Conventional Microscopy Research Articles

Fluorescent Carbon Dots for Super-Resolution Microscopy.

Conventional fluorescence microscopy is limited by the optical diffraction of light, which results in a spatial resolution of about half of the light's wavelength, approximately to 250-300 nm. The spatial resolution restricts the utilization of microscopes for studying subcellular structures. In order to improve the resolution and to shatter the diffraction limit, two general approaches were developed: a spatially patterned excitation method and a single-molecule localization strategy. The success of super-resolution imaging relies on bright and easily accessible fluorescent probes with special properties. Carbon dots, due to their unique properties, have been used for super-resolution imaging. Considering the importance and fast development of this field, this work focuses on the recent progress and applications of fluorescent carbon dots as probes for super-resolution imaging. The properties of carbon dots for super-resolution microscopy (SRM) are analyzed and discussed. The conclusions and outlook on this topic are also presented.

Single Molecule Localization Microscopy for Studying Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) are 30-200nm nanovesicles enriched with unique cargoes of nucleic acids, lipids, and proteins. sEVs are released by all cell types and have emerged as a critical mediator of cell-to-cell communication. Although many studies have dealt with the role of sEVs in health and disease, the exact mechanism of sEVs biogenesis and uptake remain unexplored due to the lack of suitable imaging technologies. For sEVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200-300nm resolution, thereby generating blurred images. To break this resolution limit, recent developments in super-resolution microscopy techniques, specifically single-molecule localization microscopy (SMLM), expanded the understanding of subcellular details at the few nanometer level. SMLM success relies on the use of appropriate fluorophores with excellent blinking properties. In this review, the basic principle of SMLM is highlighted and the state of the art of SMLM use in sEV biology is summarized. Next, how SMLM techniques implemented for cell imaging can be translated to sEV imaging is discussed by applying different labeling strategies to study sEV biogenesis and their biomolecular interaction with the distant recipient cells.

Bio-mineralized tin/bismuth oxide nanoparticles with silk fibroins for efficient electrochemical detection of 2-nitroaniline in river water samples

In recent years, the usage of nitroaniline has played a vital role in pharmaceutical formulations as it is a crucial ingredient in the synthesis of pesticides and dyes. However, the level of nitroaniline existing in industrial waste keeps rising the environmental contamination. Thus, monitoring of active nitro-residuals becomes more significant in reducing the toxicity of the ecosystem. Therefore, we have taken an attempt to evaluate the hazardous pollutant 2-nitroaniline (2-NA) using the electrocatalyst viz., tin-doped bismuth oxide inserted on a biopolymer silk fibroin composite modified glassy carbon electrode (Sn–Bi2O3/SF@GCE). The Sn–Bi2O3/SF nanocomposite was synthesized through hydrothermal and co-precipitation methods. The physicochemical properties of the prepared Sn–Bi2O3/SF hybrid composite were examined by conventional microscopy and spectroscopic techniques like FE-SEM, HR-TEM, XRD, FTIR, Raman, and XPS. Furthermore, the bio-mineralized Sn–Bi2O3/SF@GCE displayed a wide linear range (0.009 μM–785.7 μM) and a lower detection limit (3.5 nM) with good sensitivity for 2-NA detection under the optimum conditions. The result shows that the Sn–Bi2O3/SF-modified GCE has good reproducibility, repeatability, and excellent selectivity for 2-NA detection in the presence of other co-interfering compounds. Moreover, the practical applicability of Sn–Bi2O3/SF@GCE sensors was investigated for the effective detection of 2-NA in real river water samples, revealing good recovery results.

High Sensitivity of Mobile Phone Microscopy Screening for Schistosoma haematobium in Azaguié, Côte d'Ivoire.

Schistosomiasis infections continue to impact African settings disproportionately, and there is an urgent need for novel tools to evaluate infection control and elimination strategies at the community level. Mobile phone microscopes are portable and semiautomated devices with multiple applications for screening neglected tropical diseases. In a community-based schistosomiasis screening program in Azaguié, Côte d'Ivoire, mobile phone microscopy demonstrated a sensitivity of 85.7% (95% CI: 69.7-95.2%) and specificity of 93.3% (95% CI: 87.7-96.9%) for Schistosoma haematobium identification compared with conventional light microscopy, and 95% sensitivity (95% CI: 74.1-99.8%) with egg concentrations of five or more per 10 mL of urine. Mobile phone microscopy is a promising tool for schistosomiasis control and elimination efforts.

Field-portable seawater toxicity monitoring platform using lens-free shadow imaging technology

The accidental spill of hazardous and noxious substances (HNSs) in the ocean has serious environmental and human health consequences. Assessing the ecotoxicity of seawater exposed to various HNS is challenging due to the constant development of new HNS or mixtures, and assessment methods are also limited. Microalgae viability tests are often used among the various biological indicators for ecotoxicity testing, as they are the primary producers in aquatic ecosystems. However, since the conventional cell growth rate test measures cell viability over three to four days using manual inspection under a conventional optical microscope, it is labor- and time-intensive and prone to subjective errors. In this study, we propose a rapid and automated method to evaluate seawater ecotoxicity by quantification of the morphological changes of microalgae exposed to more than 30 HNSs. This method was further validated using conventional growth rate test results. Dunaliella tertiolecta, a microalgae species without rigid cell walls, was selected as the test organism. Its morphological changes in response to HNS exposure were measured at the single cell level using a custom-developed device that uses lens-free shadow imaging technology. The ecotoxicity evaluation induced by the morphological change could be available in as little as 5 min using the proposed method and device, and it could be effective for 20 HNSs out of 30 HNSs tested. Moreover, the test results of six selected HNSs with high marine transport volume and toxicity revealed that the sensitivity of the proposed method extends to half the maximum effective concentration (EC50) and even to the lowest observed effective concentration (LOEC). Furthermore, the average correlation index between the growth inhibition test (three to four days) and the proposed morphology changes test (5 min) for the six selected HNSs was 0.84, indicating great promise in the field of various point-of-care water quality monitoring. Thus, the proposed equipment and technology may provide a viable alternative to traditional on-site toxicity testing, and the potential of rapid morphological analysis may replace traditional growth inhibition testing.

Highly Thermostable Interphase Enables Boosting High-Temperature Lifespan for Metallic Lithium Batteries.

In consideration of high specific capacity and low redox potential, lithium metal anodes have attracted extensive attention. However, the cycling performance of lithium metal batteries generally deteriorates significantly under the stringent conditions of high temperature due to inferior heat tolerance of the solid electrolyte interphase (SEI). Herein, controllable SEI nanostructures with excellent thermal stability are established by the (trifluoromethyl)trimethylsilane (TMSCF3 )-induced interface engineering. First, the TMSCF3 regulates the electrolyte decomposition, thus generating an SEI with a large amount of LiF, Li3 N, and Li2 S nanocrystals incorporated. More importantly, the uniform distributed nanocrystals have endowed the SEI with enhanced thermostability according to the density functional theory simulations. Particularly, the sub-angstrom visualization on SEI through a conventional transmission electron microscope (TEM) is realized for the first time and the enhanced tolerance to the heat damage originating from TEM imaging demonstrates the ultrahigh thermostability of SEI. As a result, the highly thermostable interphase facilitates a substantially prolonged lifespan of full cells at a high temperature of 70°C. As such, this work might inspire the universal interphase design for high-energy alkali-metal-based batteries applicated in a high-temperature environment.

Development of Automatic Portable Pathology Scanner and Its Evaluation for Clinical Practice.

Digital pathological scanners transform traditional glass slides into whole slide images (WSIs), which significantly improve the efficiency of pathological diagnosis and promote the development of digital pathology. However, the huge economic burden limits the spread and application of general WSI scanners in relatively remote and backward regions. In this paper, we develop an automatic portable cytopathology scanner based on mobile internet, Landing-Smart, to avert the above problems. Landing-Smart is a tiny device with a size of 208mm × 107mm × 104mm and a weight of 1.8kg, which integrates four main components including a smartphone, a glass slide carrier, an electric controller, and an optical imaging unit. By leveraging a simple optical imaging unit to substitute the sophisticated but complex conventional light microscope, the cost of Landing-Smart is less than $3000, much cheaper than general WSI scanners. On the one hand, Landing-Smart utilizes the built-in camera of the smartphone to acquire field of views (FoVs) in the section one by one. On the other hand, it uploads the images to the cloud server in real time via mobile internet, where the image processing and stitching method is implemented to generate the WSI of the cytological sample. The practical assessment of 209 cervical cytological specimens has demonstrated that Landing-Smart is comparable to general digital scanners in cytopathology diagnosis. Landing-Smart provides an effective tool for preliminary cytological screening in underdeveloped areas.

Nondestructive 3D Pathology Image Atlas of Barrett Esophagus With Open-Top Light-Sheet Microscopy.

Anatomic pathologists render diagnosis on tissue samples sectioned onto glass slides and viewed under a bright-field microscope. This approach is destructive to the sample, which can limit its use for ancillary assays that can inform patient management. Furthermore, the subjective interpretation of a relatively small number of 2D tissue sections per sample contributes to low interobserver agreement among pathologists for the assessment (diagnosis and grading) of various lesions. To evaluate 3D pathology data sets of thick formalin-fixed Barrett esophagus specimens imaged nondestructively with open-top light-sheet (OTLS) microscopy. Formalin-fixed, paraffin-embedded Barrett esophagus samples (N = 15) were deparaffinized, stained with a fluorescent analog of hematoxylin-eosin, optically cleared, and imaged nondestructively with OTLS microscopy. The OTLS microscopy images were subsequently compared with archived hematoxylin-eosin histology sections from each sample. Barrett esophagus samples, both small endoscopic forceps biopsies and endoscopic mucosal resections, exhibited similar resolvable structures between OTLS microscopy and conventional light microscopy with up to a ×20 objective (×200 overall magnification). The 3D histologic images generated by OTLS microscopy can enable improved discrimination of cribriform and well-formed gland morphologies. In addition, a much larger amount of tissue is visualized with OTLS microscopy, which enables improved assessment of clinical specimens exhibiting high spatial heterogeneity. In esophageal specimens, OTLS microscopy can generate images comparable in quality to conventional light microscopy, with the advantages of providing 3D information for enhanced evaluation of glandular morphologies and enabling much more of the tissue specimen to be visualized nondestructively.

Exceeding the limits of algorithmic self-calibrated aberration recovery in Fourier ptychography

Fourier ptychographic microscopy is a computational imaging technique that provides quantitative phase information and high resolution over a large field-of-view. Although the technique presents numerous advantages over conventional microscopy, model mismatch due to unknown optical aberrations can significantly limit reconstruction quality. A practical way of correcting for aberrations without additional data capture is through algorithmic self-calibration, in which a pupil recovery step is embedded into the reconstruction algorithm. However, software-only aberration correction is limited in accuracy. Here, we evaluate the merits of implementing a simple, dedicated calibration procedure for applications requiring high accuracy. In simulations, we find that for a target sample reconstruction error, we can image without any aberration corrections only up to a maximum aberration magnitude of λ/40. When we use algorithmic self-calibration, we can tolerate an aberration magnitude up to λ/10 and with our proposed diffuser calibration technique, this working range is extended further to λ/3. Hence, one can trade off complexity for accuracy by using a separate calibration process, which is particularly useful for larger aberrations.

Digital cytology: Current status and future prospects.

In this paper, we reviewed the basic principle and the currentstatus of digital cytopathology. We reviewed the published papers on digitalcytology and analysed its future prospects. Virtualcytology using digital platform is being increasingly used to render diagnosisrather than conventional glass slide microscopy. Whole slide imaging (WSI)offers the prospect of true virtual microscopy and in the near future, may evenreplace glass slides in routine practice. It may be pivotal in diagnosing andtraining pathology graduates faster and more accurately. The digital cytopathology is a promising field and may have great impact indiagnosis, research and teaching.

Magnify is a universal molecular anchoring strategy for expansion microscopy

Expansion microscopy enables nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a crosslinked water-swellable hydrogel. Current expansion microscopy protocols require prior treatment with reactive anchoring chemicals to link specific labels and biomolecule classes to the gel. We describe a strategy called Magnify, which uses a mechanically sturdy gel that retains nucleic acids, proteins and lipids without the need for a separate anchoring step. Magnify expands biological specimens up to 11 times and facilitates imaging of cells and tissues with effectively around 25-nm resolution using a diffraction-limited objective lens of about 280 nm on conventional optical microscopes or with around 15 nm effective resolution if combined with super-resolution optical fluctuation imaging. We demonstrate Magnify on a broad range of biological specimens, providing insight into nanoscopic subcellular structures, including synaptic proteins from mouse brain, podocyte foot processes in formalin-fixed paraffin-embedded human kidney and defects in cilia and basal bodies in drug-treated human lung organoids.

Recording of cellular physiological histories along optically readable self-assembling protein chains

Observing cellular physiological histories is key to understanding normal and disease-related processes. Here we describe expression recording islands—a fully genetically encoded approach that enables both continual digital recording of biological information within cells and subsequent high-throughput readout in fixed cells. The information is stored in growing intracellular protein chains made of self-assembling subunits, human-designed filament-forming proteins bearing different epitope tags that each correspond to a different cellular state or function (for example, gene expression downstream of neural activity or pharmacological exposure), allowing the physiological history to be read out along the ordered subunits of protein chains with conventional optical microscopy. We use expression recording islands to record gene expression timecourse downstream of specific pharmacological and physiological stimuli in cultured neurons and in living mouse brain, with a time resolution of a fraction of a day, over periods of days to weeks.

FOLDOSCOPE SCOPE IN NEUROSURGERY A FEASIBLE AND NOVEL TECHNIQUE

Background: Intra operative squash cytology in identifying CNS lesions and availability of conventional microscope and trained pathologist remains a global health problem especially in remote areas of developing countries which have limited resources .In this study, we evaluated the performance of a low- cost, smartphone attachable paper-based microscope when used for classifying images of Squash cytology of CNS lesions in remote areas and it impact on intraoperative consultation. Samples Methodology: collected during operating CNS pathology From Month Of March,2018 To September 2020 Were Included. Total 22 Cases Were Included but 15 cases were operated in our institute. Intraoperative Squash is done at operating table with the sample tissue and images through foldscope were sent to pathologist electronically and time noted to get the result. Same tissue slides images acquired through conventional microscope are compared with images acquired by foldscope. Mobile phone-based microscope acquired ima Results: ges were observed and compared with a conventional microscope and found the morphology of the tissue sections was signicantly similar to that of conventional light microscope images. The intraoperative time to get the report signicantly reduced in remote settings. With its clear limitations, improvements Conclusion: can be made to transform it into a cost-effective use device exclusively in the diagnosis of cancer in remote and emergency situations.

Relevance of Conventional Microscopy in the Diagnosis of Mucormycosis during COVID-19 Pandemic.

Mucormycosis is a lethal disease which bewildered the health-care community of India during the ongoing second wave of the COVID-19 pandemic. The diagnosis is challenging considering the poor isolation in culture. The aim of the study was to emphasize the utility of potassium hydroxide (KOH) mount examination using conventional light microscopy for early diagnosis of mucormycosis in resource-limited settings. A retrospective analysis of results for all the samples including tissue biopsies, swabs, and pus received in the laboratory for KOH microscopy was done, and results were recorded. The clinical and demographic details of the patients were collected from the hospital information system. A total of 75 samples from 50 patients were received in the laboratory. Out of these, 43 samples from 35 patients showed fungal hyphae (38 patients with only nonseptate hyaline hyphae, 2 with septate hyaline hyphae, and 3 samples with mixed infections). All patients except one were positive for severe acute respiratory syndrome coronavirus 2 infection. The most common age group was 45-59 years (40%), followed by 30-44 years (34.28%) with a male predominance. There was a significant difference in hemoglobin A1C (P = 0.005) and ferritin (P = 0.017) levels between laboratory-confirmed mucormycosis patients and clinically suspected mucormycosis patients without confirmation. Early diagnosis and initiation of targeted therapy is the cornerstone for treating mucormycosis patients. Hence, a rapid and reliable mode of diagnosis is the need of the hour. Conventional microscopy is such a tool that may be used, especially in resource-limited settings.

Dual-channel structured illumination super-resolution quantitative fluorescence resonance energy transfer imaging

The Structured illumination (SI)-based super resolution fluorescence resonance energy transfer (SR-FRET) imaging technique, known as SISR-FRET, enables the investigation of molecular structures and functions in cellular organelles by resolving sub-diffraction FRET signals within living cells. The FRET microscopy offers unique advantages for quantitatively detecting dynamic interactions and spatial distribution of biomolecules within living cells. The spatial resolution of conventional FRET microscopy is limited by the diffraction limit, and it can only capture the average behavior of these events within the resolution limits of conventional fluorescence microscopy. The SISR-FRET performs sequential linear reconstruction of the three-channel SIM images followed by FRET quantitative analysis by using a common localization mask-based filtering approach. This two-step process ensures the fidelity of the reconstructed SR-FRET signals while effectively removing false-positive FRET signals caused by SIM artifacts. However, the slow imaging speed resulting from the switching of excitation-emission channels in SISR-FRET imaging limits its application in fast imaging scenarios. To address this issue, this study proposes a dual-channel structured illumination super-resolution quantitative FRET imaging system and method. By incorporating an FRET dual-channel imaging and registration module into the imaging pathway, the spatial switching and channel multiplexing of the SISR-FRET excitation-emission channels are achieved. Combining the image reconstruction algorithm with channel sub-pixel registration correction, the dual-channel SISR-FRET technique enhances the temporal resolution by 3.5 times while preserving the quantitative super-resolution FRET analysis. Experimental results are obtained by using a multi-color SIM system to perform super-resolution imaging of living cells expressing mitochondria outer membrane FRET standard plasmids. These experiments validate the improved spatial and temporal resolution of dual-channel SISR-FRET and the fidelity of FRET quantification analysis. In summary, this research presents a novel dual-channel structured illumination super-resolution FRET imaging system and method. It overcomes the limitations of slow imaging speed in SISR-FRET by realizing the spatial switching and channel multiplexing of excitation-emission channels. The proposed technique enhances the temporal resolution while maintaining quantitative analysis of super-resolution FRET. Experimental validation demonstrates the increased spatial and temporal resolution of dual-channel SISR-FRET and the accuracy of FRET quantification analysis. This advancement contributes to the study of molecular structures and functions in cellular organelles, providing valuable insights into the intricate mechanisms of living cells.

Three-Dimensional Video Microscopy: Potential for Improved Ergonomics without Increased Operative Time?

Three-dimensional (3D) video exoscopes are high-magnification stereo cameras that project onto monitors mounted in the operating room, viewable from different angles. Outside of plastic surgery, exoscopes have been shown to successfully improve the ergonomics of microsurgery, though sometimes with prolonged operating times. We compare a single surgeon's early experience performing free flap procedures from 2020 to 2021 using either a binocular microscope or a 3D video exoscope. Ten procedures were performed with the standard operating microscope and 8 procedures with the 3D exoscope. The microsurgeon, having minimal prior experience using an exoscope, reported less neck discomfort following the free flap procedures performed with the exoscope compared with the binocular surgical microscope. Total average operating time was comparable between the standard surgical microscope and the 3D exoscope (13.7 vs. 13.4 hours, p = 0.34). Our early experience using a 3D exoscope in place of a standard optical microscope demonstrated that the exoscope shows promise, offering an ergonomic alternative during microvascular reconstruction without increasing overall operating times. Future studies will compare free flap ischemia time between cases performed using the exoscope and the conventional binocular microscope. Medical Subject Headings authorized following words: free tissue flaps; operating rooms; ergonomics; microsurgery.

The Impact of a Robotic Digital Microscope on the Ergonomics in a Neurosurgical Operating Theatre (A Single-Centre Experience).

Ergonomics is an emerging concept in the neurosurgical operating theatre, where neurosurgeons work under great concentration and frequently neglect their uncomfortable body posture. Work-related musculoskeletal disorders (WMSD) are becoming a widespread burden in the neurosurgical community. WMSD have a negative impact on surgical performance and decrease the surgeons' quality of life. Here we present our single-centre 2-month experience with the Aesculap AEOS® Robotic Digital Microscope (RDM) and prospectively calculate the REBA (Rapid Entire Body Assessment) scores and compare them with similar neurosurgical cases operated with a conventional operative microscope (OPMI).Materials and Methods: For a period of 2months at the Department of Neurosurgery of the University Hospital Pirogov, Sofia, Bulgaria, 41 consecutive patients were operated on using RDM. The REBA employee assessment worksheets were filled in prospectively to assess the ergonomics of the senior author (N.G.) while using the RDM and the OPMI.Results and Conclusion: The ergonomics during neurosurgical operations could be substantially improved with the implementation of the exoscope. For challenging cranial approaches, where the operator must frequently "look around corners" the exoscope has a major advantage compared with the OPMI-the REBA score is 2.6 times lower for the exoscope and reaches a low risk for WMSD. For spinal operations, the neck score as part of the REBA score is three times lower for the exoscope.

Through-drop imaging of moving contact lines and contact areas on opaque water-repellent surfaces.

A myriad of natural surfaces such as plant leaves and insect wings can repel water and remain unwetted inspiring scientists and engineers to develop water-repellent surfaces for various applications. Those natural and artificial water-repellent surfaces are typically opaque, containing micro- and nano-roughness, and their wetting properties are determined by the details at the actual liquid-solid interface. However, a generally applicable way to directly observe moving contact lines on opaque water-repellent surfaces is missing. Here, we show that the advancing and receding contact lines and corresponding contact area on micro- and nano-rough water-repellent surfaces can be readily and reproducibly quantified using a transparent droplet probe. Combined with a conventional optical microscope, we quantify the progression of the apparent contact area and apparent contact line irregularity in different types of superhydrophobic silicon nanograss surfaces. Contact angles near 180° can be determined with an uncertainty as low as 0.2°, that a conventional contact angle goniometer cannot distinguish. We also identify the pinning/depinning sequences of a pillared model surface with excellent repeatability and quantify the progression of the apparent contact interface and contact angle of natural plant leaves with irregular surface topography.

Direct Observation of Archaellar Motor Rotation by Single-Molecular Imaging Techniques.

Single-molecular techniques have characterized dynamics of molecular motors such as flagellum in bacteria and myosin, kinesin, and dynein in eukaryotes. We can apply these techniques to a motility machine of archaea, namely, the archaellum, composed of a thin helical filament and a rotary motor. Although the size of the motor hinders the characterization of its motor function under a conventional optical microscope, fluorescence-labeling techniques allow us to visualize the architecture and function of the archaellar filaments in real time. Furthermore, a tiny polystyrene bead attached to the filament enables the visualization of motor rotation through the bead rotation and quantification of biophysical properties such as speed and torque produced by the rotary motor imbedded in the cell membrane. In this chapter, I describe the details of the above biophysical method based on an optical microscope.

Dynamic nano-imaging via a microsphere compound lens integrated microfluidic device with a 10× objective lens.

Optical microscopic imaging techniques are essential in biology and chemistry fields to observe and extract dynamic information of micro/nano-scale samples in microfluidic devices. However, the current microfluidic optical imaging schemes encounter dilemmas in simultaneously possessing high spatial and temporal resolutions. Recently, microsphere nanoscope has emerged as a competitive nano-imaging tool due to its merits like high spatial resolution, real-time imaging abilities, and cost-effectiveness, which make it a potential solution to address the aforementioned challenges. Here, a microsphere compound lens (MCL) integrated microfluidic imaging device is proposed for real-time super-resolution imaging. The MCL consists of two vertically stacked microspheres, which can resolve nano-objects with size beyond the optical diffraction limit and generate an image of the object with a magnification up to 10×. Exploiting the extraordinary nano-imaging and magnification ability of the MCL, optically transparent 100 nm polystyrene particles in flowing fluid can be discerned in real time by the microfluidic device under a 10× objective lens. Contrary to this, the single microsphere and the conventional optical microscope are incompetent in this case regardless of the magnification of objective lenses used, which demonstrates the superiority of the MCL imaging scheme. Besides, applications of the microfluidic device in nanoparticle tracing and live-cell monitoring are also experimentally demonstrated. The MCL integrated microfluidic imaging device can thus be a competent technique for diverse biology and chemistry applications.