Polarization‐sensitive scanned fiber confocal microscope

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.

In this paper, we propose an optical comb filter based on polarization-diversified-loop (PDL) configuration that offers switching operation between dual transmission spectra with different channel spacings by controlling the state of polarization (SOP) of input light with orthogonal relation. As shown in Fig. 1(a), the PDL-based filter consists of a four-port polarization beam splitter, combination of three half-wave plates (HWPs), one Faraday rotator (FR), and two bow-tie polarization-maintaining fiber (PMF) segments with different lengths (L and 2L) and equal birefringence (B). By controlling an input polarizer located before port 1 of the PBS, ASE light is linearly polarized such as linear horizontal polarization (LHP) and linear vertical polarization (LVP) components which propagate along clockwise (CW) and counterclockwise (CCW) directions in the PDL loop, respectively [1]. Fig. 1(b) represents principal optical paths (CW and CCW paths) divided by orthogonal SOPs of input light. Generally, when PMF segments are utilized as birefringence elements, channel spacings (Δλ) of output transmission spectra are determined by the product of their effective length (L eff ) and birefringence expressed as the equation Δλ=λ2/B-L eff where λ is wavelength in vacuum. In the proposed filter, the effective length of the two PMF segments can be the sum of or difference between their two lengths according to their angular combination of the orientation angles of the three HWPs for orthogonal input SOPs. That is, by properly adjusting angle sets of the three HWPs, the proposed filter can create two sinusoidal transmittance functions with different channel spacings determined by LB and 3LB for orthogonal input SOPs of LHP and LVP, and vice versa. According to theoretical analysis, the resulting filter transmittance is the algebraic sum of two sub transmittances along the CW and CCW paths calculated by Jones matrix formulation, given by (1-a)t CW + at CCW where a is a splitting ratio of optical power coupled to x- and y-axes at the PBS and ranges from 0 to 1 for LHP and LVP input components, respectively. Fig. 1(c) shows the switching operation between dual transmission spectra with different channel spacings for orthogonal input signals at different angle set of three HWPs, measured in a wavelength range from 1548 nm to 1552 nm. In the upper figure of Fig. 1(c), two measured transmission spectra with channel spacings of ∼0.75 nm and ∼0.25 nm were obtained for LHP and LVP input SOPs, indicated as blue solid and red dotted lines, respectively. In a similar manner, two transmission spectra with channel spacings of ∼0.25 nm and ∼0.75 nm were achieved for LHP and LVP input SOPs, indicated as blue solid and red dotted lines in the lower figure of Fig. 1(c), respectively. Consequently, the fabricated filter could demonstrate the input polarization-controlled switching operation between two transmission spectra with different channel spacings of −0.25 nm and −0.75 nm without modifying the filter structure.

Polarisation rotators are used to set defined launch polarisation states in systems designed to measure the polarisation properties of optical fibre and optical fibre components. In order to predictably set polarisation states the properties of the control elements must be well known. This paper describes the calibration of a polarisation rotator based on three rotating optical elements: a linear polariser, a quarter wave plate and a half wave plate. The calibrated polarisation rotator was used to reliably set polarisation states.

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.

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.

Light-sheet microscopy has been developed as a powerful tool for live imaging in biological studies. The efficient illumination of specimens using light-sheet microscopy makes it highly amenable to high-speed imaging. We therefore applied this technology to the observation of amoeboid movements, which are too rapid to capture with conventional microscopy. To simplify the setup of the optical system, we utilized the illumination optics from a conventional confocal laser scanning microscope. Using this set-up we achieved high-speed imaging of amoeboid movements. Three-dimensional images were captured at the recording rate of 40 frames/s and clearly outlined the fine structures of fluorescent-labeled amoeboid cellular membranes. The quality of images obtained by our system was sufficient for subsequent quantitative analysis for dynamics of amoeboid movements. This study demonstrates the application of light-sheet microscopy for high-speed imaging of biological specimens.

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.

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.

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.

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.

A new technique for the measurement of 3D crystal morphology and identification of its polymorph using tomographic images is proposed. Confocal microscopy is used for the first time to obtain tomographic images of crystals that are coated with a suitable fluorescent dye. A convex polyhedron is fitted through a stack of tomographic images of a crystal to obtain the normal vectors of each facet and their corresponding perpendicular distances from the center of the crystal. The angular patterns are generated from the measured normal vectors and are matched with the master angular patterns of each polymorph. It is shown that the matching of the angular patterns is unique and provides a simpler way to identify polymorphs. An image-analysis program that can be integrated with conventional confocal microscopes was created to sequentially perform image processing, morphology measurement, and polymorph detection. This program was used to measure morphologies and identify polymorphs of 2D and 3D acetaminophen crystals. Detailed directions are provided to enable the application of the methodology without the need for special-purpose software. The image-analysis program is also suitable for repeated measurements to produce morphology distributions. This technique will provide an effective platform for measuring the 3D shapes of materials of interest to many applications.

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.