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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 -
Indian Journal of Pediatrics Aug 2020Hematuria is one of the alarming manifestations of a renal disease. It can present as macroscopic hematuria or microscopic hematuria due to either glomerular or... (Review)
Review
Hematuria is one of the alarming manifestations of a renal disease. It can present as macroscopic hematuria or microscopic hematuria due to either glomerular or non-glomerular disorders. Clinical presentation and urine microscopy can differentiate glomerular from non-glomerular hematuria. In the majority, a good clinical examination and basic investigations including a urine microscopic examination with sophisticated tools like phase contrast and automated microscopes can help differentiate glomerular from non-glomerular causes for hematuria. Drug induced hematuria, especially secondary to use of analgesics needs to be recognized in routine clinical practice. Rarer causes of hematuria may need more detailed evaluation with a renal biopsy, electron microscopy, urine biochemical testing and imaging. There is no specific treatment to resolve or prevent hematuria. Resolution of hematuria usually occurs with appropriate management of the underlying disorder. Persistent microscopic hematuria indicates the presence of a renal disease that warrants close monitoring and evaluation. Prompt referral to a pediatric nephrologist is indicated in situations when hematuria does not resolve within 2 weeks of onset of glomerulonephritis, there is a need for a renal biopsy, in the presence of persistent microscopic hematuria and need for specific urine biochemistry testing or imaging studies.
Topics: Child; Glomerulonephritis; Hematuria; Humans; Kidney Diseases; Microscopy; Urinalysis
PubMed: 32026313
DOI: 10.1007/s12098-020-03184-4 -
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 -
Viruses May 2023Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications... (Review)
Review
Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.
Topics: Dependovirus; Genetic Vectors; Viruses; Microscopy, Fluorescence
PubMed: 37243260
DOI: 10.3390/v15051174 -
Eye (London, England) Feb 2024This short review begins with the theories of Airy, Rayleigh and Abbe on microscope resolution. Next, the principal developments in microscopy in the last half-century... (Review)
Review
This short review begins with the theories of Airy, Rayleigh and Abbe on microscope resolution. Next, the principal developments in microscopy in the last half-century are examined for relevance to ophthalmology: confocal microscopy, photoactivation light microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), structured illumination (SI), 2-photon and multiphoton excitation microscopy with a focused beam. Except for confocal, these are difficult to apply to the eye in vivo, as are the interference methods available in microscopes. However, interferometry in the form of coherence tomography is now a major ophthalmic method which has diverged from microscopy. Multiphoton excitation microscopy with an unfocussed beam is a new, low-damage microscope method so-far not exploited in ophthalmoscopy. The Mesolens, which throws off the historic limitations in microscopy set by the human eye, is described as a possible future aid to ophthalmology of the anterior eye.
PubMed: 38374367
DOI: 10.1038/s41433-024-02970-0 -
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 -
Light, Science & Applications Nov 2022EDITORIAL: "When all those around me are drunk, I alone am sober," lamented an ancient Chinese poet on fighting a lone and helpless cause. In the world of science a few...
EDITORIAL: "When all those around me are drunk, I alone am sober," lamented an ancient Chinese poet on fighting a lone and helpless cause. In the world of science a few decades ago, there was also a lone researcher who pursued the field of optical resolution despite suspicions and derisions. Unlike those who made their names young, this scientist only succeeded thanks to his own perseverance. He developed the 4Pi microscope and successfully increased the vertical resolution of traditional optical microscopes by 3-7 times. Once he sold the patent to a company, he invested the little personal money gained from the patent in breaking the Abbe diffraction limit. Despite all his hard work, his papers kept getting rejected by respected journals, and his work was subjected to mistrust and even criticism. This scientist is Stefan Hell, someone who is never afraid of difficulties and ever determined to forge ahead.Hell was awarded the Nobel Prize in Chemistry in 2014 for being the first to propose and demonstrate that the optical diffraction limit can be broken, and for successfully developing the STED super-resolution fluorescence microscope. Later Hell and his colleagues proposed and commercialized MINFLUX, which brought optical microscopy technology to the three-dimensional single-nanometer scale, opening the "post-superresolution era".Frank and sincere, persistent and humble, boldly innovative and resolute, Hell is a scientist, an entrepreneur, a mentor, and an everyday man who enjoys life.In this interview, we will reacquaint ourselves with the Nobel laureate, Stefan Hell.
PubMed: 36418300
DOI: 10.1038/s41377-022-01034-w