Pinhole Closure Improves Spatial Resolution in Confocal Scanning Microscopy.

Lacrimal Gland in Sjögren's Syndrome

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.

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.

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.

Laser assisted keratoplasty is nowadays largely used to perform minimally invasive surgery and partial thickness keratoplasty [1-3]. The use of the femtosecond laser enables to perform a customized surgery, solving the specific problem of the single patient, designing new graft profiles and partial thickness keratoplasty (PTK). The common characteristics of the PTKs and that make them eligible respect to the standard penetrating keratoplasty, are: the preservation of eyeball integrity, a reduced risk of graft rejection, a controlled postoperative astigmatism. On the other hand, the optimal surgical results after these PTKs are related to a correct comprehension of the deep stroma layers morphology, which can help in the identification of the correct cleavage plane during surgeries. In the last years some studies were published, giving new insights about the posterior stroma morphology in adult subjects [4,5]. In this work we present a study performed on two groups of tissues: one group is from 20 adult subjects aged 59 ± 18 y.o., and the other group is from 15 young subjects, aged 12±5 y.o.. The samples were from tissues not suitable for transplant in patients. Confocal microscopy and Environmental Scanning Electron Microscopy (ESEM) were used for the analysis of the deep stroma. The preliminary results of this analysis show the main differences in between young and adult tissues, enabling to improve the knowledge of the morphology and of the biomechanical properties of human cornea, in order to improve the surgical results in partial thickness keratoplasty.

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.

We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.

Topographical as well as microscopic imaging of nanoscale surfaces plays a pivotal role across various disciplines. Nevertheless, achieving fast, label-free, and accurate characterization of laterally expanded structures below the diffraction limit remains challenging. Recent studies highlight the use of microsphere assistance for resolution improvement. Confocal, conventional, and interference microscopy, augmented by microspheres, enable the imaging of small structures that were previously inaccessible. In this contribution, results of microsphere-assisted confocal and conventional bright-field microscopy (MAM) are compared to underline the decisive role of the confocal effect. Furthermore, an extensive comparison of simulated confocal results is presented to highlight the experimental results.

Objective To investigate the microscopic features of common hypopigmentation disorders using confocal laser scanning microscopy (CLSM). Methods Totally, 1915 child patients with hypopigmentation disorders were enrolled in this study, including 181 cases of vitiligo, 38 idiopathic guttate hypomelanosis, 370 amelanotic nevus, 304 lichen striatus, 712 pityriasis alba, and 310 postinflammatory hypopigmentation. CLSM was performed to observe all layers of depigmented lesions, their borders, and normal-appearing perilesional skin in these patients. Statistical analysis was done by chi-square test and Fisher's exact test. Results Grade 1 and 2 hypopigmentation was observed in 984 (51.4% ) and 684 (35.7% ) out of the 1915 patients, respectively. The proportion of patients with grade 3 hypopigmentation was 77.9% (141/181) in patients with vitiligo, significantly higher than that in those with the other diseases (0 - 10.3% ). Among the 1915 patients, 815 showed mild epidermal hyperkeratosis, 1060 focal edema of stratum spinosum, 79 changes in dermal papillary rings, and 1133 sparse inflammatory cells or dendritic cells in the superficial dermis. There were significant differences in the degree of hypopigmentation and frequencies of pathologic changes in different skin layers on CLSM images among the six hypopigmentation disorders (all P < 0.05). As CLSM showed, idiopathic guttate hypomelanosis was characterized by complete absence of dermal papillary rings; lichen striatus, pityriasis alba and postinflammatory hypopigmentation were all characterized by nonspecific inflammatory changes; lichen striatus manifested as punctiform or focal liquifaction degeneration of basal cells, and amelanotic nevus as only hypopigmentation and refractive attenuation; vitiligo was characterized by complete depigmentation and lack of integrity of dermal papillary rings in white patches. Conclusions CLSM images obviously differ between vitiligo, idiopathic guttate hypomelanosis, amelanotic nevus, lichen striatus, pityriasis alba and postinflammatory hypopigmentation, and can serve as a basis for their differential diagnosis. Key words: Microscopy, confocal; Hypopigmentation; Vitiligo; Child; Melanins; Dermal papillary rings; Pityriasis alba; Lichen striatus; Amelanotic nevus

Cell Biological Applications of Confocal Microscopy

Background: The diagnosis of melanoma at an early, curable stage is an important challenge for clinicians. Confocal scanning laser microscopy (CSLM) is a high-resolution, noninvasive technology that may facilitate improved diagnostic accuracy over clinical examination. The aim of this study was to evaluate the diagnostic accuracy of CSLM compared to dermoscopy in a prospective examination of benign and malignant melanocytic lesions. Methods: 125 patients with suspicious pigmented lesions were prospectively recruited to undergo a clinical, dermoscopic and CSLM examination. A diagnosis was made preoperatively with each technique, and the lesion was then excised and diagnosed using histopathology. Results: 125 patients with 125 lesions were studied comprising 88 melanocytic nevi and 37 melanomas. Dermoscopy had a sensitivity of 89.2%, a specificity of 84.1%, a positive predictive value of 70.2% and a negative predictive value of 94.9%. CSLM was found to have a sensitivity of 97.3%, a specificity of 83.0%, a positive predictive value of 70.6% and a negative predictive value of 98.6%. No melanomas were misidentified when both techniques were used together. Conclusions: CSLM had a relatively higher sensitivity than dermoscopy; however, the specificity was similar with CSLM and dermoscopy. These results suggest that dermoscopy and CSLM are complementary.

Objective: Control the refractive index in fixed specimens of mammalian inner ear to reduce spherical aberrations that limit our ability to obtain 3-dimensional images of fluorescently labeled inner ear specimens by conventional laser scanning confocal microscopy. Study Design: Mouse inner ear specimens were fixed with minimal dissection, rapidly decalcified and fluorescently labeled by immunohistochemistry then impregnated by epoxy resin or a clearing agent composed of 5 parts methyl salicylate:3 parts benzyl benzoate. The specimens were imaged by both confocal microscopy and by widefield epi-fluorescent microscopy, with additional processing by deconvolution. Results: Rapid decalcification preserved tissue morphology and antigenicity for the antibodies tested. Although the epoxy allowed some reduction of spherical aberration, the clearing agent enabled optical volumes of high quality and resolution to be collected through the inner ear. The conditions for immunolabeling are important to ensure a...

Background: Endodontic treatment involves the removal of the vital and necrotic contents of the root canal through chemo-mechanical means followed by obturation of the prepared root canal to prevent the ingress of fluids and avoid bacterial infection or regrowth. Root canal sealers and core filling materials are used together to fill the irregularities in the root. Penetration into the dentinal tubules also results in the inhibition of bacterial regrowth and increases the success of root canal therapy. Aim: This study aimed to evaluate the penetration depth of various sealers into the dentinal tubules using a confocal microscope. Materials and methods: A total of 65 specimens were decoronated to standardize the root length of 13mm. Working length was determined, and Biomechanical preparation for all the samples was done with a rotary ProTaper file till F4. Samples were randomly divided into five groups containing 13 teeth in each group based on the sealer used, namely Group 1: Endomethasone (n=13), Group 2: AH-Plus (n=13), Group 3: Roekoseal (n=13), Group 4: MTA Fillapex (n=13), Group 5: Endosequence BC (n=13). All the sealers were labelled with Rhodamine-B dye, and samples were obturated using cold lateral compaction technique. The specimens were sectioned orthogonally at coronal, middle, and apical thirds. All the samples were examined with a Zeiss Pascal Laser Scanning Microscope to examine the sealer penetration depth into the dentinal tubules. The data were subjected to statistical analysis using one- way Analysis of Variance (ANOVA) and Tukey's Honest Significant Difference (HSD) tests. Results: Endosequence BC showed the highest penetration into dentinal tubules, followed by MTA Fillapex and Roekoseal, AH-Plus, and Endomethasone exhibited the least penetration. Conclusion: Endosequence BC sealer exhibited maximum penetration. All the groups showed maximum penetration at coronal third, followed by the middle and apical third.