Standard Microscopy Research Articles

Diagnosis of Plasmodium infections using artificial intelligence techniques versus standard microscopy in a reference laboratory.

Diagnosing malaria using standard techniques is time-consuming. With limited staffing in many laboratories, this may lead to delays in reporting. Innovative technologies are changing the diagnostic landscape and may help alleviate staffing shortages. The miLab MAL, an automated artificial intelligence-driven instrument was compared with standard microscopy at LabCorp reference laboratories. Four hundred eight samples submitted for parasitic examination were prepared with thick and thin smears and Noul's malaria platform miLab MAL, and evaluated for positivity, negativity, percent positivity, and species identification. Of 408 samples, 399 samples were manually negative, while 397 were negative by miLab MAL. Two samples initially classified as negative manually were found positive by miLab MAL. In all nine cases, Plasmodium falciparum was identified by both methods. Percentage parasitemia was higher in the manually calculated method, especially when >1%. miLab MAL was accurate in identifying the absence of Plasmodium falciparum and exhibited higher sensitivity than the manual method. All positive samples detected by microscopy were also identified with miLab MAL. All positive Plasmodium cases were correctly identified by miLab MAL. However, the number of positive samples was limited to only Plasmodium falciparum. Although parasitemia by the manual method was on average six times higher than with miLab MAL, this may be due to sampling variability. The findings show that miLab MAL can be used to screen out negative Plasmodium falciparum samples. Further studies assessing parasitemia between methods and identification of non-falciparum samples are necessary to assess the reliability of this new technology.

One step 4× and 12× 3D-ExM enables robust super-resolution microscopy of nanoscale cellular structures.

Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared with traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4× and 12× 3D-ExM). 3D-ExM is a straightforward expansion microscopy technique featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12× 3D-ExM achieves a lateral resolution of <30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.

High-throughput expansion microscopy enables scalable super-resolution imaging.

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2 x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by the Hoechst signal across the nucleus. We show a dose-dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

High-throughput expansion microscopy enables scalable super-resolution imaging

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2 x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by the Hoechst signal across the nucleus. We show a dose-dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

Autoantibodies immuno-mechanically modulate platelet contractile force and bleeding risk

Altered mechanotransduction has been proposed as a putative mechanism for disease pathophysiology, yet evidence remains scarce. Here we introduce a concept we call single cell immuno-mechanical modulation, which links immunology, integrin biology, cellular mechanics, and disease pathophysiology and symptomology. Using a micropatterned hydrogel-laden coverslip compatible with standard fluorescence microscopy, we conduct a clinical mechanobiology study, specifically focusing on immune thrombocytopenia (ITP), an autoantibody-mediated platelet disorder that currently lacks a reliable biomarker for bleeding risk. We discover that in pediatric ITP patients (n = 53), low single platelet contraction force alone is a “physics-based” biomarker of bleeding (92.3% sensitivity, 90% specificity). Mechanistically, autoantibodies and monoclonal antibodies drive increases and decreases of cell force by stabilizing integrins in different conformations depending on the targeted epitope. Hence, immuno-mechanical modulation demonstrates how antibodies may pathologically alter mechanotransduction to cause clinical symptoms and this phenomenon can be leveraged to control cellular mechanics for research, diagnostic, and therapeutic purposes.

Contribution of amyloid deposition from oligodendrocytes in a mouse model of Alzheimer’s disease

BackgroundThe accumulation of β-amyloid (Aβ) peptides into insoluble plaques is an early pathological feature of Alzheimer’s disease (AD). BACE1 is the sole β-secretase for Aβ generation, making it an attractive therapeutic target for AD therapy. While BACE1 inhibitors have been shown to reduce Aβ levels in people with AD, clinical trials targeting BACE1 have failed due to unwanted synaptic deficits. Understanding the physiological role of BACE1 in individual cell types is essential for developing effective BACE inhibitors for the treatment of AD. Recent single-cell RNA transcriptomic assays revealed that oligodendrocytes are enriched with genes required for generating Aβ. However, the contribution of oligodendrocytes to amyloid plaque burden in AD and the side effects of oligodendrocyte-specific Bace1 deletion remain to be explored.MethodsWe generated an oligodendrocyte-specific Bace1 knockout model (Bace1fl/fl;Olig2-Cre) to monitor potential disruptions in myelination using standard electron microscopy. Long-term potentiation (LTP) was monitored to measure synaptic integrity. We crossed the Bace1fl/fl;Olig2-Cre model with heterozygous AppNL−G−F/wt knock-in AD mice to generate AD mice lacking oligodendrocyte Bace1 (Bace1fl/fl;Olig2-Cre; AppNL−G−F/wt) and examined amyloid plaque number and insoluble Aβ levels and gliosis in these animals. Single nuclei RNA sequencing experiments were conducted to examine molecular changes in response to Bace1 deficiency in oligodendrocytes in the wild type or APP knock-in background.ResultsBace1 deletion in oligodendrocytes caused no change in myelin thickness in the corpus callosum but a marginal reduction in myelin sheath thickness of the optic nerve. Synaptic strength measured by LTP was not different between Bace1fl/fl;Olig2-Cre and age-matched Bace1fl/fl control animals, suggesting no major effect on synaptic plasticity. Intriguingly, deletion of Bace1 in 12-month-old heterozygous AD knock-in mice (Bace1fl/fl;Olig2-Cre; AppNL−G−F/wt mice) caused a significant reduction of amyloid plaques by ~ 33% in the hippocampus and ~ 29% in the cortex compared to age-matched AD mice (Bace1fl/fl;AppNL−G−F/wt). Insoluble Aβ1–40 and Aβ1–42 levels were reduced comparably while more astrocytes and microglia were observed in surrounding amyloid plaques. Unbiased single-nuclei RNA sequencing results revealed that deletion of oligodendrocyte Bace1 in APPNL−G−F/wt knock-in mice increased expression of genes associated with Aβ generation and clearance such as ADAM10, Ano4, ApoE, Il33, and Sort1.ConclusionOur results provide compelling evidence that the amyloidogenic pathway in oligodendrocytes contributes to Aβ plaque formation in the AD brain. While specifically targeting BACE1 inhibition in oligodendrocytes for reducing Aβ pathology in AD is likely challenging, this is a potentially explorable strategy in future studies.

Glutaraldehyde-enhanced autofluorescence as a general tool for 3D morphological imaging.

Routine histochemical techniques are capable of producing vast amount of information from diverse sample types, but these techniques are limited in their ability to generate 3D information. Autofluorescence imaging can be used to analyse samples in 3D but it suffers from weak/low signal intensities. Here, we describe a simple chemical treatment with glutaraldehyde to enhance autofluorescence for 3D fluorescence imaging and to generate detailed morphological images on whole-mount samples. This methodology is straightforward and cost-effective to implement, suitable for a wide range of organisms and sample types. Furthermore, it can be readily integrated with standard confocal and fluorescence microscopes for analysis. This approach has the potential to facilitate the analysis of biological 3D structures and research in developmental biology, including studies on model and non-model organisms.

Prevalence of asymptomatic parasitaemia among household members of children under seasonal malaria chemoprevention coverage and comparison of the performance of standard rapid diagnostic tests versus ultrasensitive RDT for the detection of asymptomatic parasitaemia in Nanoro, Burkina Faso

Asymptomatic carriers of Plasmodium falciparum represent important parasite reservoirs maintaining malaria transmission in the community. This study aimed on the one hand to screen the other household members living with children under seasonal malaria chemoprevention (SMC) coverage in order to determine the level of malaria infection in this population and on the other hand to determine the appropriate type of rapid diagnostic test (RDT) for this screening to detect these asymptomatic carriers in the community. During the 2022 SMC campaign (July to October), a cross-sectional survey was carried out in 745 participants who were screened by ultrasensitive rapid diagnostic test (usRDT), standard rapid diagnostic test (rRDT) and microscopy. Out of them, 395 had microscopy results available and were included in the data analysis. The prevalence of asymptomatic carriers of asexual forms of Plasmodium falciparum was 26.58% (105/395) while sexual forms were found in 5.32% (21/395) of the study population. Children from 5 to 15 years had the highest prevalence of P. falciparum asexual forms 35.76% (59/165) compared with older participants. Malaria positivity rate for rRDT and usRDT was 29.40% (219/745) and 40.49% (305/745) respectively. The usRDT had a higher sensitivity than the rRDT (72.38% (95% CI 62.8–80.66) vs. 60.95% (95% CI 50.94–70.33)). In terms of specificity, rRDT had a higher specificity 82.41% (95% CI 77.53–86.62) versus 69.66% (95% CI 64.01–74.89) for usRDT. This study reports a high prevalence of parasite carriers in household members of children under SMC coverage in Nanoro, Burkina Faso. In conclusion, usRDT seems more appropriate for strategies based on detection and treatment of parasite carriers within the community.

Genetically-encoded markers for confocal visualization of single dense core vesicles.

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

Joint subarray acoustic tweezers enable controllable cell translation, rotation, and deformation

Contactless microscale tweezers are highly effective tools for manipulating, patterning, and assembling bioparticles. However, current tweezers are limited in their ability to comprehensively manipulate bioparticles, providing only partial control over the six fundamental motions (three translational and three rotational motions). This study presents a joint subarray acoustic tweezers platform that leverages acoustic radiation force and viscous torque to control the six fundamental motions of single bioparticles. This breakthrough is significant as our manipulation mechanism allows for controlling the three translational and three rotational motions of single cells, as well as enabling complex manipulation that combines controlled translational and rotational motions. Moreover, our tweezers can gradually increase the load on an acoustically trapped cell to achieve controllable cell deformation critical for characterizing cell mechanical properties. Furthermore, our platform allows for three-dimensional (3D) imaging of bioparticles without using complex confocal microscopy by rotating bioparticles with acoustic tweezers and taking images of each orientation using a standard microscope. With these capabilities, we anticipate the JSAT platform to play a pivotal role in various applications, including 3D imaging, tissue engineering, disease diagnostics, and drug testing.

SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue.

Postmortem human brain tissue is a critical resource for studying neurodegenerative disease, providing critical insights into cellular morphology, pathology, and network connectivity. To improve standard microscopy and enable high-resolution, three-dimensional (3D) images of tissues at the subcellular level, tissue-clearing methods have been developed. These 3D images allow for the analysis of large regions of interest and can be used to study structural and spatial changes that occur during neurodegeneration. Additionally, 3D imaging facilitates the visualization of whole-cell morphology, especially in cells with long processes that would otherwise be truncated in single-plane images. Human brain tissue is especially challenging for tissue clearing due to the abundance of lipids in myelin and the need for optimal fixation and low postmortem intervals. Formaldehyde-based fixatives, commonly used in preserving tissue, hinder antibody binding by crosslinking important antibody epitopes, and fluorescent microscopy requires the incorporation of fluorescent labels through passive diffusion or electrophoresis. Recent studies have focused on optimally fixed human brain tissue with short postmortem intervals, limiting the general applicability of these methods. To address these challenges, we developed SHARD (SHIELD, antigen retrieval, and delipidation), a simple and widely applicable method for clearing and labeling human brain tissue, which can be applied to long-term banked human brain tissue preserved in formaldehyde. SHARD is a novel addition to the SHIELD tissue clarification method, combining antigen retrieval, tissue clearing, and staining of 200-μm sections from long-term banked human brain tissue. The SHARD method is effective for postmortem intervals (PMIs) ranging from 10 to 72 h in multiple neurodegenerative diseases and control samples. In this study, we demonstrate that the SHARD method significantly enhances the immunostaining of glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal marker. Overall, the combination of antigen retrieval and tissue delipidation holds great potential for achieving detailed 3D immunostaining in long-term formaldehyde-fixed postmortem human brain tissue, opening new avenues for research and discovery.

Genetically-encoded markers for confocal visualization of single dense core vesicles.

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

An optical system for cellular mechanostimulation in 3D hydrogels

We introduce a method utilizing single laser-generated cavitation bubbles to stimulate cellular mechanotransduction in dermal fibroblasts embedded within 3D hydrogels. We demonstrate that fibroblasts embedded in either amorphous or fibrillar hydrogels engage in Ca2+ signaling following exposure to an impulsive mechanical stimulus provided by a single 250 µm diameter laser-generated cavitation bubble. We find that the spatial extent of the cellular signaling is larger for cells embedded within a fibrous collagen hydrogel as compared to those embedded within an amorphous polyvinyl alcohol polymer (SLO-PVA) hydrogel. Additionally, for fibroblasts embedded in collagen, we find an increased range of cellular mechanosensitivity for cells that are polarized relative to the radial axis as compared to the circumferential axis. By contrast, fibroblasts embedded within SLO-PVA did not display orientation-dependent mechanosensitivity. Fibroblasts embedded in hydrogels and cultured in calcium-free media did not show cavitation-induced mechanotransduction; implicating calcium signaling based on transmembrane Ca2+ transport. This study demonstrates the utility of single laser-generated cavitation bubbles to provide local non-invasive impulsive mechanical stimuli within 3D hydrogel tissue models with concurrent imaging using optical microscopy. Statement of significanceCurrently, there are limited methods for the non-invasive real-time assessment of cellular sensitivity to mechanical stimuli within 3D tissue scaffolds. We describe an original approach that utilizes a pulsed laser microbeam within a standard laser scanning microscope system to generate single cavitation bubbles to provide impulsive mechanostimulation to cells within 3D fibrillar and amorphous hydrogels. Using this technique, we measure the cellular mechanosensitivity of primary human dermal fibroblasts embedded in amorphous and fibrillar hydrogels, thereby providing a useful method to examine cellular mechanotransduction in 3D biomaterials. Moreover, the implementation of our method within a standard optical microscope makes it suitable for broad adoption by cellular mechanotransduction researchers and opens the possibility of high-throughput evaluation of biomaterials with respect to cellular mechanosignaling.

A Simple Model to Study Mosaic Gene Expression in 3D Endothelial Spheroids.

The goal of this study was to establish a simple model of 3D endothelial spheroids with mosaic gene expression using adeno-associated virus (AAV) transduction, with a future aim being to study the activity of post-zygotic mutations common to vascular malformations. In this study, 96-well U-bottom plates coated with a commercial repellent were seeded with two immortalized human endothelial cell lines and aggregation monitored using standard microscopy or live-cell analysis. The eGFP expression was used to monitor the AAV transduction. HUVEC-TERT2 could not form spheroids spontaneously. The inclusion of collagen I in the growth medium could stimulate cell aggregation; however, these spheroids were not stable. In contrast, the hCMEC/D3 cells aggregated spontaneously and formed reproducible, robust 3D spheroids within 3 days, growing steadily for at least 4 weeks without the need for media refreshment. The hCMEC/D3 spheroids spontaneously developed a basement membrane, including collagen I, and expressed endothelial-specific CD31 at the spheroid surface. Serotypes AAV1 and AAV2QUADYF transduced these spheroids without toxicity and established sustained, mosaic eGFP expression. In the future, this simple approach to endothelial spheroid formation combined with live-cell imaging could be used to rapidly assess the 3D phenotypes and drug and radiation sensitivities arising from mosaic mutations common to brain vascular malformations.

Empowering High-Throughput High-Content Analysis of Microphysiological Models: Open-Source Software for Automated Image Analysis of Microvessel Formation and Cell Invasion.

The primary aim of this study was to develop an open-source Python-based software for the automated analysis of dynamic cell behaviors in microphysiological models using non-confocal microscopy. This research seeks to address the existing gap in accessible tools for high-throughput analysis of endothelial tube formation and cell invasion in vitro, facilitating the rapid assessment of drug sensitivity. Our approach involved annotating over 1000 2mm Z-stacks of cancer and endothelial cell co-culture model and training machine learning models to automatically calculate cell coverage, cancer invasion depth, and microvessel dynamics. Specifically, cell coverage area was computed using focus stacking and Gaussian mixture models to generate thresholded Z-projections. Cancer invasion depth was determined using a ResNet-50 binary classification model, identifying which Z-planes contained invaded cells and measuring the total invasion depth. Lastly, microvessel dynamics were assessed through a U-Net Xception-style segmentation model for vessel prediction, the DisPerSE algorithm to extract an embedded graph, then graph analysis to quantify microvessel length and connectivity. To further validate our software, we reanalyzed an image set from a high-throughput drug screen involving a chemotherapy agent on a 3D cervical and endothelial co-culture model. Lastly, we applied this software to two naive image datasets from coculture lumen and microvascular fragment models. The software accurately measured cell coverage, cancer invasion, and microvessel length, yielding drug sensitivity IC50 values with a 95% confidence level compared to manual calculations. This approach significantly reduced the image processing time from weeks down to h. Furthermore, the software was able to calculate cell coverage, microvessel length, and invasion depth from two additional microphysiological models that were imaged with confocal microscopy, highlighting the versatility of the software. Our free and open source software offers an automated solution for quantifying 3D cell behavior in microphysiological models assessed using non-confocal microscopy, providing the broader Cellular and Molecular Bioengineering community with an alternative to standard confocal microscopy paired with proprietary software.This software can be found in our GitHub repository: https://github.com/fogg-lab/tissue-model-analysis-tools. The online version contains supplementary material available at 10.1007/s12195-024-00821-2.

Unveiling the apoptotic potential of antioxidant-rich Bangladeshi medicinal plant extractives and computational modeling to identify antitumor compounds

Nowadays, there has been a significant surge in the exploration of anticancer compounds derived from medicinal plants due to their perceived safety and efficacy. Therefore, our objective was to investigate the antioxidant and antiproliferative properties, along with the phytoconstituents, of methanol extracts from various parts of 15 selected Bangladeshi medicinal plants. Standard spectrophotometric methods and confocal microscopy were utilized to assess the antioxidant and antiproliferative potential of these extracts. Additionally, phytochemical profiling was executed through gas chromatography-mass spectrometry (GC-MS) analysis. Among the extractives, Bombax ceiba bark exhibited the highest scavenging capacity against DPPH (IC50: 10.3 ± 0.7 μg/mL) and hydroxyl (IC50: 3.9 ± 0.1 μg/mL) free radicals. Furthermore, the total antioxidants, reducing power, and polyphenols of B. ceiba bark were higher than those of other extracts. B. ceiba bark also showed significant antiproliferative capacity against MCF-7 cells (86.67 %) in the MTT assay, followed by Cocos nucifera roots (83.92 %), Bixa orellana leaves (44.09 %), and Leea macrophylla roots (25 %). Moreover, B. ceiba bark, L. macrophylla roots, C. nucifera roots, and B. orellana leaves-treated Ehrlich ascites carcinoma (EAC) cells demonstrated growth inhibition rates of 87.27 %, 80.45 %, 42.9 %, and 37.27 %, respectively. Fluorescence microscopic analysis of EAC cells treated with these extracts revealed apoptotic features such as condensed chromatin, cell shrinkage, nucleus fragmentation, and membrane blebbing compared to untreated EAC cells. The GC-MS analysis of B. ceiba bark identified 18 compounds, including various alcohols, alkenes, and esters. Additionally, a molecular docking study revealed oxalic acid, cyclohexyl dodecyl ester as the most potent compound (−6.5) active against breast cancer. In summary, our results demonstrate that B. ceiba bark possesses robust antioxidant and antiproliferative properties, along with potent antitumor compounds, which could be utilized in the treatment of carcinoma.

SCIP: A scalable, reproducible and open-source pipeline for morphological profiling of image cytometry and microscopy data.

Imaging flow cytometry (IFC) provides single-cell imaging data at a high acquisition rate. It is increasingly used in image-based profiling experiments consisting of hundreds of thousands of multi-channel images of cells. Currently available software solutions for processing microscopy data can provide good results in downstream analysis, but are limited in efficiency and scalability, and often ill-adapted to IFC data. In this work, we propose Scalable Cytometry Image Processing (SCIP), a Python software that efficiently processes images from IFC and standard microscopy datasets. We also propose a file format for efficiently storing IFC data. We showcase our contributions on two large-scale microscopy and one IFC datasets, all of which are publicly available. Our results show that SCIP can extract the same kind of information as other tools, in a much shorter time and in a more scalable manner.

HiExM: high-throughput expansion microscopy enables scalable super-resolution imaging.

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by Hoechst signal across the nucleus. We show a dose dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

Design of an FPGA-Based Controller for Fast Scanning Probe Microscopy.

Atomic-scale imaging using scanning probe microscopy is a pivotal method for investigating the morphology and physico-chemical properties of nanostructured surfaces. Time resolution represents a significant limitation of this technique, as typical image acquisition times are on the order of several seconds or even a few minutes, while dynamic processes-such as surface restructuring or particle sintering, to be observed upon external stimuli such as changes in gas atmosphere or electrochemical potential-often occur within timescales shorter than a second. In this article, we present a fully redesigned field programmable gate array (FPGA)-based instrument that can be integrated into most commercially available standard scanning probe microscopes. This instrument not only significantly accelerates the acquisition of atomic-scale images by orders of magnitude but also enables the tracking of moving features such as adatoms, vacancies, or clusters across the surface ("atom tracking") due to the parallel execution of sophisticated control and acquisition algorithms and the fast exchange of data with an external processor. Each of these measurement modes requires a complex series of operations within the FPGA that are explained in detail.

Optical detection and enumeration of Escherichia coli and Salmonella enterica using a low-magnification light microscope

A rapid and cost-effective method for detecting bacterial cells from surfaces is critical to food safety, clinical hygiene, and pharmacy quality. Herein, we established an optical detection method based on a gold chip coating with 3-mercaptophenylboronic acid (3-MPBA) to capture bacterial cells, which allows for the detection and quantification of bacterial cells with a standard light microscope under low-magnification (10×) objective lens. Then, integrate the developed optical detection method with swab sampling to detect bacterial cells loading on stainless-steel surfaces. Using Salmonella enterica (SE1045) and Escherichia coli (E. coli OP50) as model bacterial cells, we achieved a capture efficiency of up to 76.0 ± 2.0 % for SE1045 cells and 81.1 ± 3.3 % for E. coli OP50 cells at 103 CFU/mL upon the optimized conditions, which slightly decreased with the increasing bacterial concentrations. Our assay showed good linear relationships between the concentrations of bacterial cells with the cell counting in images in the range of 103 -107 CFU/mL for SE1045, and 103 -108 CFU/mL for E. coli OP50 cells. The limit of detection (LOD) was 103 CFU/mL for both SE1045 and E. coli OP50 cells. A further increase in sensitivity in detecting E. coli OP50 cells was achieved through a heat treatment, enabling the LOD to be reduced as low as 102 CFU/mL. Furthermore, a preliminary application succeeded in assessing bacterial contamination on stainless-steel surfaces following integration with the approximately 40 % recovery rate, suggesting prospects for evaluating the bacteria from surfaces. The entire process was completed within around 2 h, costing merely a few dollars per sample. Considering the low cost of standard light microscopes, our method holds significant potential for practical industrial applications in bacterial contamination control on surfaces, especially in low-resource settings.