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Journal of Microscopy Mar 2021Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add...
Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume-EM with high-resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high-resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. LAY DESCRIPTION: As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high-resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra-structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra-structural detail.
Topics: Animals; Electrons; Imaging, Three-Dimensional; Liver; Mice; Microscopy, Electron, Scanning; Workflow
PubMed: 33034376
DOI: 10.1111/jmi.12967 -
Nanoscale Advances Feb 2021Direct visualization and imaging of nanomaterials under ambient conditions is of great significance for their characterization and application. In most cases, the... (Review)
Review
Direct visualization and imaging of nanomaterials under ambient conditions is of great significance for their characterization and application. In most cases, the observation of individual nanomaterials usually requires high-resolution electron microscopes under high vacuum. In comparison, an optical microscope is much more convenient due to its facile operation and open space. However, the resolution of optical microscopes is much lower than that of electron microscope-based tools. Therefore, effective visualization and imaging strategies for nanomaterials are required to realize their direct observation, accurate location and controllable manipulation. In this review, we summarized the progress of optical visualization and imaging strategies for nanomaterials in recent years, including vapor-condensation-assisted optical visualization, nanoparticle-assisted optical visualization, substrate-assisted optical visualization and fluorescence visualization, and the applications of these techniques were also introduced. We believe that this review will inspire further improvement in optical visualization of nanomaterials and drive the application of nanomaterials in a broader domain.
PubMed: 36133288
DOI: 10.1039/d0na00945h -
Indian Journal of Ophthalmology Oct 2020Microscopes play an important role in the diagnosis of microorganisms and pathological lesions in ophthalmology guiding us to the appropriate management. The current...
Microscopes play an important role in the diagnosis of microorganisms and pathological lesions in ophthalmology guiding us to the appropriate management. The current trend of collecting samples and examination is mostly laboratory-based which consume time, labor, and are costly. Smartphones are being used in different fields of ophthalmology with great ubiquity. The good quality photographs obtained by smartphones along with the ease of mobility has made it possible to warrant its use in the microscopic world. This article describes a simple novel technique of preparing an intraocular lens system which can be used in conjunction with a smartphone to detect microorganisms and pathological lesions.
Topics: Eye, Artificial; Humans; Lenses, Intraocular; Microscopy; Photography; Smartphone
PubMed: 32971646
DOI: 10.4103/ijo.IJO_2032_19 -
World Neurosurgery Jan 2022Operating microscopes and adjunctive technologies are continually refined to advance microneurosurgical care. How frequently these advances are used is unknown. In the...
BACKGROUND
Operating microscopes and adjunctive technologies are continually refined to advance microneurosurgical care. How frequently these advances are used is unknown. In the present study, we assessed the international adoption of microneurosurgical technologies and discussed their value.
METHODS
A 27-question electronic survey was distributed to cerebrovascular neurosurgeon members of U.S., European, and North American neurosurgical societies and social media networks of cerebrovascular and skull base neurosurgeons. The survey encompassed the surgeons' training background, surgical preferences, and standard microneurosurgical practices.
RESULTS
Of the respondents, 56% (53 of 95) were attendings, 74% (70 of 95) were in their first 10 years of practice, and 67% (63 of 94) practiced at an academic teaching hospital. Vascular, endovascular, and skull base fellowships had been completed by 38% (36 of 95), 27% (26 of 95), and 32% (30 of 95) of the respondents, respectively. Most respondents did not use an exoscope (78%; 73 of 94), a mouthpiece (61%; 58 of 95), or foot pedals (56%; 55 of 94). All 95 respondents used a microscope, and 71 (75%) used Zeiss microscopes. Overall, 57 neurosurgeons (60%) used indocyanine green for aneurysms (n = 54), arteriovenous malformations (n = 43), and dural arteriovenous fistulas (n = 42). Most (80%; 75 of 94) did not use fluorescence. The respondents with a vascular-focused practice more commonly used indocyanine green, Yellow 560 fluorescence, and intraoperative 2-dimensional digital subtraction angiography. The respondents with a skull base-focused practice more commonly used foot pedals and an endoscope-assist device.
CONCLUSIONS
The results from the present survey have characterized the current adoption of operative microscopes and adjunctive technologies in microneurosurgery. Despite numerous innovations to improve the symbiosis between neurosurgeon and microscope, their adoption has been underwhelming. Future advances are essential to improve surgical outcomes.
Topics: Adult; Biomedical Technology; Female; Humans; Internationality; Male; Microsurgery; Middle Aged; Neurosurgeons; Neurosurgical Procedures; Surveys and Questionnaires
PubMed: 34687936
DOI: 10.1016/j.wneu.2021.10.128 -
Journal of Biomedical Optics Jan 2021Surgical microscopes provide adjustable magnification, bright illumination, and clear visualization of the surgical field and have been increasingly used in operating... (Review)
Review
SIGNIFICANCE
Surgical microscopes provide adjustable magnification, bright illumination, and clear visualization of the surgical field and have been increasingly used in operating rooms. State-of-the-art surgical microscopes are integrated with various imaging modalities, such as optical coherence tomography (OCT), fluorescence imaging, and augmented reality (AR) for image-guided surgery.
AIM
This comprehensive review is based on the literature of over 500 papers that cover the technology development and applications of surgical microscopy over the past century. The aim of this review is threefold: (i) providing a comprehensive technical overview of surgical microscopes, (ii) providing critical references for microscope selection and system development, and (iii) providing an overview of various medical applications.
APPROACH
More than 500 references were collected and reviewed. A timeline of important milestones during the evolution of surgical microscope is provided in this study. An in-depth technical overview of the optical system, mechanical system, illumination, visualization, and integration with advanced imaging modalities is provided. Various medical applications of surgical microscopes in neurosurgery and spine surgery, ophthalmic surgery, ear-nose-throat (ENT) surgery, endodontics, and plastic and reconstructive surgery are described.
RESULTS
Surgical microscopy has been significantly advanced in the technical aspects of high-end optics, bright and shadow-free illumination, stable and flexible mechanical design, and versatile visualization. New imaging modalities, such as hyperspectral imaging, OCT, fluorescence imaging, photoacoustic microscopy, and laser speckle contrast imaging, are being integrated with surgical microscopes. Advanced visualization and AR are being added to surgical microscopes as new features that are changing clinical practices in the operating room.
CONCLUSIONS
The combination of new imaging technologies and surgical microscopy will enable surgeons to perform challenging procedures and improve surgical outcomes. With advanced visualization and improved ergonomics, the surgical microscope has become a powerful tool in neurosurgery, spinal, ENT, ophthalmic, plastic and reconstructive surgeries.
Topics: Industrial Development; Microscopy; Neurosurgical Procedures; Surgery, Computer-Assisted; Tomography, Optical Coherence
PubMed: 33398948
DOI: 10.1117/1.JBO.26.1.010901 -
Medicine and Pharmacy Reports Jan 2021Magnification devices improve direct and indirect vision and precision being significantly higher in microscope use compared to the loupes. Dental loupes are the most... (Review)
Review
Magnification devices improve direct and indirect vision and precision being significantly higher in microscope use compared to the loupes. Dental loupes are the most commonly used devices for magnification, due to the more affordable prices and the ease of use without major changes in the working protocol and ergonomics. Loupes primary benefits reported refers to ergonomics and posture, restoration evaluation/detection and overall treatment quality. There are some disadvantages that limit the use of loupes among dentists: lack of fixed position (fine movements of the dentist's head disturb the image of the magnified operating field); the need to change the loupes to achieve different magnification. On the contrary, the use of a dental microscope require minimum adjustment and effort so as to reduce postural deviation while working. It has been more than 30 years since dental practitioners raised the issue of using dental operating microscopes (DOM) in restorative dentistry, identifying the benefits of magnification in diagnosis and treatment steps. The growth is evident in recent years, maybe because of the familiarity of dentists with dental microscopes which have already become implicit endowment for endodontic specialists. Fiber optic lighting is the key factor that complements the magnification offered by the microscope so that treatments can now be performed under increased safety conditions and in conditions of significantly higher quality than in the past. Since the beginning of the 2000s the principles of minimally invasive dentistry have been widely promoted in dentistry. These principles are major impetus for the use of microscope by dentists. Working with magnification leads dentists to be more conservative with dental tissues.
PubMed: 33629044
DOI: 10.15386/mpr-1662 -
Lab on a Chip Nov 2021We report the Muscope, a miniature lensless holographic microscope suitable for on-chip integration. The prototype of the Muscope measured approximately only 7 mm × 4...
We report the Muscope, a miniature lensless holographic microscope suitable for on-chip integration. The prototype of the Muscope measured approximately only 7 mm × 4 mm × 4 mm, and was capable of offering a sub-micron half-pitch resolution. We have used, for the first time, a microLED display as the light source in a microscope. The individual pixels of a microLED display chip are used as programmable, microscopic and intense LEDs which can be spatially moved in a two-dimensional plane with a 5 μm pitch. This unique feature set of the display was used to implement computational super-resolution and wide-field imaging without any extra hardware, unlike many other lensless microscopes. We also report a new method to evaluate the magnification in our setting. The Muscope surpasses the existing lensless microscopes in compactness, scalability for production, automated operation and system integration. It provides exciting opportunities for a new class of devices with in-built optical imaging and monitoring and/or sensing capabilities.
Topics: Holography; Microscopy; Optical Imaging
PubMed: 34723299
DOI: 10.1039/d1lc00792k -
Journal of Cell Science Oct 2021Custom-built microscopes often require control of multiple hardware devices and precise hardware coordination. It is also desirable to have a solution that is scalable...
Custom-built microscopes often require control of multiple hardware devices and precise hardware coordination. It is also desirable to have a solution that is scalable to complex systems and that is translatable between components from different manufacturers. Here we report Python-Microscope, a free and open-source Python library for high-performance control of arbitrarily complex and scalable custom microscope systems. Python-Microscope offers simple to use Python-based tools, abstracting differences between physical devices by providing a defined interface for different device types. Concrete implementations are provided for a range of specific hardware, and a framework exists for further expansion. Python-Microscope supports the distribution of devices over multiple computers while maintaining synchronisation via highly precise hardware triggers. We discuss the architectural features of Python-Microscope that overcome the performance problems often raised against Python and demonstrate the different use cases that drove its design: integration with user-facing projects, namely the Microscope-Cockpit project; control of complex microscopes at high speed while using the Python programming language; and use as a microscope simulation tool for software development.
Topics: Computer Simulation; Gene Library; Software
PubMed: 34448002
DOI: 10.1242/jcs.258955 -
IScience Jun 2020Optical microscope is one of the most widely used imaging tools for its great flexibility, reliable design, and low cost. Optical microsphere nanoscope (OMN) is invented... (Review)
Review
Optical microscope is one of the most widely used imaging tools for its great flexibility, reliable design, and low cost. Optical microsphere nanoscope (OMN) is invented as a method that can greatly enhance the observation power of conventional optical microscopes. In this perspective, the promising outlook for this approach is briefly discussed. There exists a great freedom to apply this method in various applications. OMN has been successfully commercialized. Our past experience and strategies are summarized in this perspective, which serves as a good reference for the future technology entrepreneurs. Based on our story and model, the factors for success are listed. It can be used to evaluate other commercialization projects and find out the directions that require further improvement.
PubMed: 32534443
DOI: 10.1016/j.isci.2020.101211