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Current Protocols in Mouse Biology Jun 2020The light (or optical) microscope is the icon of science. The aphorism "seeing is believing" is often quoted in scientific papers involving microscopy. Unlike many...
The light (or optical) microscope is the icon of science. The aphorism "seeing is believing" is often quoted in scientific papers involving microscopy. Unlike many scientific instruments, the light microscope will deliver an image however badly it is set up. Fluorescence microscopy is a widely used research tool across all disciplines of biological and biomedical science. Most universities and research institutions have microscopes, including confocal microscopes. This introductory paper in a series detailing advanced light microscopy techniques explains the foundations of both electron and light microscopy for biologists and life scientists working with the mouse. An explanation is given of how an image is formed. A description is given of how to set up a light microscope, whether it be a brightfield light microscope on the laboratory bench, a widefield fluorescence microscope, or a confocal microscope. These explanations are accompanied by operational protocols. A full explanation on how to set up and adjust a microscope according to the principles of Köhler illumination is given. The importance of Nyquist sampling is discussed. Guidelines are given on how to choose the best microscope to image the particular sample or slide preparation that you are working with. These are the basic principles of microscopy that a researcher must have an understanding of when operating core bioimaging facility instruments, in order to collect high-quality images. © 2020 The Authors. Basic Protocol 1: Setting up Köhler illumination for a brightfield microscope Basic Protocol 2: Aligning the fluorescence bulb and setting up Köhler illumination for a widefield fluorescence microscope Basic Protocol 3: Generic protocol for operating a confocal microscope.
Topics: Animals; Humans; Microscopy; Microscopy, Confocal
PubMed: 32497416
DOI: 10.1002/cpmo.76 -
European Journal of Dentistry 2015This article reviews the diagnosis and treatment of cracked teeth, and explores common clinical examples of cracked teeth, such as cusp fractures, fractures into tooth... (Review)
Review
This article reviews the diagnosis and treatment of cracked teeth, and explores common clinical examples of cracked teeth, such as cusp fractures, fractures into tooth furcations, and root fractures. This article provides alternative definitions of terms such as cracked teeth, complete and incomplete fractures and crack lines, and explores the scientific rationale for dental terminology commonly used to describe cracked teeth, such as cracked tooth syndrome, structural versus nonstructural cracks, and vertical, horizontal, and oblique fractures. The article explains the advantages of high magnification loupes (×6-8 or greater), or the surgical operating microscope, combined with co-axial or head-mounted illumination, when observing teeth for microscopic crack lines or enamel craze lines. The article explores what biomechanical factors help to facilitate the development of cracks in teeth, and under what circumstances a full coverage crown may be indicated for preventing further propagation of a fracture plane. Articles on cracked tooth phenomena were located via a PubMed search using a variety of keywords, and via selective hand-searching of citations contained within located articles.
PubMed: 26038667
DOI: 10.4103/1305-7456.156840 -
Reports on Progress in Physics.... May 2023The weak equivalence principle (WEP) is the cornerstone of general relativity (GR). Testing it is thus a natural way to confront GR to experiments, which has been... (Review)
Review
The weak equivalence principle (WEP) is the cornerstone of general relativity (GR). Testing it is thus a natural way to confront GR to experiments, which has been pursued for four centuries with increasing precision. MICROSCOPE is a space mission designed to test the WEP with a precision of 1 in 10parts, two orders of magnitude better than previous experimental constraints. After completing its two-year mission, from 2016 to 2018, MICROSCOPE delivered unprecedented precise constraintsη(Ti,Pt)=[-1.5±2.3 (stat)±1.5 (syst)]×10-15(at 1in statistical errors) on the Eötvös parameter between one proof mass made of titanium and another made of platinum. This bound allowed for improved constraints on alternative theories of gravitation. This review discusses the science beyond MICROSCOPE-GR and its alternatives, with an emphasis on scalar-tensor theories-before presenting the experimental concept and apparatus. The mission's science returns are then discussed before future tests of the WEP are introduced.
PubMed: 37137301
DOI: 10.1088/1361-6633/acd203 -
Nature Protocols May 2020When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues. The confocal microscope's... (Review)
Review
When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues. The confocal microscope's ability to block out-of-focus light and thereby perform optical sectioning through a specimen allows the researcher to quantify fluorescence with very high spatial precision. However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. In this tutorial, the researcher is guided through all aspects of acquiring quantitative confocal microscopy images, including optimizing sample preparation for fixed and live cells, choosing the most suitable microscope for a given application and configuring the microscope parameters. Suggestions are offered for planning unbiased and rigorous confocal microscope experiments. Common pitfalls such as photobleaching and cross-talk are addressed, as well as several troubling instrumentation problems that may prevent the acquisition of quantitative data. Finally, guidelines for analyzing and presenting confocal images in a way that maintains the quantitative nature of the data are presented, and statistical analysis is discussed. A visual summary of this tutorial is available as a poster (https://doi.org/10.1038/s41596-020-0307-7).
Topics: Microscopy, Confocal; Microscopy, Fluorescence; Tissue Fixation
PubMed: 32235926
DOI: 10.1038/s41596-020-0313-9 -
Biomedical Optics Express Nov 2018By integrating a phase-only Spatial Light Modulator (SLM) into the illumination arm of a cylindrical-lens-based Selective Plane Illumination Microscope (SPIM), we have...
By integrating a phase-only Spatial Light Modulator (SLM) into the illumination arm of a cylindrical-lens-based Selective Plane Illumination Microscope (SPIM), we have created a versatile system able to deliver high quality images by operating in a wide variety of different imaging modalities. When placed in a Fourier plane, the SLM permits modulation of the microscope's light-sheet to implement imaging techniques such as structured illumination, tiling, pivoting, autofocusing and pencil beam scanning. Previous publications on dedicated microscope setups have shown how these techniques can deliver improved image quality by rejecting out-of-focus light (structured illumination and pencil beam scanning), reducing shadowing (light-sheet pivoting), and obtaining a more uniform illumination by moving the highest-resolution region of the light-sheet across the imaging Field of View (tiling). Our SLM-SPIM configuration is easy to build and use, and has been designed to allow all of these techniques to be employed on an easily reconfigurable optical setup, compatible with the OpenSPIM design. It offers the possibility to choose between three different light-sheets, in thickness and height, which can be selected according to the characteristics of the sample and the imaging technique to be applied. We demonstrate the flexibility and performance of the system with results obtained by applying a variety of different imaging techniques on samples of fluorescent beads, zebrafish embryos, and optically cleared whole mouse brain samples. Thus our approach allows easy implementation of advanced imaging techniques while retaining the simplicity of a cylindrical-lens-based light-sheet microscope.
PubMed: 30460137
DOI: 10.1364/BOE.9.005419 -
Indian Journal of Otolaryngology and... Oct 2022To assess the parameters' setting of the microscope during parotidectomy and the impact of microscopic parotidectomy on facial nerve functional status. A prospective...
To assess the parameters' setting of the microscope during parotidectomy and the impact of microscopic parotidectomy on facial nerve functional status. A prospective study was conducted on 28 patients in a tertiary care center, who underwent microscopic parotidectomy. Microscope's settings' like magnification, focal length, diameter of the visualized field, and clock position were recorded. Facial nerve functional status was also recorded. All surgeries were performed by right-handed surgeons using Leica F 20 M525 microscope. Clock position of microscope for right parotidectomy ranged between 7 and 10 o clock and for left, it ranged between 7 and 12 o clock. Magnification ranged between 1.3 × and 3.2 ×; magnifications of 1.3 × and 1.8 × were preferred from incision to separation of parotid from sternocleidomastoid muscle, 1.8 × and 2 × for dissection of the facial nerve trunk, and 2 × and 3.2 × for individual branches of the facial nerve. Focal length ranged between 251 and 410 mm and the diameter of the visualized field ranged between 7 and 14.7 cm. Out of 24 cases of benign lesions, 2 (8.3%) developed facial paresis which resolved in 3 months. Two out of four cases of malignancy developed permanent palsy as nerve branches were sacrificed to achieve tumor clearance. Using a microscope for parotidectomy is advantageous for facial nerve dissection, reducing chances of facial paralysis. The microscope was also found to be useful for teaching. The use of appropriate microscopic parameters avoids the glitch of transition from open to microscopic approach.
PubMed: 36452574
DOI: 10.1007/s12070-020-02106-2 -
Cells Nov 2022In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the... (Review)
Review
In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the conventional bench-top microscopic imaging equipment is limited. The comprehensive multi-mode (bright/dark field imaging, fluorescence excitation imaging, polarized light imaging, and differential interference microscopy imaging, etc.) biomedical microscopy imaging systems are generally large in size and expensive. They also require professional operation, which means high labor-cost, money-cost and time-cost. These characteristics prevent them from being applied in bio-medical mobile workstations. The bio-medical mobile workstations need microscopy systems which are inexpensive and able to handle fast, timely and large-scale deployment. The development of lightweight, low-cost and portable microscopic imaging devices can meet these demands. Presently, for the increasing needs of point-of-care-test and tele-diagnosis, high-performance computational portable microscopes are widely developed. Bluetooth modules, WLAN modules and 3G/4G/5G modules generally feature very small sizes and low prices. And industrial imaging lens, microscopy objective lens, and CMOS/CCD photoelectric image sensors are also available in small sizes and at low prices. Here we review and discuss these typical computational, portable and low-cost microscopes by refined specifications and schematics, from the aspect of optics, electronic, algorithms principle and typical bio-medical applications.
Topics: Microscopy; Point-of-Care Systems; Lenses; Algorithms; Microscopy, Interference
PubMed: 36429102
DOI: 10.3390/cells11223670 -
International Journal of Laboratory... Jun 2024The manuscript discusses the historical evolution of observing blood cell morphology under an optical microscope, from the earliest microscopes in the 17th century to... (Review)
Review
The manuscript discusses the historical evolution of observing blood cell morphology under an optical microscope, from the earliest microscopes in the 17th century to the modern digital era, highlighting key advancements and contributions in the field. Blood has historically held symbolic importance in various cultures, with early medical observations dating back to Hippocrates and Galeno. The discovery of cells and subsequent advancements in microscopy by scientists like Hooke and van Leeuwenhoek paved the way for understanding blood cell morphology. Influential figures such as Hewson, Donné, and Ehrlich followed. Diagnostic cytology evolved from manual cell counting to the development of automated hematological systems. Automated complete blood counting came to support microscopic examination in diagnosing hematological disorders. Morphology is crucial in predicting disease outcomes and guiding treatment decisions, particularly hematological neoplasms. The introduction of flow cytometry and its integration with traditional morphological analysis and the new cytogenetic and molecular techniques revolutionized the classification and prognostication of hematologic disorders. Digital microscopy has emerged as a powerful tool in recent years, offering rapid acquisition and sharing of blood cell images. Integrating Artificial Intelligence with digital microscopy has further enhanced morphological analysis, improving diagnostic efficiency. We also discuss the prospects of AI in pre-classifying blood cells in bone marrow aspirate samples, potentially revolutionizing diagnostic pathways for hematologic diseases. Overall, the manuscript provides a comprehensive overview of the historical development, clinical significance and technological advancements in observing blood cell morphology, underscoring its continued relevance in modern hematology practice.
PubMed: 38898733
DOI: 10.1111/ijlh.14330 -
Quarterly Reviews of Biophysics Jan 2021Cryo-electron microscopy (cryo-EM) has become the technique of choice for structural biology of macromolecular assemblies, after the 'resolution revolution' that has... (Review)
Review
Cryo-electron microscopy (cryo-EM) has become the technique of choice for structural biology of macromolecular assemblies, after the 'resolution revolution' that has occurred in this field since 2012. With a suitable instrument, an appropriate electron detector and, last but not least, a cooperative sample it is now possible to collect images from which macromolecular structures can be determined to better than 2 Å resolution, where reliable atomic models can be built. By electron tomography and sub-tomogram averaging of cryo-samples, it is also possible to reconstruct subcellular structures to sub-nanometre resolution. This review describes the infrastructure that is needed to achieve this goal. Ideally, a cryo-EM lab will have a dedicated 300 kV electron microscope for data recording and a 200 kV instrument for screening cryo-samples, both with direct electron detectors, and at least one 120 kV EM for negative-stain screening at room temperature. Added to this should be ancillary equipment for specimen preparation, including a light microscope, carbon coater, plasma cleaner, glow discharge unit, a device for fast, robotic sample freezing, liquid nitrogen storage Dewars and a ready supply of clean liquid nitrogen. In practice, of course, the available budget will determine the number and types of microscopes and how elaborate the lab can be. The cryo-EM lab should be designed with adequate space for the electron microscopes and ancillary equipment, and should allow for sufficient storage space. Each electron microscope room should be connected to the image-processing computers by fibre-optic cables for the rapid transfer of large datasets. The cryo-EM lab should be overseen by a facility manager whose responsibilities include the day-to-day tasks to ensure that all microscopes are operating perfectly, organising service and repairs to minimise downtime, and controlling the budget. Large facilities will require additional support staff who help to oversee the operation of the facility and instruct new users.
Topics: Cryoelectron Microscopy; Humans; Image Processing, Computer-Assisted; Laboratories; Macromolecular Substances; Specimen Handling
PubMed: 33413714
DOI: 10.1017/S003358352000013X