 50 °C. The results indicate the feasibility of the proposed approach, where the temperature variations lead only to a wavelength shift of the Bragg wavelength, whereas the mechanical disturbances (the curvatures) lead only to variations in the transmitted and reflected optical powers. Thus, by analyzing the transmitted and reflected optical powers in conjunction with the Bragg wavelength shift, it is possible to estimate both the mechanical disturbance amplitude (i.e., curvature angle) and the position along each NP-doped optical fiber section. Results indicate a relative error of around 3 % and 4 % for the mechanical disturbance location and absolute value, respectively. Moreover, the temperature cross-sensitivity in this case is below 2 % considering both amplitude and location of the mechanical disturbance. The proposed approach can be applied in structural health monitoring of different types of structures by integrating the fibers in the structures themselves with the possibility of measuring the strain distribution along the fibers (instead of in different points along the fiber) using a lower cost hardware when compared with similar distributed optical fiber sensing approaches.

Substance P- and Insulin-like Growth Factor 1-derived Tetrapeptides for Neurotrophic Keratopathy Related to Leprosy: A Clinical Trial

PurposeNeurotrophic keratopathy is part of the leprosy sequelae and causes progressive deterioration of visual acuity. Although leprosy is bacteriologically curable, there is currently no efficient treatment. Eye drops containing tetrapeptides, Phenylalanine-Glycine-Leucine-Methionine-amide (FGLM-NH2) and Serine-Serine-Serine-Arginine (SSSR), derived from substance P and insulin-like growth factor 1 (IGF-1), are clinically efficacious in the treatment of corneal epithelial disorders caused by neurotrophic keratopathy. To further investigate the effect of this treatment on leprosy sequalae, we evaluated the clinical efficacy of FGLM-NH2+SSSR eye drops for treating neurotrophic keratopathy. DesignClinical trial: Interventional, multicenter, exploratory, single-arm, before and after comparison. ParticipantsThe eyes (12) of 11 patients, aged >60 years, were studied from two leprosy sanatoriums in Japan. MethodsPatients with neurotrophic keratopathy in leprosy sanatorium, specifically those with corneal perception of <40 mm, assessed by the Cochet-Bonnet corneal esthesiometer, and persistent corneal epithelial defects (PEDs) and/or corneal stromal thinning were included in this study. Those treated for infection in the acute phase were excluded from the study. Eye drops containing FGLM-NH2 0.05% and SSSR 5x10-6% were administered four times daily for up to 3 months. Fluorescein staining and optical corneal sections were photographed using a slit-lamp microscope at protocol-set intervals. Where possible, anterior segment optical coherence tomography was performed before and after the intervention. Main outcome measuresThe primary outcome measured was improvement in neurotrophic keratopathy. The patient was judged to have improved when one or more of the following criteria were met: 1) healing epithelial defects or 2) increased thickness in the thin area of the cornea. Secondary endpoints were visual acuity, subjective findings, and time to complete healing for a PED. ResultsNeurotrophic keratopathy on epithelial defects or stromal thickness improved in 83.3% of the patients (90% confidence interval 56.2%–97.0%, P<0.00001). The mean value of corrected visual acuity increased -0.16 by logarithm of the minimum angle of resolution. There were no adverse events reported in association with the treatment. ConclusionsWe confirmed that FGLM-NH2+SSSR eye drops are effective for neurotrophic keratopathy without any adverse reaction in leprosy. These results should be disseminated to any parties who could need this information.

Pixelation with Concentration‐Encoded Effective Photons for Quantitative Molecular Optical Sectioning Microscopy (Laser Photonics Rev. 18(10)/2024)

Pixelation with Concentration‐Encoded Effective Photons for Quantitative Molecular Optical Sectioning Microscopy (Laser Photonics Rev. 18(10)/2024)

Rapid full-color serial sectioning tomography with speckle illumination and ultraviolet excitation

Three-dimensional (3D) high-resolution large-volume imaging has remained a challenge. Translational rapid ultraviolet-excited sectioning tomography (TRUST) achieves rapid and cost-effective whole-organ subcellular imaging through iterative optical scanning and mechanical sectioning. However, the axial resolution is limited by the mechanical sectioning thickness or the UV light penetration depth in tissue. Here, assisted with high-and-low-frequency (HiLo) microscopy (HiLoTRUST), the optical sectioning thickness has been reduced from tens of micrometers to ~5.8 µm. In addition, HiLoTRUST has attained a finer mechanical sectioning thickness (10–15 µm) compared to TRUST (50 µm). For high-content imaging as in TRUST, we employed two additional UV light-emitting diodes (LEDs) specifically for uniform illumination. The full-color imaging ability and improved axial resolution of HiLoTRUST have been validated by two-dimensional (2D)/3D imaging of various mouse organs and human lung cancer specimens. HiLoTRUST offers a cost-effective, full-color, and high-resolution 3D imaging approach, showing its great potential in 3D histology applications.

Large-field optical sectioning structured illumination microscopy

Nowadays, fringe generation and fast-switching in structured illumination microscopy (SIM) is often performed using spatial light modulators (SLMs) and digital micromirror devices (DMDs). Yet, the field of view (FOV) or the spatial bandwidth product (SBP) is constrained by the limited pixel number of the SLMs or DMDs. In this study, we present a large-field optical-sectioning structured illumination microscopy (LF-OS-SIM) scheme, which features a large FOV and fast phase shifting. LF-OS-SIM utilizes a one-dimensional grating to generate stripe patterns and a spatial light modulator (SLM) to perform phase-shifting in the Fourier domain. Therefore, this technique can improve the spatial bandwidth product (SBP) by a factor of 2.6 compared to the conventional OS-SIM under the same experimental parameters. The superiority of LF-OS-SIM is demonstrated by imaging euro coins and fluorescent samples, and the results imply that this method has the potential to be applied for 3D imaging of industrial micro-devices and biosamples.

Model based optimization for refractive index mismatched light sheet imaging

Selective plane illumination microscopy (SPIM) is an optical sectioning imaging approach based on orthogonal light pathways for excitation and detection. The excitation pathway has an inverse relation between the optical sectioning strength and the effective field of view (FOV). Multiple approaches exist to extend the effective FOV, and here we focus on remote focusing to axially scan the light sheet, synchronized with a CMOS camera’s rolling shutter. A typical axially scanned SPIM configuration for imaging large samples utilizes a tunable optic for remote focusing, paired with air objectives focused into higher refractive index media. To quantitatively explore the effect of remote focus choices and sample space refractive index mismatch on light sheet intensity distributions, we developed an open-source computational approach for integrating ray tracing and field propagation. We validate our model’s performance against experimental light sheet profiles for various SPIM configurations. Our findings indicate that optimizing the position of the sample chamber relative to the excitation optics can enhance image quality by balancing aberrations induced by refractive index mismatch. We validate this prediction using a home-built, large sample axially scanned SPIM configuration and calibration samples. Our open-source, extensible modeling software can easily extend to explore optimal imaging configurations in diverse light sheet imaging settings.

Sub-60-nm isotropic 3D super-resolution microscopy through self-interference field excitation

Due to its unique optical sectioning capability, confocal laser scanning microscopy (CLSM) can provide highly sensitive, highly specific imaging of specimens in three dimensions and has been recognized as an indispensable tool for biological and medical studies. Nonetheless, the spatial resolution of CLSM is constrained by the diffraction nature, with λ/2 resolution laterally (xy) and 1.5λ resolution axially (z). To improve the imaging resolution beyond the diffraction limit as well as to achieve its isotropy, we present a strategy of mirror-assisted self-interference field excitation (SIEx) highly nonlinear microscopy. The imaging principle has been theoretically modeled and investigated in accordance with the Wolf vector diffraction theory. The experimental demonstration of isotropic three-dimensional SIEx nanoscopy, assisted with the ultrahigh-order optical nonlinearity of photon avalanching nanoparticles, was achieved utilizing a common laser-scanning microscope configuration, resulting in a lateral resolution of 54 nm (λ/15) and an axial resolution of 57 nm (λ/15) with one single beam from a low-power, continuous-wave, near-infrared laser (19kW⋅cm−2). We further extended the applicability of the SIEx scheme to biological imaging and demonstrated super-resolution imaging for immunolabeled actin filaments of BSC-1 cells with an isotropic full width at half maximum of ∼67nm (λ/13). Our facile SIEx methodology can, in principle, be seamlessly integrated with the existing and widely available laser-scanning fluorescence microscopes without adding any complexity, thereby enabling their capability of 3D isotropic super-resolution imaging.

Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet.

Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample. This easily adaptable novel microfluidic fabrication pipeline allows for the incorporation of reflective optics into microfluidic channels without disrupting efficient and automated solution exchange. By combining these innovations with point spread function engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets, we demonstrate whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed.

Identification of novel genes regulating the development of the palate.

The International Mouse Phenotyping Consortium (IMPC) has generated thousands of knockout mouse lines, many of which exhibit embryonic or perinatal lethality. Using micro-computed tomography (micro-CT), the IMPC has created and publicly released 3D image datasets of embryos from these lethal and subviable lines. In this study, we leveraged this dataset to screen homozygous null mutants for anomalies in secondary palate development. We analyzed optical sections from 2,987 embryos at embryonic days E15.5 and E18.5, representing 484 homozygous mutant lines. Our analysis identified 45 novel genes implicated in palatogenesis. Gene set enrichment analysis highlighted biological processes and pathways relevant to palate development and uncovered 18 genes jointly regulating the development of the eye and the palate. These findings present a valuable resource for further research, offer novel insights into the molecular mechanisms underlying palatogenesis, and provide important context for understanding the etiology of rare human congenital disorders involving simultaneous malformations of the palate and other organs, including the eyes, ears, kidneys, and lungs.

Quantitative and comparative assessment of dyes and protocols for rapid ex vivo microscopy of fresh tissues

Using ex vivo microscopy, virtual pathology can improve histological procedures by providing pathology images in near real-time without tissue destruction. Several emerging and promising approaches leverage fast-acting small-molecule fluorescent stains to replicate traditional pathology structural contrast, combined with rapid optical sectioning microscopes. However, several vital challenges must be addressed to translate virtual pathology into the clinical environment. One such challenge is selecting robust, reliable, and repeatable staining protocols that can be adopted across institutions. In this work, we addressed the effects of dye selection and staining protocol on image quality in rapid point-of-care imaging settings. For this purpose, we used structured illumination microscopy to evaluate fluorescent dyes currently used in the field of ex vivo virtual pathology, in particular, studying the effects of staining protocol and temporal and photostability on image quality. We observed that DRAQ5 and SYBR gold provide higher image quality than TO-PRO3 and RedDot1 in the nuclear channel and Eosin Y515 in the extracellular/cytoplasmic channel than Atto488. Further, we found that TO-PRO3 and Eosin Y515 are less photostable than other dyes. Finally, we identify the optimal staining protocol for each dye and demonstrate pan-species generalizability.

Comprehensive characterization of nitrogen-related defect states in β-Ga2O3 using quantitative optical and thermal defect spectroscopy methods

This study provides a comprehensive analysis of the dominant deep acceptor level in nitrogen-doped beta-phase gallium oxide (β-Ga2O3), elucidating and reconciling the hole emission features observed in deep-level optical spectroscopy (DLOS). The unique behavior of this defect, coupled with its small optical cross section, complicates trap concentration analysis using DLOS, which is essential for defect characterization in β-Ga2O3. A complex feature arises in DLOS results due to simultaneous electron emission to the conduction band and hole emission to the valence band from the same defect state, indicating the formation of two distinct atomic configurations and suggesting metastable defect characteristics. This study discusses the implications of this behavior on DLOS analysis and employs advanced spectroscopy techniques such as double-beam DLOS and optical isothermal measurements to address these complications. The double-beam DLOS method reveals a distinct hole emission process at EV+1.3 eV previously obscured in conventional DLOS. Optical isothermal measurements further characterize this energy level, appearing only in N-doped β-Ga2O3. This enables an estimate of the β-Ga2O3 hole effective mass by analyzing temperature-dependent carrier emission rates. This work highlights the impact of partial trap-filling behavior on DLOS analysis and identifies the presence of hole trapping and emission in β-Ga2O3. Although N-doping is ideal for creating semi-insulating material through the efficient compensation of free electrons, this study also reveals a significant hole emission and migration process within the weak electric fields of the Schottky diode depletion region.