Image-Analysis-Based Method for 3D Crystal Morphology Measurement and Polymorph Identification Using Confocal Microscopy

Point defects in crystals provide important building blocks for quantum applications. Since we optically address these defect qubits, having an efficient optical interface is a highly important aspect. However, conventional confocal fluorescence microscopy of high-refractive-index crystals suffers from limited photon collection efficiency and spatial resolution. Here, we demonstrate high-resolution, high-contrast imaging of defects in diamonds using microsphere-assisted confocal microscopy. A microsphere provides an excellent optical interface for point defects with a magnified virtual image that increases the spatial resolution up to λ/5, as well as the optical signal-to-noise ratio by four times. These features enable individual optical addressing of single photons and single spins of multiple defects that are spatially unresolved in conventional confocal microscopy, with improved signal contrast. Combined with optical tweezers, this system also demonstrates the possibility of positioning or scanning the microspheres. The approach does not require any complicated fabrication or additional optical systems, but uses simple, off-the-shelf micro-optics. From these distinctive advantages of microspheres, our approach provides an efficient way to image and address closely spaced defects with much better resolution and sensitivity.

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

We describe a polarization-sensitive fiber optic based confocal microscope. Polarization maintaining single mode fibers are used to illuminate a specimen with linearly polarized light. The orientation of the incident linearly polarized light is continuously variable using a Se´narmont polarization rotator. A polarization-sensitive light detector enables the microscope to record the intensity and polarization orientation of the reflected light. The microscope can thus function as a conventional confocal microscope as well as detect rotations of the polarization orientation of the incident light by a birefringent specimen. Lateral scanning of the microscope focal volume is achieved by moving the fiber tip in a reciprocal layout. Confocal operation of the system is demonstrated both theoretically and experimentally. The theoretical and measured responses of the microscope to a dielectric mirror, glass slide, quarter wave plate, linear polarizer, and calcite crystal are considered. Images of a birefringent specimen are presented.

Single-Cell Imaging

Confocal microscopy is a simple, super-resolution technique, which does not produce a marked increase in resolution compared to other advanced techniques, such as super-resolution nanoscopy. Here, we present a simple protocol to acquire "slightly, but easily resolved" images by pinhole closure (<1 airy unit) in a conventional confocal scanning microscope equipped with an avalanche photodiode, a detector with high sensitivity. We use murine neuroblastoma Neuro2a cells to demonstrate the image resolution obtained via this protocol without the use of any special software to enhance image quality.

Traditional histological processing techniques can subject tissue to distortion and artifacts and provide a limited view of cell dynamics. An evolving histological technique is the use of optical microscopy to acquire high resolution image stacks to visualize tissue in‐depth. Using the confocal laser scanning microscope (CLSM), it is possible to collect an image stack to process into a 3‐D perspective. In bone, autofluorescence is largely due to the collagen component. We aimed to use the autofluorescent properties of the collagen in the growth plate of murine femurs to characterize and evaluate the resting, proliferating and hypertrophic zones of articular cartilage. Murine distal femurs were sectioned along their longitudinal axis. The bones were allowed to incubate in refractive index mating solution (RIMS) media for two days prior to imaging. Z stacks of approximately 80–100 μm in depth were captured using ACS APO 40x/1.15 oil on Leica SPE confocal microscope. Acquisition of the z‐stacks were obtained in the blue‐green and green‐yellow emission spectrum. Incubation of tissue in RIMS solution reduced the opacity of the tissue and facilitated in‐depth optical imaging. Imaging was focused on the growth plate (Fig 1). Sequential image stacks were acquired with confocal microscope and Leica LAS software algorithms were used to compose 3‐D images. Image stacks were further manipulated in ImageJ and ICY to isolate and count the chondrocytes, respectively. 3D histology can be accomplished with minimal processing cost and can provide an alternate perspective of cell dynamics. Intrinsic biomolecules in tissue possess endogenous fluorophores that can at times be advantageous when evaluating intact tissue architecture. Confocal microscopy in conjunction with a refractive index matching solution rendered the murine femurs less opaque and allowed for greater photon penetration and thus resolution of spatial‐temporal structures in the bone matrix. Examination of the matrix and morphology of the cells within, provided an indication of the development of the cells and progression of ossification.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

This letter describes a confocal scanning beam macroscope/microscope that can image specimens up to 7 cm in diameter using both photoluminescence and reflected light. The macroscope generates digital images (512×512 pixels) with a maximum 5 μm lateral resolution and 200 μm axial resolution in under 5 s, and in combination with a conventional confocal scanning laser microscope can provide quality control at a macroscopic/microscopic level for porous silicon specimens, wafers, detectors, and similar devices. This combination of instruments can also be used as a method for evaluating preparation parameters involved in the manufacture of porous silicon. Various confocal and nonconfocal photoluminescence and reflected-light images of porous silicon are shown using both a macroscope and a conventional confocal scanning laser microscope. A 3D profile of a porous silicon structure reconstructed from confocal slices is also shown.

A recently developed micrometer-scale spatially offset Raman spectroscopy (micro-SORS) method provides a new analytical capability for investigating nondestructively the chemical composition of subsurface, micrometer-scale-thick, diffusely scattering layers at depths beyond the reach of conventional confocal Raman microscopy. Here we provide, for the first time, the theoretical foundations for the micro-SORS defocusing concept based on Monte Carlo simulations. Specifically, we investigate a defocusing variant of micro-SORS that we used in our recent proof-of-concept study in conditions involving thin, diffusely scattering layers on top of an extended, diffusely scattering substrate. This configuration is pertinent, for example, for the subsurface analysis of painted layers in cultural heritage studies. The depth of the origin of Raman signal and the relative micro-SORS enhancement of the sublayer signals reached are studied as a function of layer thickness, sample photon transport length, and absorption. The model predicts that sublayer enhancement initially rapidly increases with increasing defocusing, ultimately reaching a plateau. The magnitude of the enhancement was found to be larger for thicker layers. The simulations also indicate that the penetration depths of micro-SORS can be between one and two orders of magnitude larger than those reached using conventional confocal Raman microscopy. The model provides a deeper insight into the underlying Raman photon migration mechanisms permitting the more effective optimization of experimental conditions for specific sample parameters.

There are no reports on the autologous transplantation and patency of microvessels in living tissue. We autotransplanted microvessel fragments (Mvf) labeled with DiI-Ac-LDL into the peritoneum and then observed the peritoneum for 7 days postoperatively with a conventional fluorescence or laser scanning confocal microscope. We illustrated a neomicrovascular network of transplanted Mvf labeled with DiI-Ac-LDL in the peritoneum with both a fluorescence and a laser scanning confocal microscope. Furthermore, we demonstrated not only the existence of erythrocytes in the lumina of transplanted DiI-Ac-LDL-labeled Mvf, but also the presence of India ink perfused through the superior mesenteric artery in the lumina of the labeled Mvf. This evidence directly suggests that transplanted Mvf can survive and proliferate to connect adjacent microvascular branches of the superior mesenteric artery in the very early phase of wound healing. Moreover, these findings imply that implantation of Mvf in the microvascular ischemic circulatory tissue might accelerate angiogenesis to reconstitute a new microvascular network connecting to the nearby host microvascular system, which ultimately improves microcirculation.

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.

Hypoxia-inducible factor-1 (HIF-1), a transcriptional complex composed of an oxygen-sensitive alpha- and a beta-subunit, plays a pivotal role in cellular adaptation to low oxygen availability. Under normoxia, the alpha-subunit of HIF-1 is hydroxylated by a family of prolyl hydroxylases (PHDs) and consequently targeted for proteasomal degradation. Three different PHDs have been identified, but the difference among their in vivo roles remain unclear. PHD3 is strikingly expressed by hypoxia, displays high substrate specificity, and has been identified in other signaling pathways. PHD3 may therefore hydroxylate divergent substrates and/or connect divergent cellular responses with HIF. We identified a novel WD-repeat protein, recently designated Morg1 (MAPK organizer 1), by screening a cDNA library with yeast two-hybrid assays. The interaction between PHD3 and Morg1 was confirmed in vitro and in vivo. We found seven WD-repeat domains by cloning the full-length cDNA of Morg1. By confocal microscopy both proteins co-localize within the cytoplasm and the nucleus and display a similar tissue expression pattern in Northern blots. Binding occurs at a conserved region predicted to the top surface of one propeller blade. Finally, HIF-mediated reporter gene activity is decreased by Morg1 and reduced to basal levels when Morg1 is co-expressed with PHD3. Suppression of Morg1 or PHD3 by stealth RNA leads to a marked increase of HIF-1 activity. These results indicate that Morg1 specifically interacts with PHD3 most likely by acting as a molecular scaffold. This interaction may provide a molecular framework between HIF regulation and other signaling pathways.

Theoretical and experimental results for a new reflection-type confocal microscope with a phase-conjugate mirror (PCM) are presented. The microscope achieves better lateral and axial resolution than the conventional confocal microscope. The observation volume is reduced considerably (approximately 63%). Owing to the properties of the PCM, the system is self-aligning.

Enlarged early endosomes have been visualized in Alzheimer’s disease (AD) and Down syndrome (DS) using conventional confocal microscopy at a resolution corresponding to endosomal size (hundreds of nm). In order to overtake the diffraction limit, we used super-resolution structured illumination microscopy (SR-SIM) and transmission electron microscopies (TEM) to analyze the early endosomal compartment in DS.By immunofluorescence and confocal microscopy, we confirmed that the volume of Early Endosome Antigen 1 (EEA1)-positive puncta was 13–19% larger in fibroblasts and iPSC-derived neurons from individuals with DS, and in basal forebrain cholinergic neurons (BFCN) of the Ts65Dn mice modelling DS. However, EEA1-positive structures imaged by TEM or SR-SIM after chemical fixation had a normal size but appeared clustered. In order to disentangle these discrepancies, we imaged optimally preserved High Pressure Freezing (HPF)-vitrified DS fibroblasts by TEM and found that early endosomes were 75% denser but remained normal-sized.RNA sequencing of DS and euploid fibroblasts revealed a subgroup of differentially-expressed genes related to cargo sorting at multivesicular bodies (MVBs). We thus studied the dynamics of endocytosis, recycling and MVB-dependent degradation in DS fibroblasts. We found no change in endocytosis, increased recycling and delayed degradation, suggesting a “traffic jam” in the endosomal compartment.Finally, we show that the phosphoinositide PI (3) P, involved in early endosome fusion, is decreased in DS fibroblasts, unveiling a new mechanism for endosomal dysfunctions in DS and a target for pharmacotherapy.

Intravital two-photon laser scanning microscopy (TP-LSM) has allowed the direct observation of immune cells in intact organs of living animals. In the B cell biology field TP-LSM has detailed the movement of B cells in high endothelial venules and during their transmigration into lymph organs; described the movement and positioning of B cells within lymphoid organs; outlined the mechanisms by which antigen is delivered to B cells; observed B cell interacting with T cells, other cell types, and even with pathogens; and delineated the egress of B cells from the lymph node (LN) parenchyma into the efferent lymphatics. As the quality of TP-LSM improves and as new fluorescent probes become available additional insights into B cell behavior and function await new investigations. Yet intravital TP-LSM has some disadvantages including a lower resolution than standard confocal microscopy, a narrow imaging window, and a shallow depth of imaging. We have found that supplementing intravital TP-LSM with conventional confocal microscopy using thick LN sections helps to overcome some of these shortcomings. Here, we describe procedures for visualizing the behavior and trafficking of fluorescently labeled, adoptively transferred antigen-activated B cells within the inguinal LN of live mice using two-photon microscopy. Also, we introduce procedures for fixed thick section imaging using standard confocal microscopy, which allows imaging of fluorescently labeled cells deep in the LN cortex and in the spleen with high resolution.

We show that the focal modulation microscopy (FMM), which combines a spatial phase modulator with confocal microscopy, results in an improvement in spatial resolution. This technique was introduced to increase imaging depth into tissue and rejection of background from a thick scattering object. A theory for image formation in FMM is presented, and the effects of detecting the in-phase modulated fluorescence signal are discussed. Compared with conventional confocal microscopy, the width of the point-spread function for the in-phase fluorescence signal is improved by 16.4%. When applied to saturable fluorescence, the half-width at half-maximum is improved by 33.6%, 50.0%, and 62.9%, at demodulation frequencies 2omega, 4omega, and 8omega, respectively.