High-magnification Microscopy Research Articles

An open-source membrane stretcher for simultaneous mechanical and structural characterizations of soft materials and biological tissues

The ability to simultaneously measure material mechanics and structure is central for understanding their nonlinear relationship that underlies the mechanical properties of materials, such as hysteresis, strain-stiffening and -softening, and plasticity. This experimental capability is also critical in biomechanics and mechanobiology research, as it enables direct characterizations of the intricate interplay between cellular responses and tissue mechanics. Stretching devices developed over the past few decades, however, do not often allow simultaneous measurements of the structural and mechanical responses of the sample. In this work, we introduce an open-source stretching system that can apply uniaxial strain at a submicron resolution, report the tensile force response of the sample, and be mounted on an inverted microscope for real-time imaging. Our system consists of a pair of stepper-based linear motors that stretch the sample symmetrically, a force transducer that records the sample tensile force, and an optically clear sample holder that allows for high-magnification microscopy. Using polymer samples and cellular specimens, we characterized the motion control accuracy, force measurement robustness, and microscopy compatibility of our stretching system. We envision that this uniaxial stretching system will be a valuable tool for characterizing soft and living materials.

A Review Diagnosing Male Infertility: Beyond Conventional Semen Analysis

Infertility, defined as the inability of couples to conceive after one year of consistent, unprotected sexual contact, is a widespread concern affecting a significant portion of the population. Notably, between 14 to 17% of couples may encounter infertility at some point during their reproductive journey. This review delves into the realm of diagnosing male infertility beyond conventional semen analysis. Conventional semen analysis, which is a globally accepted tool for male infertility, primarily focuses on assessing sperm count, motility, viability, and morphology, leaving certain aspects of sperm functionality unexplored. These limitations necessitate the integration of advanced semen analysis techniques to provide a more comprehensive evaluation. Advanced semen analysis encompasses a range of sophisticated tests that probe deeper into the intricacies of male fertility. These tests include assessing sperm penetration capabilities, acrosomal discharge, and spermatozoa Reactive Oxygen Species (ROS) levels. Moreover, advanced semen analysis encompasses tests like sperm chromatin structure assay, DNA damage assessment, sperm proteomics, sperm metabolomics, and high sperm magnification microscopy, all of which shed light on various facets of sperm function and quality. While advanced semen analysis techniques offer a more comprehensive understanding of male infertility, their uptake and utilization in clinical practice have been limited. This review aims to elucidate the roles, merits, and drawbacks of both conventional and advanced semen analysis in diagnosing male infertility. This review sheds light on the strengths and weaknesses of each approach, it promotes a more nuanced approach to male infertility diagnosis, enhancing the prospects of successful conception for infertility challenged couples.

Direct dielectrophoretic characterization of particles in the high-density microwell array using optical tweezers

In this work, a quantitative and intuitive measurement of dielectrophoresis (DEP) behavior in a high-density dielectrophoretic microwell array chip was accomplished in assistance with the holographic optical tweezers. A one-dimensional force balance system with high reliability was achieved between the transverse optical trapping (TOT) force and the DEP force. The value of the DEP force was inferred by accurately calculating the TOT force, and the direction of the DEP force was observed with high-magnification microscopy. In order to estimate the availability and operability of the DEP force measurement system, we systematically investigated the influences of various external factors on the DEP behaviors of microspheres and live cells. All the quantitative measurement results, which agree well with the theory, showed that this system could serve as an accurate research tool for DEP behavior characterizations to optimize and improve the DEP-based manipulations of microparticles and live cells for a broad range of applications. The system also provides an approach to quantitatively obtain important parameters such as Clausius-Mossotti factors for cells in mediums, which could potentially be used to understand the dielectric properties of different types of cells for cell differentiation and diagnosis at the single-cell level.

Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations.

Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing. We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract. We measured tdTomato fluorescence in ex vivo organs at time points between 24h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections. Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

Both incompatible and compatible rhizobia inhabit the intercellular spaces of leguminous root nodules

ABSTRACT In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

TransFusion-net for multifocus microscopic biomedical image fusion

Background and objectiveDue to the depth of focus (DOF) limitations of the optical systems of microscopes, it is often difficult to achieve full clarity from microscopic biomedical images under high-magnification microscopy. Multifocus microscopic biomedical image fusion (MFBIF) can effectively solve this problem. Considering both information richness and visual authenticity, this paper proposes a transformer network for MFBIF called TransFusion-Net. MethodsTransFusion-Net consists of two modules. One module is an interlayer cross-attention module, which is used to obtain feature mappings under the long-range dependencies observed among multiple nonfocus source images. The other module is a spatial attention upsampling network (SAU-Net) module, which is used to obtain global semantic information after further spatial attention is applied. Thus, TransFusion-Net can simultaneously receive multiple input images from a nonfull-focus microscope and make full use of the strong correlations between the source images to output accurate fusion results in an end-to-end manner. ResultsThe fusion results were quantitatively and qualitatively compared with those of eight state-of-the-art algorithms. In the quantitative experiments, five evaluation metrics, QAB/F, QMI, QAVG, QCB, and PSNR, were used to evaluate the performance of each method, and the proposed method achieved values of 0.6574, 8.4572, 5.6305, 0.7341, and 89.5685, respectively, which are higher than those of the current state-of-the-art algorithms. In the qualitative experiments, a differential image was used for further validation, and the near-zero residuals visually verified the adequacy of the proposed method for fusion. Furthermore, we showed some fusion results of multifocused biomedical microscopy images to verify the reliability of the proposed method, which shows high-quality fusion results. ConclusionMultifocus biomedical microscopic image fusion can be accurately and effectively achieved by devising a deep convolutional neural network with joint cross-attention and spatial attention mechanisms.

Insight on Bacterial Newborn Meningitis Using a Neurovascular-Unit-on-a-Chip.

Understanding the pathogenesis of bacterial infections is critical for combatting them. For some infections, animal models are inadequate and functional genomic studies are not possible. One example is bacterial meningitis, a life-threatening infection with high mortality and morbidity. Here, we used the newly developed, physiologically relevant, organ-on-a-chip platform integrating the endothelium with neurons, closely mimicking in vivo conditions. Using high-magnification microscopy, permeability measurements, electrophysiological recordings, and immunofluorescence staining, we studied the dynamic by which the pathogens cross the blood-brain barrier and damage the neurons. Our work opens up possibilities for performing large-scale screens with bacterial mutant libraries for identifying the virulence genes involved in meningitis and determining the role of these genes, including various capsule types, in the infection process. These data are essential for understanding and therapy of bacterial meningitis. Moreover, our system offers possibilities for the study of additional infections-bacterial, fungal, and viral. IMPORTANCE The interactions of newborn meningitis (NBM) with the neurovascular unit are very complex and are hard to study. This work presents a new platform to study NBM in a system that enables monitoring of multicellular interactions and identifies processes that were not observed before.

Additional fossil specimens of Eophylica (Rhamnaceae) in mid-Cretaceous amber from the Hukawng Valley, Myanmar

The recently described fossil angiosperm genus Eophylica (Rhamnaceae) was based on 18 amber-embedded specimens derived from two sites in northern Myanmar. A vegetatively similar fossil, which came from the same amber mines but which lacked flowers, had been previously described as a putative green alga and given the name Electrophycus. The dimensions and vegetative aspect of that taxon’s holotype specimen are identical with those of the Eophylica fossils. Four additional specimens were acquired by the senior author after the publication of Electrophycus, all of which have a single terminal flower that is either immature or essentially fully formed. Using techniques of floral dissection, high magnification microscopy, and microphotography of the better-developed flowers, we present a more detailed picture of the floral organs, especially the stamens and style, of Eophylica. These flowers differ from the holotype specimen of Eophylica and 3 of its paratypes in that they possess involucral bracts of a so-called ‘pseudanthial head,’ a structure that the authors of Eophylica had only ascribed to certain other paratype specimens of the genus.

Human Sperm Morphology as a Marker of Its Nuclear Quality and Epigenetic Pattern.

Background: Human sperm chromatin condensation is a sum of epigenetic events that allows for the near-complete replacement of histones with protamines. Under high-magnification microscopy, nuclear vacuoles have been described as thumbprints with poor chromatin condensation. The objective of this study is to examine whether vacuolated spermatozoa carry specific epigenetic marks, which may influence embryo development. Methods: The presence and three-dimensional distribution of ten epigenetic marks (protamine-P2, histone-H3, H3K4me1/me2/me3, H3K9me1/me2/me3, H3K27me3, H4k20me2) were evaluated and compared in morphometrically normal spermatozoa according to the presence or absence of a large vacuole occupying more than 15% of the head surface (n = 4193). Results: Vacuolated spermatozoa were significantly more frequently labelled with H3 and H3K4me3 than normal spermatozoa (88.1% ± 2.7 and 78.5% ± 5.2 vs. 74.8% ± 4.8 and 49.1% ± 7.4, respectively; p = 0.009 and p < 0.001) and significantly less marked by P2 and H3K27me3 (50.2% ± 6.2 and 63.9% ± 6.3 vs. 82.1% ± 4.4 and 73.6% ± 5.1, respectively; p < 0.001 and p = 0.028). In three dimensions, vacuoles are nuclear concavities filled with DNA carrying the H3K4me3 marker. Conclusion: High-magnification microscopy is a simple tool to estimate in real time the sperm epigenetic profile. The selection of normal spermatozoa without vacuoles and the deselection of spermatozoa with vacuoles appear to be epigenetically favorable to embryo development and safe offspring.

Positioning Van Leeuwenhoek's microscopes in 17th-century microscopic practice.

The self-made nature of Antoni van Leeuwenhoek's discoveries and microscopes tends to obscure the rich and dynamic 17th-century culture of microscopy that preceded his work. Rather than being limited by available magnifications, 17th-century microscopy was shaped by philosophical paradigms, visual and preparation techniques, and observation conditions. Taking into account new insights into his lens making methods, a comparison of Van Leeuwenhoek's methodology with 17th-century predecessors reveals, on the one hand, how his work was rooted in existing traditions, while on the other hand it makes us appreciate his innovations better. Van Leeuwenhoek elegantly turned the viewing practice associated with high-magnification microscopy into a convincing narrative. In doing so, he reaffirmed the central role of the microscope in the 17th-century inquiry of nature. This allowed him to open up new vistas and become the founder of microbiology.

Subcellular Detection of SARS-CoV-2 RNA in Human Tissue Reveals Distinct Localization in Alveolar Type 2 Pneumocytes and Alveolar Macrophages.

ABSTRACTThe widespread coronavirus disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have limited understanding of which cells become infected with SARS-CoV-2 in human tissues and where viral RNA localizes on the subcellular level. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA fluorescence in situ hybridization (FISH) with amplification by hybridization chain reaction. We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N), as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type-specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO) but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE (formalin fixation and paraffin embedding) autopsy specimens. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues, as well as extended for other applications, including investigating the viral life cycle, viral diagnostics, and drug screening.

E-Cadherin Modulation and Inter-Cellular Trafficking in Tubular Gastric Adenocarcinoma: A High-Resolution Microscopy Pilot Study.

Despite the numerous advances in tumor molecular biology and chemotherapy options, gastric adenocarcinoma is still the most frequent form of gastric cancer. One of the core proteins that regulates inter-cellular adhesion, E-cadherin plays important roles in tumorigenesis as well as in tumor progression; however, the exact expression changes and modulation that occur in gastric cancer are not yet fully understood. In an attempt to estimate if the synthesis/degradation balance matches the final membrane expression of this adhesion molecule in cancer tissue, we assessed the proportion of E-cadherin that is found in the Golgi vesicles as well as in the lysosomal pathway We utilized archived tissue fragments from 18 patients with well and poorly differentiated intestinal types of gastric cancer and 5 samples of normal gastric mucosa, by using high-magnification multispectral microscopy and high-resolution fluorescence deconvolution microscopy. Our data showed that E-cadherin is not only expressed in the membrane, but also in the cytoplasm of normal and tumor gastric epithelia. E-cadherin colocalization with the Golgian vesicles seemed to be increasing with less differentiated tumors, while co-localization with the lysosomal system decreased in tumor tissue; however, the membrane expression of the adhesion molecule clearly dropped from well to poorly differentiated tumors. Thus E-cadherin seems to be more abundantly synthetized than eliminated via lysosomes/exosomes in less differentiated tumors, suggesting that post-translational modifications, such as cleavage, conformational inactivation, or exocytosis, are responsible for the net drop of E-cadherin at the level of the membrane in more anaplastic tumors. This behavior is in perfect accordance with the concept of partial epithelial-to-mesenchymal transition (P-EMT), when the E-cadherin expression of tumor cells is in fact not downregulated but redistributed away from the membrane in recycling vesicles. Moreover, our high-resolution deconvolution microscopy study showed for the first time, at the tissue level, the presence of Lysosome-associated membrane glycoprotein 1 (LAMP1)-positive exosomes/multivesicular bodies being trafficked across the membranes of tumor epithelial cells. Altogether, a myriad of putative modulatory pathways is available as a treatment turning point, even if we are to only consider the metabolism of membrane E-cadherin regulation. Future super-resolution microscopy studies are needed to clarify the extent of lysosome/exosome exchange between tumor cells and with the surrounding stroma, in histopathology samples or even in vivo.

Fracture toughness properties and characteristics of Friction stir welded high strength aluminium alloy by post weld heat treatment (PWHT)

Abstract This paper presents the PWHTed effects on fracture toughness properties on stir-welded AA7075 aluminum alloy joints. 12-mm thick rolled AA7075-T651 plates were joined. The joints were given post-weld heat treated by STA and AA. Compact tension (CT) specimens were prepared to evaluate the plane strain fracture toughness of the welded joints. The fracture toughness properties were evaluated under uniaxial tensile loading conditions (stress ratio = 0.1, Frequency = 10 Hz) at room temperature using a servo-hydraulic controlled machine. The welded joints microstructures were examined by low magnification microscopy (OM) and high magnification microscopy (TEM) techniques. It is found that the joint treated with the solutionizing followed by artificial aging cycle exhibited superior fracture toughness properties compared to as-welded joint and artificially aged joints.

Textural Characteristics of Barren and Mineralized Colloform Quartz Bands at the Low-Sulfidation Epithermal Deposits of the Omu Camp in Hokkaido, Japan: Implications for Processes Resulting in Bonanza-Grade Precious Metal Enrichment

Abstract The Miocene low-sulfidation epithermal deposits of the Omu camp in northeastern Hokkaido, Japan, are small past-producers of precious metals and represent significant exploration targets for high-grade Au and Ag ores. The quartz textures of ore samples and the distribution of ore minerals within quartz veins were studied to identify the processes that resulted in the bonanza-grade precious metal enrichment in these deposits. In the high-grade vein samples, which are crustiform or brecciated in hand specimen, ore minerals exclusively occur within colloform quartz bands. High-magnification microscopy reveals that ore-bearing colloform bands consist of fine-grained quartz exhibiting relic microsphere textures and quartz having a mosaic texture that formed through recrystallization of the microspheres. The presence of relic microspheres is evidence that the microcrystalline quartz hosting the ore minerals formed through recrystallization of a noncrystalline silica precursor phase. The ore-hosting colloform bands composed of agglomerated microspheres alternate with barren colloform quartz bands that are composed of fibrous chalcedonic quartz and mosaic quartz formed through recrystallization of the chalcedony. The findings of this study are consistent with previous models linking bonanza-grade precious metal enrichment and the formation of bands of noncrystalline silica in low-sulfidation epithermal veins to episodic vigorous boiling or flashing of the hydrothermal system in the near-surface environment.

Pacific Barkcloth Under the Microscope – Characterisation of Condition, Decoration and Structure

The creation of barkcloth begins with harvesting the inner bark of certain types of trees followed by soaking and beating with grooved beaters, a process which often leaves undulations on the surface of the bark, also known as the beater mark. The cloth can then be decorated using colorants, applied as particulate pigments, dyes or paints. The resulting material is highly ornate with varying surface textures and colours. The usefulness of stereo and standard light microscopy, ranging from around x10 for low and up to x200 for high-magnification microscopy, and macro photography to examine the subtleties of the surface of the cloth is highlighted in this research, with examples of barkcloth from the Hunterian, University of Glasgow, Glasgow and Royal Botanic Gardens, Kew, London collections. Examples of beater marks, particulate dye material, cracking painted surfaces, as well as the presence of two species used to create one cloth are shown, aspects that are not immediately obvious when examining the cloths with the naked eye. To further enhance the analysis from microscopy XRF and FTIR were used when appropriate. Observing the material in this way can increase appreciation for the aesthetic aspects of barkcloth and can further knowledge of the materials used in production. This can inform condition reports, storage requirements, and potential conservation treatments.

Tool wear monitoring in micromilling using Support Vector Machine with vibration and sound sensors

Cutting tool wear is inevitable and becomes even more critical in micromachining processes, due to the small size of the microtools, which makes it impossible to detect any damage or break in the microtool without the use of high magnification microscopy. Therefore, monitoring the wear conditions of microtools is essential to guarantee the quality of the surfaces generated by micromachining processes. Even with the use of sensors, because of the complexity and similarity of the signals, identifying changes related to variation in wear is not a simple task. To overcome these problems, this paper presents a new approach to monitor the wear of cutting tools used in the micromilling process using SVM (Support Vector Machine) artificial intelligence model, vibration and sound signals. The signals were acquired for microchannels manufactured using carbide microtools coated with (Al, Ti) N, with a cutting diameter of 400 μm. The input features for the model were selected using the RFE method (Recursive Feature Elimination). In addition to the main objective, the behavior of the wear curve of the microtool in relation to the wear curve of the conventional machining process was studied. The results showed that the behavior of the curves were similar and the microtool with shorter cutting length had a longer life. The proposed classification methodology obtained a classification accuracy of up to 97.54%, showing that it is possible to use it to monitor the cutting tool wear.

Tissue Coagulation in Laser Hemorrhoidoplasty - An Experimental Study.

BackgroundLaser hemorrhoidoplasty (LHP) is a new technique for treatment of hemorrhoids. The exact extent of coagulation is not visible at the time of the procedure. There currently is no experimental or clinical data on the subject.ObjectiveTo evaluate the length of coagulation defect according to power and activation time of 1470nm diode laser on the perianal tissue model.MethodsFresh anorectal tissue of twenty-four pigs was used to produce 54 experimental samples. Each sample was randomly assigned to the laser power of 6, 8 and 10 W and 1, 2 or 3-second pulses. The procedure was performed using Biolitec Ceralas © diode laser with 1.85 mm optical fiber. The fiber was inserted in a manner, similar to intrahemorrhoidal laser application. Samples were evaluated using low-power and high-power light microscopy by a single pathologist. The length of tissue injury was measured on high-magnification microscopy.ResultsThe longest tissue injury (mean 3.93 mm) was caused by the longest laser exposure time (3 sec) with no significant difference between laser power used.Conclusions8 W 3-second application of the 1470nm diode laser results in coagulation area approximately 4 mm, and further coagulation should be initiated approximately 5 mm from the first one.

Optimization of in vitro trophoblast assay for real-time impedimetric sensing of trophoblast-erythrocyte interactions in Plasmodium falciparum malaria.

Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) is responsible for the pathophysiology of placental malaria, leading to serious complications such as intrauterine growth restriction and low birth weight. However, it is an experimental challenge to study the biology of human placenta. Conventional cell culture-based in vitro placental models rely on immunostaining techniques and high-magnification microscopy is limited in providing real-time quantitative analysis. Impedimetric sensing in combination with cell culture may offer a useful tool. In this paper, we report that real-time label-free measurement of cellular electrical impedance using xCELLigence technology can be used to quantify the proliferation, syncytial fusion, and long-term response of BeWo cells to IEs cytoadhesion. Specifically, we optimized key experimental parameters of cell seeding density and concentration of forskolin, a compound used to promote cell syncitiation, based on electrical signals and immunostaining results. Prolonged time of infection with IEs that led to cell-cell junction vanishment in BeWo cells and release of inflammatory cytokines were monitored in real time by continuous change in electrical impedance. The results suggest that the impedimetric technique is sensitive and can offer new opportunities for the study of cellular responses of trophoblast cells to IEs. The developed system can provide potentially a high-throughput screening tool of anti-adhesion or anti-inflammatory drugs for placental malaria infections.

Click chemistry-based amplification and detection of endogenous RNA and DNA molecules in situ using clampFISH probes.

Direct labeling and measurement of gene expression in single cells show the tremendous variability otherwise hidden in bulk measurements. Single-molecule RNA fluorescence in situ hybridization (FISH) has become a mainstay in laboratories worldwide for measuring gene expression with precision. However, this method remains relatively low throughput because the total fluorescent signal produced is weak and requires long exposure times and high magnification microscopy, which limits the total number of cells sampled in each image. As such, it is experimentally difficult and time-consuming to sample a large enough population of cells to visualize and quantify specific gene expression of rare cells directly. Several FISH-based tools were recently developed that retain single-molecule sensitivity and specificity while greatly amplifying the fluorescent signal, thus making FISH-based analysis possible using standard microscopes with low magnification objectives. These tools have also enabled the detection of smaller and more specific targets like splice junctions or single nucleotide polymorphisms. Here we will describe one such tool, clampFISH, an oligonucleotide-based fluorescence amplification strategy for visualizing genomic loci and individual RNA transcripts in fixed cells. ClampFISH maintains specificity while amplifying fluorescent signals, making it amenable to high throughput assays such as low magnification microscopy, spatial transcriptomics, and flow sorting. The clampFISH technique involves probing the target RNA or DNA using a series of C-shaped oligonucleotide probes, each with a 3' azide and a 5' alkyne. Hybridization of the probe with the target nucleic acid brings the azide and the alkyne in close proximity, allowing for ligation via bioorthogonal click chemistry (CuAAC). As a result, the probe forms a closed loop around the target sequence, thus enabling stringent washes to remove nonspecific binding in further rounds of amplification and retention of signal throughout liquid handling steps. Iterative rounds of hybridization with C-shaped, fluorescently labeled probes exponentially amplify the fluorescent signal. ClampFISH is simple to implement and expands the utility of in situ hybridization for multiple high throughput techniques such as low magnification microscopy, flow cytometry, and sorting based on RNA expression levels.

Quantitative Multiparameter Evaluation of Vacuoles in Intraocular Lenses Employing a High-Magnification Digital Microscopy Method.

As small imperfections with micrometric sizes, fluid-filled vacuoles, also referred to as glistenings, in intraocular lenses (IOLs) have been known to induce significant unwanted light scattering that in several cases presumably cause complaints and sometimes lead to IOL explantation and replacement. This unwanted scatter is of particular concern for patients viewing bright light in reduced-light conditions such as when driving at night, as the scattered light toward the retina can cause temporary blindness. In this study, we have developed and implemented an accurate test methodology based on a high-magnification digital microscopy approach for quantitative multiparameter evaluation and classification of IOL vacuoles depending on their critical optical characteristics including vacuole size, density, shape, and orientation within the IOL material. Using the multiparameter database developed by evaluating vacuole characteristics, we established a classification grading system that can be used to evaluate vacuole effects on light scattering.