Optical Sectioning Microscopy Research Articles

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)

Pixelation with Concentration‐Encoded Effective Photons for Quantitative Molecular Optical Sectioning Microscopy

AbstractIrreproducibility in molecular optical sectioning microscopy has hindered the transformation of acquired digital images from qualitative descriptions to quantitative data. Although numerous tools, metrics, and phantoms have been developed, accurate quantitative comparisons of data from different microscopy systems with diverse acquisition conditions remains a challenge. Here, they develop a simple tool based on an absolute measurement of bulk fluorophore solutions with related Poisson photon statistics, to overcome this obstacle is developed. Demonstrated in a prototypical multiphoton microscope, this tool unifies the unit of pixelated measurement to enable objective comparison of imaging performance across different modalities, microscopes, components/settings, and molecular targets. The application of this tool in live specimens identifies an attractive methodology for quantitative imaging, which rapidly acquires low signal‐to‐noise frames with either gentle illumination or low‐concentration fluorescence labeling.

Smartphone-based optical sectioning (SOS) microscopy with a telecentric design for fluorescence imaging.

We propose a smartphone-based optical sectioning (SOS) microscope based on the HiLo technique, with a single smartphone replacing a high-cost illumination source and a camera sensor. We built our SOS with off-the-shelf optical, mechanical cage systems with 3D-printed adapters to seamlessly integrate the smartphone with the SOS main body. The liquid light guide can be integrated with the adapter, guiding the smartphone's LED light to the digital mirror device (DMD) with neglectable loss. We used an electrically tuneable lens (ETL) instead of a mechanical translation stage to realise low-cost axial scanning. The ETL was conjugated to the objective lens's back pupil plane (BPP) to construct a telecentric design by a 4f configuration to maintain the lateral magnification for different axial positions. SOS has a 571.5µm telecentric scanning range and an 11.7µm axial resolution. The broadband smartphone LED torch can effectively excite fluorescent polystyrene (PS) beads. We successfully used SOS for high-contrast fluorescent PS beads imaging with different wavelengths and optical sectioning imaging of multilayer fluorescent PS beads. To our knowledge, the proposed SOS is the first smartphone-based HiLo optical sectioning microscopy (£1965), which can save around £7035 compared with a traditional HiLo system (£9000). It is a powerful tool for biomedical research in resource-limited areas.

A qualitative comparison of 3D visualization in Xenopus laevis using a traditional method and a non-destructive method

Many tools are currently available to investigate and visualize soft and hard tissues in animals both in high-resolution and three dimensions. The most popular and traditional method is based on destructive histological techniques. However, these techniques have some specific limitations. In order to avoid those limitations, various non-destructive approaches have surfaced in the last decades. One of those is micro-CT-scanning. In the best conditions, resolution achieved in micro-CT currently approaches that of standard histological protocols. In addition to bone, soft tissues can also be made visible through micro-CT-scanning. However, discriminating between structures of the same tissue and among different tissue types remains a challenge. An alternative approach, which has not yet been explored to its full potential for comparative anatomy studies, is Orthogonal-Plane Fluorescence Optical Sectioning (OPFOS) microscopy or tomography, also known as (Laser) Light Sheet based Fluorescence Microscopy (LSFM). In this study, we compare OPFOS with light microscopy, applying those techniques to the model organism Xenopus laevis. The potential of both methods for discrimination between different types of tissues, as well as different structures of the same tissue type, is tested and illustrated. Since the histological sections provided a better resolution, adjacent structures of the same tissue type could be discerned more easily compared to our OPFOS images. However, we obtained a more naturally-shaped 3D model of the musculoskeletal system of Xenopus laevis with OPFOS. An overview of the advantages and disadvantages of both techniques is given and their applicability for a wider scope of biological research is discussed.

Adaptive structured illumination optical-sectioning microscopy based on the prior knowledge of sample structure

Fast and accurate online detection is of great significance to improve mass production efficiency and reduce the costs of semiconductor chips and other micro-nano devices. Compared with other 3D imaging detection techniques, structured illumination optical-sectioning microscopy has faster 3D imaging because it uses wide-field sinusoidal fringe structured illumination. However, commercial structured illumination optical-sectioning microscopes are designed for general needs, and the orientations of the fringe patterns they project are not optimized for the structures of the sample. To obtain an isotropic result, they need to project multiple sinusoidal fringe patterns with different orientations and perform multiple optical-sectioning imaging, and then average the optical-sectioning results, resulting in low imaging efficiency. This paper proposes an adaptive structured illumination optical-sectioning microscopy. It exploits prior knowledge of the sample structure to design the sinusoidal fringe orientation for each local structure and generates an adaptive structured illumination. In this way, we can achieve the best imaging effect with only one optical-sectioning imaging, thereby improving the imaging efficiency, which is beneficial to the improvement of the mass production efficiency of products. The method proposed in this paper will provide useful guidance on how to correctly and efficiently use structured illumination optical-sectioning microscopy to detect defects of micro-nano devices such as semiconductor chips with known designed structures.

Three-Dimensional Phase Interface Reconstruction of Micro-Grooved Heat Pipe Based on Optical Sectioning Microscopy

Three-Dimensional Phase Interface Reconstruction of Micro-Grooved Heat Pipe Based on Optical Sectioning Microscopy

3D Optical Sectioning Microscopy with Sparse Structured Illumination

3D Optical Sectioning Microscopy with Sparse Structured Illumination

Rapid slide-free and non-destructive histological imaging using wide-field optical-sectioning microscopy.

Histopathology based on formalin-fixed and paraffin-embedded tissues has long been the gold standard for surgical margin assessment (SMA). However, routine pathological practice is lengthy and laborious, failing to guide surgeons intraoperatively. In this report, we propose a practical and low-cost histological imaging method with wide-field optical-sectioning microscopy (i.e., High-and-Low-frequency (HiLo) microscopy). HiLo can achieve rapid and non-destructive imaging of freshly-excised tissues at an extremely high acquisition speed of 5 cm2/min with a spatial resolution of 1.3 µm (lateral) and 5.8 µm (axial), showing great potential as an SMA tool that can provide immediate feedback to surgeons and pathologists for intraoperative decision-making. We demonstrate that HiLo enables rapid extraction of diagnostic features for different subtypes of human lung adenocarcinoma and hepatocellular carcinoma, producing surface images of rough specimens with large field-of-views and cellular features that are comparable to the clinical standard. Our results show promising clinical translations of HiLo microscopy to improve the current standard of care.

Single-shot optical sectioning microscopy based on structured illumination.

In this Letter, we present a single-shot 3D-resolved structured illumination microscopy (SIM) based on a digital micromirror device (DMD), a galvanometric mirror, and the HiLo algorithm. During imaging, the DMD rapidly generates sinusoidal and plane illuminations in the focal region. By synchronizing the DMD with a galvanometric scanner and exploiting the unique data readout process of the camera, the emissions from the specimen under two different illuminations, i.e., structured and uniform illumination, are projected to different regions on a camera, achieving high-resolution single-exposure optical sectioning at the camera's limiting speed, i.e., 200 Hz, without sacrificing the resolution. A model has been developed to guide the design and optimization of the optical system. Imaging experiments on pollen and mouse kidney samples have been performed to verify the predicted performance. The results show that the single-shot SIM with the HiLo algorithm achieves comparable resolution to the standard two-shot HiLo method with a twofold speed enhancement, which may find important applications in biophotonics, e.g., visualizing high-speed biological events in vivo.

Comprehensive histological imaging of native microbiota in human glioma.

Mounting evidence suggests that distinct microbial communities reside in tumors and play important roles in tumor physiology. Recently, a previous study profiled the composition and localization of intratumoral bacteria using 16S ribosomal DNA (rDNA) sequencing and histological visualization methods across seven tumor types, including human glioblastoma. However, their results based on traditional histological examinations should be further validated considering potential sources of contamination originating from sample collection and processing. Here, we aim to propose a three-dimensional (3D) in situ intratumoral microbiota visualization and quantification protocol avoiding surface contamination and provide a comprehensive histological investigation on local bacteria within human glioma samples. We develop a 3D quantitative in situ intratumoral microbiota imaging strategy, combining tissue clearing, immunofluorescent labeling, optical sectioning microscopy, and image processing, to visualize bacterial lipopolysaccharide (LPS) within gliomas in a direct, contaminant-free, and unambiguous manner. Through an automated statistical algorithm, reliable signals can be distinguished for further analysis of their sizes, distribution, and fluorescence intensities. In tandem, we also combined 2D images obtained from thin-section histological methods, including immunohistochemistry and fluorescence in situ hybridization, to provide comprehensive histological imaging for local bacterial components within human glioma samples. We have, for the first time, achieved 3D quantitative imaging of bacterial LPS colonized in gliomas in a contamination-free manner within human glioma samples. We also built the multiple histological evidence chain demonstrating the irregular shapes and sparse distribution of bacterial components within human glioma samples, mostly localized near nuclear membranes or in the intercellular space. This study provides favorable evidence for the presence of microbiota in human gliomas and provides information on the feature and distribution of bacterial components. The results, along with the integrated 3D quantitative intratumoral microbiota imaging method, are promising to provide insightful information into the direct interactions between the microbial community and the host in the tumor microenvironment.

Wide-field-of-view and high-resolution HiLo optical sectioning microscopy system

The predecessor of China Optics was China Optics and Applied Optics Digest, which was founded in 1985. At that time, it was the only retrieval journal in the field of optics in China. At the end of 2008, China Optics and Applied Optics Digest was renamed

Rapid histological imaging of bone without microtome sectioning using nonlinear microscopy.

Tissue preparation for histologic evaluation of bone is particularly lengthy, limiting its use in intraoperative or intraprocedural histological evaluation. Nonlinear microscopy (NLM) is an optical sectioning microscopy method that can visualize pathology in freshly excised tissue without requiring physical microtome sectioning. This study describes a rapid protocol for NLM imaging of bone and associated cartilage. NLM imaging was performed on 71 specimens of normal bone as well as arthritic, malignant and inflammatory bone tissue from 40 patients who underwent joint replacement, amputation, bone marrow biopsy or autopsy. Specimens ranged in size from core needle biopsies to transections of entire femoral heads. Specimens were stained with acridine orange and sulforhodamine 101, nuclear and cytoplasmic/stromal fluorescent dyes, for 5min, then rinsed for 30s. NLM fluorescent images were displayed using colors analogous to hematoxylin and eosin (H&E) to facilitate interpretation. Pathologists examined NLM images of the specimens in real time by rapidly translating the specimen to areas of interest, similar to a standard transmission light microscope. By adjusting the NLM focus depth, images from a few-μm-thick layer could be obtained down to ~100μm below the tissue surface, analogous to serial sectioning. Following real-time NLM imaging, the tissue was processed for conventional paraffin histology, and H&E slides were compared to recorded NLM images. Similarities and differences between NLM and paraffin H&E were assessed. NLM enabled visualization of normal bone architecture, including the lamellar matrix and osteocytes of trabecular bone, articular cartilage, as well as pathological bone features such osteoarthritis, osteomyelitis, and malignancy with an appearance resembling the paraffin H&E. Differences such as changes in cell border sharpness, cellular and nucleolar size, and color patterns were noted, suggesting that training is required for accurate evaluation of bone pathology with NLM. Irregular surface contours and debris generated by gross tissue preparation of bone can make some regions difficult to evaluate with NLM, but the ability to perform rapid three-dimensional translation and sub-surface imaging reduced these problems. NLM is a promising technique for rapid evaluation of bone pathology. Further studies assessing diagnostic performance are warranted.

Deep learning 2D and 3D optical sectioning microscopy using cross-modality Pix2Pix cGAN image translation.

Structured illumination microscopy (SIM) reconstructs optically-sectioned images of a sample from multiple spatially-patterned wide-field images, but the traditional single non-patterned wide-field images are more inexpensively obtained since they do not require generation of specialized illumination patterns. In this work, we translated wide-field fluorescence microscopy images to optically-sectioned SIM images by a Pix2Pix conditional generative adversarial network (cGAN). Our model shows the capability of both 2D cross-modality image translation from wide-field images to optical sections, and further demonstrates potential to recover 3D optically-sectioned volumes from wide-field image stacks. The utility of the model was tested on a variety of samples including fluorescent beads and fresh human tissue samples.

DeepS: a web server for image optical sectioning and super resolution microscopy based on a deep learning framework.

Microscopy technology plays important roles in many biological research fields. Solvent-cleared brain high-resolution (HR) 3D image reconstruction is an important microscopy application. However, 3D microscopy image generation is time-consuming and expensive. Therefore, we have developed a deep learning framework (DeepS) for both image optical sectioning and super resolution microscopy. Using DeepS to perform super resolution solvent-cleared mouse brain microscopy 3D image yields improved performance in comparison with the standard image processing workflow. We have also developed a web server to allow online usage of DeepS. Users can train their own models with only one pair of training images using the transfer learning function of the web server. http://deeps.cibr.ac.cn. Supplementary data are available at Bioinformatics online.

Correlative fluorescence and atomic force microscopy to advance the bio-physical characterisation of co-culture of living cells

An understanding of the cell mechanical properties involved in numerous cellular processes including cell division, cell migration/invasion, and cell morphology, is crucial in developing and informing cell physiology and function. Atomic force microscopy (AFM) offers a powerful biophysical technique that facilitates the imaging of living cells under physiological buffer conditions. However, AFM in isolation cannot discriminate between different cell types within heterogeneous samples for example in a solid biopsy. The current studies demonstrate the potential of AFM in combination with correlative fluorescence optical sectioning microscopy for live cell imaging. Furthermore, this work establishes the advantage of fluorescence-AFM imaging to distinguish and analyse single-cell bio-physical properties in mixed human cell populations, in real-time. Critically, our results show that correlative fluorescence-AFM imaging allows the simultaneous co-localised detection of fluorescence coupled with nano-mechanical mapping. The findings from this work contribute to the promotion and dissemination of correlative multimodal imaging in life sciences, providing a platform for further investigations in biological and pre-clinical research.

Ca2+-Daptomycin targets cell wall biosynthesis by forming a tripartite complex with undecaprenyl-coupled intermediates and membrane lipids

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca2+-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.

Metallic Nanofilm Enhanced Fluorescence Cell Imaging: A Study of Distance-Dependent Intensity and Lifetime by Optical Sectioning Microscopy.

Simple, stable, easily-fabricated smooth metallic nanofilm can improve the imaging intensity and imaging contrast. However, its application in micrometer-scale cells has not been popularized due to the lack of full understanding of their related fluorescence properties. In this study, fluorescence enhancement of cell imaging on smooth Au nanofilm was investigated over a micrometer-scale range via employment of the optical sectioning method available with a laser scanning confocal fluorescence microscope. The fluorescence enhancement reduced with the distance away from the surface of metallic nanofilm, and this distance dependence was determined by the factors of numerical aperture, dye-substrate distance, and emission wavelength. In addition, distance-dependent fluorescence lifetime images of cells were also measured to study the interaction between fluorophores and metallic film. The enhancement effect of Au nanofilm on fluorescence cell imaging can be induced not only by the standing wave formed by the reflected light and exciting light but also by the interaction between fluorophore and surface plasmons on the metallic nanofilm. Our study on smooth metallic nanofilm should pave the way for utilizing its uniform fluorescence enhancement characteristic for biological imaging.

Optical Sectioning Microscopy Through Single-Shot Lightfield Protocol

Optical sectioning microscopy is usually performed by means of a scanning, multi-shot procedure in combination with non-uniform illumination. In this paper, we change the paradigm and report a method that is based in the lightfield concept, and that provides optical sectioning for 3D microscopy images after a single-shot capture. To do this we first capture multiple orthographic perspectives of the sample by means of Fourier-domain integral microscopy (FiMic). The second stage of our protocol is the application of a novel refocusing algorithm that is able to produce optical sectioning in real time, and with no resolution worsening, in the case of sparse fluorescent samples. We provide the theoretical derivation of the algorithm, and demonstrate its utility by applying it to simulations and to experimental data.

Long-term monitoring in a microfluidic system to study tumour spheroid response to chronic and cycling hypoxia

We demonstrate the application of a microfluidic platform combining spatiotemporal oxygen control and long-term microscopy monitoring to observe tumour spheroid response to hypoxia. The platform is capable of recreating physiologically-relevant low and cycling oxygen levels not attainable in traditional cell culture environments, while image-based monitoring visualizes cell response to these physiologically-relevant conditions. Monitoring spheroid cultures during hypoxic exposure allows us to observe, for the first time, that spheroids swell and shrink in response to time-varying oxygen profiles switching between 0% and 10% O2; this swelling-shrinkage behaviour appears to be driven by swelling of individual cells within the spheroids. We also apply the system to monitoring tumour models during anticancer treatment under varying oxygen conditions. We observe higher uptake of the anticancer agent doxorubicin under a cycling hypoxia profile than under either chronic hypoxia or in vitro normoxia, and the two-photon microscopy monitoring facilitated by our system also allows us to observe heterogeneity in doxorubicin uptake within spheroids at the single-cell level. Combining optical sectioning microscopy with precise spatiotemporal oxygen control and 3D culture opens the door for a wide range of future studies on microenvironmental mechanisms driving cancer progression and resistance to anticancer therapy. These types of studies could facilitate future improvements in cancer diagnostics and treatment.

Cameraless high-throughput three-dimensional imaging flow cytometry

Today’s imaging flow cytometer (IFC) systems are limited by the projection problem: collapsing three-dimensional (3D) information onto a two-dimensional (2D) image causes a lack of tomographic 3D resolution and reduced information content, limiting the reliability of spot counting or co-localization crucial to cell phenotyping. We present 3D imaging flow cytometry as a solution to the problem. Our high-throughput 3D cell imager based on optical sectioning microscopy combines orthogonal light-sheet scanning illumination with our previous spatiotemporal transformation detection to produce 3D cell image reconstruction from a cameraless single-pixel photodetector readout. We demonstrate this capability by cocapturing 3D fluorescence and label-free side-scattering images of single cells in flow with a throughput of approximately 500 cells per second.