Conventional Confocal Microscopy Research Articles

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 Framework of Multi-View Machine Learning for Biological Spectral Unmixing of Fluorophores with Overlapping Excitation and Emission Spectra.

The accuracy in assigning fluorophore identity and abundance, termed spectral unmixing, in biological fluorescence microscopy images remains challenging due to the unavoidable and significant overlap in emission spectra among fluorophores. In conventional laser scanning confocal spectral microscopy, fluorophore information is acquired by recording emission spectra with a single combination of discrete excitation wavelengths. As a matter of fact, organic fluorophores have not only unique emission spectral signatures but also have unique and characteristic excitation spectra. In this paper, we propose a generalized multi-view machine learning approach, which makes use of both excitation and emission spectra to greatly improve the accuracy in differentiating multiple highly overlapping fluorophores in a single image. By recording emission spectra of the same field with multiple combinations of excitation wavelengths, we obtain data representing these different views of the underlying fluorophore distribution in the sample. We then propose a framework of multi-view machine learning methods, which allows us to flexibly incorporate noise information and abundance constraints, to extract the spectral signatures of fluorophores from their reference images and to efficiently recover their corresponding abundances in unknown mixed images. Numerical experiments on simulated image data demonstrate the method's efficacy in improving accuracy, allowing for the discrimination of 100 fluorophores with highly overlapping spectra. Furthermore, validation on images of mixtures of fluorescently labeled E. coli demonstrates the power of the proposed multi-view strategy in discriminating fluorophores with spectral overlap in real biological images.

Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

The muscle and neural architecture of Taenia crassiceps cysticerci revisited; implications on head-tail polarization of the larvae.

Taenia crassiceps has been used for decades as an experimental model for the study of human and porcine cysticercosis. Even though, its life cycle, tissue organization, ultrastructure and immune response elicited in the host, have been extensively described, there are many other biological questions remaining to be addressed. In the present study we revisited the muscle and neural architecture of cysticerci in two of the most frequently used strains (WFU and ORF), using conventional staining and confocal microscopy imaging, aiming to assemble an updated anatomy. Differences between both strains, including polarization processes during development of the young budding larvae, are emphasized. We also performed a search for genes that have been related to peptidergic neural processes in other related flatworms. These findings can help to understand the anatomical and molecular consequences of the scolex presence or absence in both strains.

Single image Fourier ring correlation

We address resolution assessment for (light super-resolution) microscopy imaging. In modalities where imaging is not diffraction limited, correlation between two noise independent images is the standard way to infer the resolution. Here we take away the need for two noise independent images by computationally splitting one image acquisition into two noise independent realizations. This procedure generates two Poisson noise distributed images if the input is Poissonian distributed. As most modern cameras are shot-noise limited this procedure is directly applicable. However, also in the presence of readout noise we can compute the resolution faithfully via a correction factor. We evaluate our method on simulations and experimental data of widefield microscopy, STED microscopy, rescan confocal microscopy, image scanning microscopy, conventional confocal microscopy, and transmission electron microscopy. In all situations we find that using one image instead of two results in the same computed image resolution.

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye.

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca2+ entry and mitochondrial Ca2+ homeostasis in cardiomyocytes

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca2+ sensors that trigger store-operated Ca2+ entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca2+ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the ISOC current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca2+ store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the ISOC current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca2+ cycling, we observed, using Rhod-2/AM Ca2+ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca2+ (mCa2+) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IP3Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca2+ uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa2+ uptake capacity possibly via the STIM2-IP3Rs-VDAC-MCU and MNF2 complex.

Understanding the anatomy of dermoscopy of melanocytic skin tumours: Correlation invivo with line-field optical coherence tomography.

Early melanoma detection is the main factor affecting prognosis and survival. For that reason, non-invasive technologies have been developed to provide a more accurate diagnosis. Recently, line-field confocal optical coherence tomography (LC-OCT) was developed to provide an invivo, imaging device, with deep penetration and cellular resolution in three dimensions. Combining the advantages of conventional OCT and reflectance confocal microscopy, this tool seems to be particularly suitable for melanocytic lesions. The objective of this study was to identify and describe the correlation between specific dermoscopic criteria and LC-OCT features in three dimensions associated with melanocytic lesions. Dermoscopic and LC-OCT images of 126 melanocytic lesions were acquired in three different centres. The following dermoscopic criteria have been considered: reticular pattern, dots and globules, structureless areas, blue-whitish veil, regression structures, negative network, homogeneous pattern, streaks and blotches. 69 (55%) benign and 57 (45%) malignant lesions were analysed. A regular reticular pattern was found associated in the 75% of the cases with the presence of elongated rete ridges with pigmented cells along the basal layer, while atypical reticular pattern showed an irregular organization of rete ridges with melanocytic hyperplasia, broadened and fused ridges and elongated nests. Both typical and atypical dots and globules were found associated with melanocytic nests in the dermis or at the dermoepidermal junction (DEJ), as well as with keratin cysts/pseudocysts. Grey globules corresponded to the presence of melanin-containing dermal inflammatory cells (melanophages) within the papillae. Structureless brown/black areas correlated with alterations of the DEJ. We observed the same DEJ alterations, but with the presence of dermal melanophages, in 36% of the cases of blue/white/grey structureless areas. A description of each LC-OCT/dermoscopy correlation was made. LC-OCT permitted for the first time to perform an invivo, 3D correlation between dermoscopic criteria and pathological-like features of melanocytic lesions.

In situ FRET measurement of cellular tension using conventional confocal laser microscopy in newly established reporter mice expressing actinin tension sensor

FRET-based sensors are utilized for real-time measurements of cellular tension. However, transfection of the sensor gene shows low efficacy and is only effective for a short period. Reporter mice expressing such sensors have been developed, but sensor fluorescence has not been measured successfully using conventional confocal microscopy. Therefore, methods for spatiotemporal measurement of cellular tension in vivo or ex vivo are still limited. We established a reporter mouse line expressing FRET-based actinin tension sensors consisting of EGFP as the donor and mCherry as the acceptor and whose FRET ratio change is observable with confocal microscopy. Tension-induced changes in FRET signals were monitored in the aorta and tail tendon fascicles, as well as aortic smooth muscle cells isolated from these mice. The pattern of FRET changes was distinctive, depending on tissue type. Indeed, aortic smooth muscle cells exhibit different sensitivity to macroscopic tensile strain in situ and in an isolated state. This mouse strain will enable novel types of biomechanical investigations of cell functions in important physiological events.

Observations on the embryonic development of the mud crab, Scylla paramamosain

To investigate the embryonic development of the mud crab Scylla paramamosain, we analyzed three critical parameters: egg color of, embryo morphology (through conventional and laser scanning confocal microscopy), and the distribution of cell divisions. During embryonic development, the egg color exhibited a progressive transition, shifting from orange to reddish-orange, then to brown, before ultimately darkening to black. Each embryo displayed a spherical shape, measuring approximately 280 μm in diameter, characterized by a smooth surface devoid of any depressions. The embryonic cell division was in the form of mixed oogenesis, comprised of complete division in the early stage, spiral oogenesis in the middle stage and surface division in the late stage. It is noteworthy that the blastopore appeared at the position where the transparent area and cell aggregation just appeared under the microscope, and the blastomere was a characteristic of the embryo entering the gastrulation stage. After entering the gastrulation stage, the cells aggregated towards the blastopore and formed two symmetrical cell clusters, which formed a V-shape with the void of the classic blastopore. When the transparent region occupied approximately 1/5 of the embryo’s volume, the embryo entered the nauplius stage, and the thoracic and abdominal armor, as well as the optic lobe and abdominal limb primordia, could be clearly distinguished. The appearance of the compound eye pigment band indicated the stage of compound eye pigment formation. At this time, the transparent area accounted for 1/4 of the embryo and a large number of ganglia appeared. The change of the compound eye pigment band from red to black was also one of the reasons for the blackening of the egg color of the crabs. The data obtained through this study have potential applications in the determination of embryonic development status and obtaining of high-quality seeds for S. paramamosain culture.

The interwoven fibril-like structure of amyloid-beta plaques in mouse brain tissue visualized using super-resolution STED microscopy

BackgroundStandard neuropathologic analysis of Alzheimer’s brain relies on traditional fluorescence microscopy, which suffers from limited spatial resolution due to light diffraction. As a result, it fails to reveal intricate details of amyloid plaques. While electron microscopy (EM) offers higher resolution, its extensive sample preparation, involving fixation, dehydration, embedding, and sectioning, can introduce artifacts and distortions in the complex brain tissue. Moreover, EM lacks molecular specificity and has limited field of view and imaging depth.ResultsIn our study, we employed super-resolution Stimulated Emission Depletion (STED) microscopy in conjunction with the anti-human APP recombinant antibody 1C3 fluorescently labelled with DyLightTM633 (1C3-DyLight633). This combination allowed us to visualize amyloidogenic aggregates in vitro and in brain sections from a 17-month-old 3×Tg-AD mouse with sub-diffraction limited spatial resolution. Remarkably, we achieved a spatial resolution of 29 nm in vitro and 62 nm in brain tissue sections, surpassing the capabilities of conventional confocal microscopy by 5–10 times. Consequently, we could discern individual fibrils within plaques, an achievement previously only possible with EM.ConclusionsThe utilization of STED microscopy represents a groundbreaking advancement in the field, enabling researchers to delve into the characterization of local mechanisms that underlie Amyloid (Aβ) deposition into plaques and their subsequent clearance. This unprecedented level of detail is especially crucial for comprehending the etiology of Alzheimer’s disease and developing the next generation of anti-amyloid treatments. By facilitating the evaluation of drug candidates and non-pharmacological interventions aiming to reduce amyloid burden, STED microscopy emerges as an indispensable tool for driving scientific progress in Alzheimer’s research.

Cells-in-Touch: 3D Printing in Reconstruction and Modelling of Microscopic Biological Geometries for Education and Future Research Applications.

Additive manufacturing (3D printing) and computer-aided design (CAD) still have limited uptake in biomedical and bioengineering research and education, despite the significant potential of these technologies. The utility of organ-scale 3D-printed models of living structures is widely appreciated, while the workflows for microscopy data translation into tactile accessible replicas are not well developed yet. Here, we demonstrate an accessible and reproducible CAD-based methodology for generating 3D-printed scalable models of human cells cultured in vitro and imaged using conventional scanning confocal microscopy with fused deposition modeling (FDM) 3D printing. We termed this technology CiTo-3DP (Cells-in-Touch for 3D Printing). As a proof-of-concept, we created dismountable CiTo-3DP models of human epithelial, mesenchymal, and neural cells by using selectively stained nuclei and cytoskeletal components. We also provide educational and research context for the presented cellular models. In the future, the CiTo-3DP approach can be adapted to different imaging and 3D printing modalities and comprehensively present various cell types, subcellular structures, and extracellular matrices. The resulting CAD and 3D printed models could be used for a broad spectrum of education and research applications.

High-resolution multi-z confocal microscopy with a diffractive optical element.

There has been recent interest in the development of fluorescence microscopes that provide high-speed volumetric imaging for life-science applications. For example, multi-z confocal microscopy enables simultaneous optically-sectioned imaging at multiple depths over relatively large fields of view. However, to date, multi-z microscopy has been hampered by limited spatial resolution owing to its initial design. Here we present a variant of multi-z microscopy that recovers the full spatial resolution of a conventional confocal microscope while retaining the simplicity and ease of use of our initial design. By introducing a diffractive optical element in the illumination path of our microscope, we engineer the excitation beam into multiple tightly focused spots that are conjugated to axially distributed confocal pinholes. We discuss the performance of this multi-z microscope in terms of resolution and detectability and demonstrate its versatility by performing in-vivo imaging of beating cardiomyocytes in engineered heart tissues and neuronal activity in c. elegans and zebrafish brains.

Pressure induced changes in endothelial cell morphology and Ca2+ signaling in ex vivo pressurized resistance arteries revealed using 4D diSPIM myography and AI deep-learning

The vascular endothelium is critical in promoting vasodilation of resistance-size arteries through Ca 2+ dependent pathways. Vascular endothelial cells (ECs) are exposed to mechanical stimulation through changing intravascular pressure and flow, yet it is unclear how these forces impact EC morphology and Ca 2+ signaling in pressurized arteries. Visualizing how the endothelium of an intact resistance-size vessel responds to changing physical forces requires isometric, sub-micron, resolution. Conventional confocal fluorescent microscope systems are limited by poor axial resolution. 4D dual inverted selective plane of illumination microscopy (diSPIM; 3i) uniquely provides isometric resolution at ~330 nm with high temporal resolution and minimal photobleaching or phototoxicity. We designed custom pressure myography chambers to allow simultaneous 4D diSPIM fluorescent imaging and vessel diameter tracking measurements via transmitted IR light (diSPIM Myography). We captured fluorescent images of ex vivo pressurized resistance-size mouse mesenteric arteries at 80, 60, 40, and 20 mm Hg before and after the generation of myogenic tone and in the absence of extracellular Ca 2+ . Representative images were annotated for ECs, nuclei, myoendothelial projections (MEPS), and Ca 2+ signals. Annotations were used as ground truth for CNN AI model generation and validation using Apeer (Zeiss). Arivis Vision4D (Zeiss) was used to run the AI model and quantify data. As static intravascular pressure is increased in arteries without tone (with or without Ca 2+ ), ECs increase in width as the vessel dilates. Myogenic tone minimizes pressure dilation and changes in EC width, however, ECs compress by up to 1 micron as pressure increases from 20 to 80 mm Hg. Additionally, increasing intravascular pressure in arteries with tone decreases local EC Ca 2+ signaling. The data demonstrate a relationship between intravascular pressure and EC shape and Ca 2+ signaling in intact resistance arteries under conditions of physiologic temperature, pressure, pH, and tone. diSPIM myography provides a method of studying the effects of physiologically relevant mechanical stimuli and the roles of various mechanosensitive mechanisms on vascular function. AI image annotation reduces analysis bias and increases the breadth of quantifiable microscopy data. DMC is supported by NIH R00HL133451 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging.

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged molecules within complex biological samples. While traditional confocal microscopes allow one to resolve two adjacent fluorescent point sources located a few hundred nanometers apart, observing the finer details of subcellular biology requires the ability to resolve signals in the order of tens of nanometers. Numerous hardware-based methods for super-resolution microscopy have been developed to allow researchers to sidestep such resolution limits, although these methods require specialized microscopes that are not available to all researchers. An alternative method for increasing resolving power is to isotropically enlarge the sample itself through a process known as expansion microscopy (ExM), which was first described by the Boyden group in 2015. ExM is not a type of microscopy per se but is rather a method for swelling a sample while preserving the relative spatial organization of its constituent molecules. The expanded sample can then be observed at an effectively increased resolution using a traditional confocal microscope. Here, we describe a protocol for implementing ExM in whole-mount Drosophila embryos, which is used to examine the localization of Par-3, myosin II, and mitochondria within the surface epithelial cells. This protocol yields an approximately four-fold increase in sample size, allowing for the detection of subcellular details that are not visible with conventional confocal microscopy. As proof of principle, an anti-GFP antibody is used to distinguish distinct pools of myosin-GFP between adjacent cell cortices, and fluorescently labeled streptavidin is used to detect endogenous biotinylated molecules to reveal the fine details of the mitochondrial network architecture. This protocol utilizes common antibodies and reagents for fluorescence labeling, and it should be compatible with many existing immunofluorescence protocols.

Abstract 6613: Label-free multimodal multiphoton microscopy for predicting DNA damage response in patient derived non-small cell lung cancer organoids

Abstract Background: DNA damage response (DDR) is a fundamental mechanism for evaluating efficacy of cancer therapeutics, many of which target DNA or associated processes (e.g., etoposide: inhibits DNA synthesis, cisplatin: crosslinks DNA). Current techniques to evaluate DDR rely on immunostaining for gamma H2AX foci (γH2AX), which is an indicator of DNA double-strand break formation. While γH2AX immunostaining can provide a snapshot of DDR in fixed cellular and tissue samples, this method is technically cumbersome as: 1) temporal monitoring of DDR requires timepoint replicates; 2) extensive assay development efforts for 3D samples such as organoids, and 3) protocols for γH2AX immunostaining and its evaluation are time-consuming. The goal of this project is to reduce overall burden on assay duration and development in NSCLC organoids by leveraging label-free multiphoton imaging. Method: To predict DDR in 3D organoid samples, label-free multimodal multiphoton techniques such as simultaneous label-free autofluorescence multiharmonic (SLAM) imaging can provide intracellular information based on endogenous contrast mechanisms. SLAM makes use of a single laser to excite autofluorescence in multiple cellular components including FAD and NAD(P)H, enabling the calculation of the optical redox ratio, which estimates the equilibrium of electron donating versus accepting species in cells. Moreover, label-free, multiphoton systems enable imaging of live samples without the need to add labels or generate sacrificial samples and have improved image acquisition in 3D space over conventional confocal microscopy. Using SLAM, we propose to predict DDR in non-small cell lung cancer (NSCLC) organoids. NSCLC organoids recapitulate histological and genetic features of their derived patient tissue, retaining tumor variability. Results: Predictive modeling via gradient boost regressor between handcrafted SLAM image features and γH2AX immunostained images on weakly-paired images confirmed strong correlation between SLAM image features and γH2AX speckles/nuclei. Across multiple patient derived NSCLC organoid lines and multiple DNA targeting chemotherapeutics, the optical redox ratio was demonstrated to robustly predict DDR. Conclusion: Label-free imaging via SLAM can be used to predict DDR in live 3D NSCLC organoids with minimal sample handling and without the need to add labelled reporters, enabling quicker and less technically involved DDR assaying. Citation Format: Terrence T. Roh, Aneesh Alex, Janet E. Sorrells, Prasanna Chandramouleeswaran, Marina Marjanovic, Steve R. Hood, BanuPriya Sridharan, Stephen A. Boppart. Label-free multimodal multiphoton microscopy for predicting DNA damage response in patient derived non-small cell lung cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6613.

Rcell2: Microscopy‐Based Cytometry in R

This article describes a method for quantifying various cellular features (e.g., volume, curvature, total and sub-cellular fluorescence localization) of individual cells from sets of microscope images, and for tracking them over time-course microscopy experiments. One purposely defocused transmission image (sometimes referred to as bright-field or BF) is used to segment the image and locate each cell. Fluorescence images (one for each of the color channels or z-stacks to be analyzed) may be acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses a set of R packages called rcell2. Relative to the original release of Rcell (Bush etal., 2012), the updated version bundles, into a single software suite, the image-processing capabilities of Cell-ID, offers new data analysis tools for cytometry, and relies on the widely used data analysis and visualization tools of the statistical programming framework R. © 2023 Wiley Periodicals LLC. Basic Protocol: Extracting quantitative information from single cells Support Protocol 1: Obtaining and installing Cell-ID and R Support Protocol 2: Preparing cells for imaging.

Compound super-oscillation lens for reflective confocal imaging

The super-oscillation lens (SOL) can achieve super-resolution focusing but have to trade-off with weaker hotspots and higher sidebands. We propose a single compound SOL to achieve reflective confocal imaging in principle without additional lenses. The designed SOL consists of an outer lens and an inner lens which play the role of focusing lens and collective lens respectively. As a result, focusing and collecting functions can be simultaneously realized. The improved system can achieve excellent imaging performance with an ultra-high resolution (<0.314λ/NA, NA stands for numerical aperture), and almost negligible side lobe ratio and no side bands, which proved superior to conventional laser scanning confocal microscopy and single SOL. This technology can be attractive for a variety of applications in super-resolution imaging and biomedical sciences.

P071 T-cell, B-cell and monocytic infiltrations of myenteric plexus in IBD

Abstract Background Patients suffering from IBD frequently suffer from chronic visceral pain, severely affecting quality of life. To better understand factors impacting pain signaling by enteric nervous system (ENS) neuronal cells, we analyzed the infiltration of the myenteric plexus (MP) of the ENS by inflammatory cell populations. In detail, we characterized (i) the immune cell populations infiltrating the MP, (ii) neurotransmitter expression by MP cells and (iii) neuronal cell survival of MP in Crohn’s disease (CD) and ulcerative colitis (UC). Methods We included 12 UC and nine CD patients that received colectomies or ileocecal resections, respectively, and 10 control patients with surgeries for non-inflammatory reasons. FFPE material was used for immunohistochemistry analysis of the MP. Immunostaining was performed for the detection of ENS neurons (beta-III tubulin, PGP9.5), for T-cells (CD3, CD4, CD8 and FOXP3), for B-cells (CD20), for monocytes (CD68, CD163) and apoptosis (Annexin V) and the neurotransmitters CGRP and substance P. Intraganglionic and periganglionic immune cell populations were determined by conventional LSM confocal microscopy. Results CD3+ and CD8+ intraganglionic T-cells for CD (CD3+: 1.7 ± 0.6 cells/10.000 µm²; CD8+: 4.8 ± 1.1 cells/10.000 µm²) were found to be increased compared to control patients (CD3+: 0.2 ± 0.1 cells/10.000 µm², p=0.04; CD8+: 0.4 ± 0.2 cells/10.000 µm², p=0.006), Figure 1. CD20+ B-cells and CD68+ monocytes were significantly increased in CD MP as well (CD20+ B-cells in CD: 0.8 cells/10.000 µm², CD20+ B-cells in controls: 0 cells/10.000 µm, p=0.02), CD68+ monocytes in CD: 12.3 cells/10.000 µm², CD68+ monocytes in controls: 3.5 cells/10.000 µm², p=0.001). CD8+ cytotoxic T-cells infiltrating the periganglionic area of the MP were significantly increased for UC. 1.6-fold increased expression of the neurotransmitter CGRP were detected for UC MP, p=0.01. Substance P was found to be 0.7-fold reduced for CD MP, p=0.01. MP-cell survival (Annexin V) was found to be reduced for UC MP, p=0.048. Figure 2 shows representative neurotransmitter immunostainings in two patients per condition. Conclusion In conclusion, MP immune cell infiltrations in CD are comprised of CD3+, CD8+ T-cells as well as CD68+ monocytes and CD20+ B-cells. Specifically periganglionically, CD8+ T-cells are found in UC. The expression of the neurotransmitter CGRP is increased in CD-MPs whereas levels of substance P are reduced in UC MP. UC-affected MPs revealed higher levels of apoptosis.

Novel Chromatic Differential Confocal Matrix Sensor Using Multiband Spectral Images

In this letter, a novel chromatic differential confocal matrix (CDCM) sensor is proposed and demonstrated for accurate and fast three-dimensional (3D) profiling without mechanical scanning. In contrast to conventional chromatic confocal microscopy (CCM) methods that require spectral expansion and a peak-intensity-wavelength detection algorithm for axial positioning, our CDCM employs the intensity difference between a pair of spectral confocal images to accurately discriminate surface height. Due to the elimination of spectral expansion required by a conventional CCM, the CDCM has an at least one order of magnitude higher axial sensitivity and surface profiling efficiency than a conventional CCM. In the CDCM, axial scanning is achieved by residual chromatic aberration of an achromatic objective and by constructing a difference intensity signal between two spectral confocal images. Spectral confocal images are acquired in a parallel manner by using a digital micromirror device as an array of illumination pinholes and digitally applying the virtual detection pinhole concept. Quantitative evaluation experiments with the proof-of-principle sensor system indicate that with a <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$10\times $ </tex-math></inline-formula> NA 0.25 objective, the CDCM can achieve better than <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$0.1~\mu \text{m}$ </tex-math></inline-formula> axial accuracy with an axial measurement range of approximately <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$28~\mu \text{m}$ </tex-math></inline-formula> , a lateral resolution of <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$0.87~\mu \text{m}$ </tex-math></inline-formula> .