Development of a low-cost robotized 3D-prototype for automated optical microscopy diagnosis: An open-source system.

In a clinical context, conventional optical microscopy is commonly used for the visualization of biological samples for diagnosis. However, the availability of molecular techniques and rapid diagnostic tests are reducing the use of conventional microscopy, and consequently the number of experienced professionals starts to decrease. Moreover, the continuous visualization during long periods of time through an optical microscope could affect the final diagnosis results due to induced human errors and fatigue. Therefore, microscopy automation is a challenge to be achieved and address this problem. The aim of the study is to develop a low-cost automated system for the visualization of microbiological/parasitological samples by using a conventional optical microscope, and specially designed for its implementation in resource-poor settings laboratories. A 3D-prototype to automate the majority of conventional optical microscopes was designed. Pieces were built with 3D-printing technology and polylactic acid biodegradable material with Tinkercad/Ultimaker Cura 5.1 slicing softwares. The system's components were divided into three subgroups: microscope stage pieces, storage/autofocus-pieces, and smartphone pieces. The prototype is based on servo motors, controlled by Arduino open-source electronic platform, to emulate the X-Y and auto-focus (Z) movements of the microscope. An average time of 27.00 ± 2.58 seconds is required to auto-focus a single FoV. Auto-focus evaluation demonstrates a mean average maximum Laplacian value of 11.83 with tested images. The whole automation process is controlled by a smartphone device, which is responsible for acquiring images for further diagnosis via convolutional neural networks. The prototype is specially designed for resource-poor settings, where microscopy diagnosis is still a routine process. The coalescence between convolutional neural network predictive models and the automation of the movements of a conventional optical microscope confer the system a wide range of image-based diagnosis applications. The accessibility of the system could help improve diagnostics and provide new tools to laboratories worldwide.

Innovating in a bioimaging core through instrument development.

Developing devices and instrumentation in a bioimaging core facility is an important part of the innovation mandate inherent in the core facility model but is a complex area due to the required skills and investments, and the impossibility of a universally applicable model. Here, we seek to define technological innovation in microscopy and situate it within the wider core facility innovation portfolio, highlighting how strategic development can accelerate access to innovative imaging modalities and increase service range, and thus maintain the cutting edge needed for sustainability. We consider technology development from the perspective of core facility staff and their stakeholders as well as their research environment and aim to present a practical guide to the 'Why, When, and How' of developing and integrating innovative technology in the core facility portfolio. Core facilities need to innovate to stay up to date. However, how to carry out the innovation is not very obvious. One area of innovation in imaging core facilities is the building of optical setups. However, the creation of optical setups requires specific skill sets, time, and investments. Consequently, the topic of whether a core facility should develop optical devices is discussed as controversial. Here, we provide resources that should help get into this topic, and we discuss different options when and how it makes sense to build optical devices in core facilities. We discuss various aspects, including consequences for staff and the relation of the core to the institute, and also broaden the scope toward other areas of innovation.

Towards ultra-low-cost smartphone microscopy.

The outbreak of COVID-19 exposed the inadequacy of our technical tools for home health surveillance, and recent studies have shown the potential of smartphones as a universal optical microscopic imaging platform for such applications. However, most of them use laboratory-grade optomechanical components and transmitted illuminations to ensure focus tuning capability and imaging quality, which keeps the cost of the equipment high. Here, we propose an ultra-low-cost solution for smartphone microscopy. To realize focus tunability, we designed a seesaw-like structure capable of converting large displacements on one side into small displacements on the other (reduced to ∼9.1%), which leverages the intrinsic flexibility of 3D printing materials. We achieved a focus-tuning accuracy of ∼5 𝜇m, which is 40 times higher than the machining accuracy of the 3D-printed lens holder itself. For microscopic imaging, we used an off-the-shelf smartphone camera lens as the objective and the built-in flashlight as the illumination. To compensate for the resulting image quality degradation, we developed a learning-based image enhancement method. We used the CycleGAN architecture to establish the mapping from smartphone microscope images to benchtop microscope images without pairing. We verified the imaging performance on different biomedical samples. Except for the smartphone, we kept the full costs of the device under 4 USD. We think these efforts to lower the costs of smartphone microscopes will benefit their applications in various scenarios, such as point-of-care testing, on-site diagnosis, and home health surveillance. RESEARCH HIGHLIGHTS: We propose a solution for ultra-low-cost smartphone microscopy. Utilizing the flexibility of 3D-printed material, we can achieve focusing accuracy of ∼5 𝜇m. Such a low-cost device will benefit point-of-care diagnosis and home health surveillance.

Handheld and Cost-Effective Fourier Lightfield Microscope.

In this work, the design, building, and testing of the most portable, easy-to-build, robust, handheld, and cost-effective Fourier Lightfield Microscope (FLMic) to date is reported. The FLMic is built by means of a surveillance camera lens and additional off-the-shelf optical elements, resulting in a cost-effective FLMic exhibiting all the regular sought features in lightfield microscopy, such as refocusing and gathering 3D information of samples by means of a single-shot approach. The proposed FLMic features reduced dimensions and light weight, which, combined with its low cost, turn the presented FLMic into a strong candidate for in-field application where 3D imaging capabilities are pursued. The use of cost-effective optical elements has a relatively low impact on the optical performance, regarding the figures dictated by the theory, while its price can be at least 100 times lower than that of a regular FLMic. The system operability is tested in both bright-field and fluorescent modes by imaging a resolution target, a honeybee wing, and a knot of dyed cotton fibers.

Low-cost 3D-printed inverted microscope to detect Mycobacterium tuberculosis in a MODS culture.

MODS, an assay for diagnosis of tuberculosis and drug-susceptibility, is based in the microscopic observation of the characteristic cords of Mycobacterium tuberculosis colonies grown in liquid media. An inverted optical microscope (100× magnification) is required to observe and interpret MODS cultures. Unfortunately, the cost of commercial inverted microscopes is not affordable in low resource settings. To perform a diagnosis of tuberculosis using the MODS assay, images with modest quality are enough for proper interpretation. Therefore, the use of a high cost commercial inverted optical microscope is not indispensable. In this study, we designed a prototype of an optical inverted microscope created by 3D-printing and based on a smartphone. The system was evaluated with 226 MODS TB positive and 207 MODS TB negative digital images. These images were obtained from 10 sputum samples MODS positive and 10 sputum samples MODS negative. The quality of all images was assessed by a qualified technician, in terms of adequacy to interpret and classify them as positive or negative for tuberculosis. The quality of the images was considered appropriate for MODS interpretation. All the 20 samples were correctly classified (as TB positive/negative) by reading with the prototype 3D-printed inverted microscope.

Adhering interacting cells to two opposing coverslips allows super-resolution imaging of cell-cell interfaces.

Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide dissemination for super-resolving the intricate structure and molecular organization in a variety of cell-cell interfaces.

Improving Ki67 assessment concordance by the use of an artificial intelligence-empowered microscope: a multi-institutional ring study.

AIMS: The nuclear proliferation biomarker Ki67 plays potential prognostic and predictive roles in breast cancer treatment. However, the lack of interpathologist consistency in Ki67 assessment limits the clinical use of Ki67. The aim of this article was to report a solution utilising an artificial intelligence (AI)-empowered microscope to improve Ki67 scoring concordance. METHODS AND RESULTS: We developed an AI-empowered microscope in which the conventional microscope was equipped with AI algorithms, and AI results were provided to pathologists in real time through augmented reality. We recruited 30 pathologists with various experience levels from five institutes to assess the Ki67 labelling index on 100 Ki67-stained slides from invasive breast cancer patients. In the first round, pathologists conducted visual assessment on a conventional microscope; in the second round, they were assisted with reference cards; and in the third round, they were assisted with an AI-empowered microscope. Experienced pathologists had better reproducibility and accuracy [intraclass correlation coefficient (ICC) = 0.864, mean error = 8.25%] than inexperienced pathologists (ICC = 0.807, mean error = 11.0%) in visual assessment. Moreover, with reference cards, inexperienced pathologists (ICC = 0.836, mean error = 10.7%) and experienced pathologists (ICC = 0.875, mean error = 7.56%) improved their reproducibility and accuracy. Finally, both experienced pathologists (ICC = 0.937, mean error = 4.36%) and inexperienced pathologists (ICC = 0.923, mean error = 4.71%) improved the reproducibility and accuracy significantly with the AI-empowered microscope. CONCLUSION: The AI-empowered microscope allows seamless integration of the AI solution into the clinical workflow, and helps pathologists to obtain higher consistency and accuracy for Ki67 assessment.

Open-source, cost-effective, portable, 3D-printed digital lensless holographic microscope.

In this work, the design, construction, and testing of the most cost-effective digital lensless holographic microscope to date are presented. The architecture of digital lensless holographic microscopy (DLHM) is built by means of a 3D-printed setup and utilizing off-the-shelf materials to produce a DLHM microscope costing US$52.82. For the processing of the recorded in-line holograms, an open-source software specifically developed to process this type of recordings is utilized. The presented DLHM setup has all the degrees of freedom needed to achieve different fields of view, levels of spatial resolution, and 2D scanning of the sample. The feasibility of the presented platform is tested by imaging non-bio and bio samples; the resolution test targets, a section of the head of a Drosophila melanogaster fly, red blood cells, and cheek cells are imaged on the built microscope.

MR-Eye: High-Resolution Microscopy Coil MRI for the Assessment of the Orbit and Periorbital Structures, Part 2: Clinical Applications.

In the first part of this 2-part series, we described how to implement microscopy coil MR imaging of the orbits. Beyond being a useful anatomic educational tool, microscopy coil MR imaging has valuable applications in clinical practice. By depicting deep tissue tumor extension, which cannot be evaluated clinically, ophthalmic surgeons can minimize the surgical field, preserve normal anatomy when possible, and maximize the accuracy of resection margins. Here we demonstrate common and uncommon pathologies that may be encountered in orbital microscopy coil MR imaging practice and discuss the imaging appearance, the underlying pathologic processes, and the clinical relevance of the microscopy coil MR imaging findings.

Comparative study of endoscopic and microscopic tympanoplasty performed by a single experienced surgeon.

PURPOSE: The use of endoscopes in otologic procedures has been increasing worldwide. This study aimed to compare the efficacy of microscopic tympanoplasty (MT) and endoscopic tympanoplasty (ET) for tympanic membrane and middle ear surgery. MATERIALS AND METHODS: We retrospectively analyzed 81 patients who underwent MT (n = 44) and ET (n = 37) for chronic otitis media with tympanic membrane perforation performed by a single surgeon between January 2013 and September 2019. The hearing outcomes, graft success rate, complications, operation time and hospital stay, and cost-effectiveness were recorded and compared between groups. Hearing outcomes were determined by pure tone audiometry. Cost-effectiveness was determined by the operation cost and total cost. RESULTS: There was no significant difference between the MT and ET groups regarding demographic characteristics, with the exception of the male:female ratio. There was no significant difference in the pre- and postoperative air conduction, bone conduction thresholds, and air-bone gap values between the two groups, but a significant audiologic improvement was observed in both groups (p < 0.05). In terms of recurrence of tympanic membrane perforation, postoperative otorrhea, and discomfort symptoms, there was no significant difference between groups (p > 0.05). The operation time and hospital stay were shorter in the ET group than in the MT group (p < 0.05). There were no significant differences in operation cost between the two groups (p > 0.05), but the total cost was significantly lower in the ET group than the MT group (p < 0.05). CONCLUSION: ET is as safe and medically efficacious as conventional MT, shortens the operation time and hospital stay, and is cost-effective.

Accurate assessment of nonalcoholic fatty liver disease lesions in liver allograft biopsies by a smartphone platform: A proof of concept.

Macrovesicular steatosis (MS) is a major risk factor for liver graft failure after transplantation and pathological microscopic examination of a frozen tissue section remains the gold standard for its assessment. However, the latter requires an experienced in-house pathologist for correct and rapid diagnosis as well as specific equipment that is not always available. Smartphones, which are must-have tools for everyone, are very suitable for incorporation into promising technology to generate moveable diagnostic tools as for telepathology. The study aims to compare the microscopic assessment of nonalcoholic fatty liver disease (NAFLD) spectrum in liver allograft biopsies by a smartphone microscopy platform (DIPLE device) to standard light microscopy. Forty-two liver graft biopsies were evaluated in transmitted light, using an iPhone X and the microscopy platform. A significant correlation was reported between the two different approaches for graft MS assessment (Spearman's correlation coefficient: r = .93; p < .001) and for steatohepatitis feature (r = .56; p < .001; r = .45; p < .001). Based on these findings, a smartphone integrated with a cheap microscopy platform can achieve adequate accuracy in the assessment of NAFLD in liver graft and could be used as an alternative to standard light microscopy when the latter is unavailable.

Cone-shaped optical fiber tip for cost-effective digital lensless holographic microscopy.

In this work, the development and application of a cost-effective and robust digital lensless holographic microscopy (DLHM) system is presented. In the simple architecture of DLHM based on a point source and a digital camera, the production of the former is introduced by means of an engineered step-index optical fiber with a cone-shaped end tip. The conventional and regularly expensive point source in DLHM is produced by means of a high-numerical-aperture microscope objective and a metallic wavelength-sized pinhole. The proposed replacement renders to DLHM additional simplicity of building, in addition to mechanical stability and robustness, and further reduces the cost of the microscope. The simplified cost-effective DLHM architecture is utilized for imaging resolution test targets and samples of human blood and pond water, revealing competitive mechanical stability and trustable phase images of the imaged specimens.

Costs and Complications Associated With Resection of Supratentorial Tumors With and Without the Operative Microscope in the United States.

BACKGROUND: The operative microscope, a commonly used tool in neurosurgery, is critical in many supratentorial tumor cases. However, use of operating microscope for supratentorial tumor varies by surgeon. OBJECTIVES: To assess complication rates, readmissions, and costs associated with operative microscope use in supratentorial resections. METHODS: A retrospective analysis was conducted using a national administrative database to identify patients with glioma or brain metastases who underwent supratentorial resection between 2007 and 2016. Univariate and multivariate analyses were used to assess 30-day complications, readmissions, and costs between patients who underwent resection with and without use of microscope. RESULTS: The cohort included 12,058 glioma patients and 5433 metastasis patients. Rates of microscope use varied by state from 19.0% to 68.6%. Microscope use was associated with $5228.90 in additional costs of index hospitalization among glioma patients (P <0.001), and $2824.00 among metastasis patients (P <0.001). Rates of intraoperative cerebral edema were lower among the microscope cohort than among the nonmicroscope cohort (P <0.027). Microscope use was associated with a slight reduction in 30-day rates of neurological complications (14.7% vs. 16.7%, P = 0.048), specifically in nonspecific cerebrovascular complications. There were no differences in rates of other complications, readmissions, or 30-day postoperative costs. CONCLUSIONS: Use of operative microscope for supratentorial resections varies by state and is associated with higher cost of surgery. Microscope use may be associated with lower rates of intraoperative cerebral edema and some cerebrovascular complications, but is not associated with significant differences in other complications, readmissions, or 30-day costs.

A Low-Cost Humidity Control System to Protect Microscopes in a Tropical Climate.

Introduction: A clean and functional microscope is necessary for accurate diagnosis of infectious diseases. In tropical climates, high humidity levels and improper storage conditions allow for the accumulation of debris and fungus on the optical components of diagnostic equipment, such as microscopes. Objective: Our objective was to develop and implement a low-cost, sustainable, easy to manage, low-maintenance, passive humidity control chamber to both reduce debris accumulation and microbial growth onto the optical components of microscopes. Methods: Constructed from easily-sourced and locally available materials, the cost of each humidity control chamber is approximately $2.35 USD. Relative humidity levels were recorded every 30 minutes over a period of 10 weeks from two chambers deployed at the Belize Vector and Ecology Center and the University of Belize. Results: The humidity control chamber deployed at the University of Belize maintained internal relative humidity at an average of 35.3% (SD = 4.2%) over 10 weeks, while the average external relative humidity was 86.4% (SD = 12.4%). The humidity control chamber deployed at the Belize Vector and Ecology Center effectively maintained internal relative humidity to an average of 54.5% (SD = 9.4%) over 10 weeks, while the average external relative humidity was 86.9% (SD = 12.9%). Conclusions: Control of relative humidity is paramount for the sustainability of medical equipment in tropical climates. The humidity control chambers reduced relative humidity to levels that were not conducive for fungal growth while reducing microscope contamination from external sources. This will likely extend the service life of the microscopes while taking advantage of low-cost, locally sourced components.

Visualisation of dCas9 target search in vivo using an open-microscopy framework.

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.

Rapid detection of rice disease using microscopy image identification based on the synergistic judgment of texture and shape features and decision tree-confusion matrix method.

BACKGROUND: Rice smut and rice blast are listed as two of the three major diseases of rice. Owing to the small size and similar structure of rice blast and rice smut spores, traditional microscopic methods are troublesome to detect them. Therefore, this paper uses microscopy image identification based on the synergistic judgment of texture and shape features and the decision tree-confusion matrix method. RESULTS: The distance transformation-Gaussian filtering-watershed algorithm method was proposed to separate the adherent rice blast spores, and the accuracy was increased by about 10%. Four shape features (area, perimeter, ellipticity, complexity) and three texture features (entropy, homogeneity, contrast) were selected for decision-tree model classification. The confusion-matrix algorithm was used to calculate the classification accuracy, in which global accuracy is 82% and the Kappa coefficient is 0.81. At the same time, the detection accuracy is as high as 94%. CONCLUSIONS: The synergistic judgment of texture and shape features and the decision tree-confusion matrix method can be used to detect rice disease quickly and precisely. The proposed method can be combined with a spore trap, which is vital to devise strategies early and to control rice disease effectively. © 2019 Society of Chemical Industry.

Capabilities of Gabor-domain optical coherence microscopy for the assessment of corneal disease.

To identify the microstructural modification of the corneal layers during the course of the disease, optical technologies have been pushing the boundary of innovation to achieve cellular resolution of deep layers of the cornea. Gabor-domain optical coherence microscopy (GD-OCM), an optical coherence tomography-based technique that can achieve an isotropic of ∼2-µm resolution over a volume of 1 mm × 1 mm × 1.2 mm, was developed to investigate the microstructural modifications of corneal layers in four common corneal diseases. Since individual layer visualization without cutting through several layers is challenging due to corneal curvature, a flattening algorithm was developed to remove the global curvature of the endothelial layer and display the full view of the endothelium and Descemet's membrane in single en face images. As a result, GD-OCM revealed the qualitative changes in size and reflectivity of keratocytes in Fuchs endothelial corneal dystrophy (FECD), which varied by the degree of disease. More importantly, elongated shape and hyperactivation characteristics of keratocytes, associated with the early development of guttae, appeared to start in the posterior stroma very early in the disease process and move toward the anterior stroma during disease progression. This work opens a venue into the pathogenesis of FECD.

Detection of breast cancer lymph node metastases in frozen sections with a point-of-care low-cost microscope scanner.

BACKGROUND: Detection of lymph node metastases is essential in breast cancer diagnostics and staging, affecting treatment and prognosis. Intraoperative microscopy analysis of sentinel lymph node frozen sections is standard for detection of axillary metastases but requires access to a pathologist for sample analysis. Remote analysis of digitized samples is an alternative solution but is limited by the requirement for high-end slide scanning equipment. OBJECTIVE: To determine whether the image quality achievable with a low-cost, miniature digital microscope scanner is sufficient for detection of metastases in breast cancer lymph node frozen sections. METHODS: Lymph node frozen sections from 79 breast cancer patients were digitized using a prototype miniature microscope scanner and a high-end slide scanner. Images were independently reviewed by two pathologists and results compared between devices with conventional light microscopy analysis as ground truth. RESULTS: Detection of metastases in the images acquired with the miniature scanner yielded an overall sensitivity of 91% and specificity of 99% and showed strong agreement when compared to light microscopy (k = 0.91). Strong agreement was also observed when results were compared to results from the high-end slide scanner (k = 0.94). A majority of discrepant cases were micrometastases and sections of which no anticytokeratin staining was available. CONCLUSION: Accuracy of detection of metastatic cells in breast cancer sentinel lymph node frozen sections by visual analysis of samples digitized using low-cost, point-of-care microscopy is comparable to analysis of digital samples scanned using a high-end, whole slide scanner. This technique could potentially provide a workflow for digital diagnostics in resource-limited settings, facilitate sample analysis at the point-of-care and reduce the need for trained experts on-site during surgical procedures.