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Methods in Molecular Biology (Clifton,... 2022Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have...
Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.
Topics: Fluorescent Dyes; Lasers; Microscopy, Confocal; Microscopy, Fluorescence
PubMed: 35218544
DOI: 10.1007/978-1-0716-2051-9_15 -
Analytical Chemistry Nov 2022Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging...
Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). Femtosecond lasers are typically used for MPEF and SHG due to the requirement of high peak power for excitation, while picosecond lasers are preferred for CRS due to the need for high spectral resolution. Therefore, it is challenging to integrate CRS with MPEF and SHG for chemical imaging. We develop a pulse-picking strategy based on an acousto-optic modulator that can program the duty cycle of the laser pulse train, significantly increasing the pulse peak power at low input average power. This approach offers strong enhancement of nonlinear optical signals and makes hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy compatible with MPEF and SHG for multimodal imaging at low laser average power. The pulse-picking method also enables the evaluation and comparison of phototoxicity of laser pulses at different average and peak power levels. The photo-perturbations to biological samples are evaluated using cellular dynamics and sample morphological changes, allowing the selection of optimal laser power for the best sensitivity and minimal phototoxicity.
Topics: Spectrum Analysis, Raman; Lasers; Microscopy; Nonlinear Optical Microscopy; Light
PubMed: 36282141
DOI: 10.1021/acs.analchem.2c03284 -
Current Opinion in Structural Biology Oct 2021With a strong understanding of how proteins fold in hand, it is now possible to ask how in-cell environments modulate their folding, binding and function. Studies... (Review)
Review
With a strong understanding of how proteins fold in hand, it is now possible to ask how in-cell environments modulate their folding, binding and function. Studies accessing fast (ns to s) in-cell dynamics have accelerated over the past few years through a combination of in-cell NMR spectroscopy and time-resolved fluorescence microscopies. Here, we discuss this recent work and the emerging picture of protein surfaces as not just hydrophilic coats interfacing the solvent to the protein's core and functional regions, but as critical components in cells controlling protein mobility, function and communication with post-translational modifications.
Topics: Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy; Microscopy; Protein Folding; Proteins; Solvents
PubMed: 33662744
DOI: 10.1016/j.sbi.2021.02.001 -
Annual Review of Physical Chemistry Apr 2021We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and... (Review)
Review
We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.
Topics: Caulobacter crescentus; Cryoelectron Microscopy; Electron Microscope Tomography; HEK293 Cells; Humans; Microscopy, Electron; Microscopy, Fluorescence; Nanotechnology; Single Molecule Imaging; Subcellular Fractions
PubMed: 33441030
DOI: 10.1146/annurev-physchem-090319-051546 -
Methods in Molecular Biology (Clifton,... 2022Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the...
Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of the arterial wall: the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other.
Topics: Arteries; Atherosclerosis; Humans; Imaging, Three-Dimensional; Microscopy
PubMed: 35237999
DOI: 10.1007/978-1-0716-1924-7_45 -
Current Opinion in Cell Biology Oct 2020At the time of this writing, searching Google Scholar for 'light-sheet microscopy' returns almost 8500 results; over three-quarters of which were published in the last 5... (Review)
Review
At the time of this writing, searching Google Scholar for 'light-sheet microscopy' returns almost 8500 results; over three-quarters of which were published in the last 5 years alone. Searching for other advanced imaging methods in the last 5 years yields similar results: 'super-resolution microscopy' (>16 000), 'single-molecule imaging' (almost 10 000), SPIM (Single Plane Illumination Microscopy, 5000), and 'lattice light-sheet' (1300). The explosion of new imaging methods has also produced a dizzying menagerie of acronyms, with over 100 different species of 'light-sheet' alone, from SPIM to UM (Ultra microscopy) to SiMView (Simultaneous MultiView) to iSPIM (inclined SPIM, not to be confused with iSPIM, inverted SPIM). How then is the average biologist, without an advanced degree in physics, optics, or computer science supposed to make heads or tails of which method is best suited for their needs? Let us also not forget the plight of the optical physicist, who at best might need help with obtaining healthy samples and keeping them that way, or at worst may not realize the impact their newest technique could have for biologists. This review will not attempt to solve all these problems, but instead highlight some of the most recent, successful mergers between biology and advanced imaging technologies, as well as hopefully provide some guidance for anyone interested in journeying into the world of live-cell imaging.
Topics: Animals; Cell Survival; Fluorescence; Humans; Imaging, Three-Dimensional; Microscopy; Staining and Labeling
PubMed: 32470820
DOI: 10.1016/j.ceb.2020.04.008 -
Advances in Experimental Medicine and... 2021Not only is fabrication important for research in materials science, but also materials characterization and analysis. Special microscopes capable of ultra-high...
Not only is fabrication important for research in materials science, but also materials characterization and analysis. Special microscopes capable of ultra-high magnification are more essential for observing and analyzing nanoparticles than for macro-size particles. Recently, electron microscopy (EM) and scanning probe microscopy (SPM) are commonly used for observing and analyzing nanoparticles. In this chapter, the basic principles of various techniques in optical and electron microscopy are described and classified. In particular, techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are explained.
Topics: Microscopy, Atomic Force; Microscopy, Electron, Scanning; Microscopy, Scanning Probe; Nanoparticles; Nanostructures
PubMed: 33782877
DOI: 10.1007/978-981-33-6158-4_12 -
Journal of Microscopy Nov 2021Microscopic observation of biological specimen smears is the mainstay of diagnostic pathology, as defined by the Digital Pathology Association. Though automated systems... (Review)
Review
Microscopic observation of biological specimen smears is the mainstay of diagnostic pathology, as defined by the Digital Pathology Association. Though automated systems for this are commercially available, their bulky size and high cost renders them unusable for remote areas. The research community is investing much effort towards building equivalent but portable, low-cost systems. An overview of such research is presented here, including a comparative analysis of recent reports. This paper also reviews recently reported systems for automated staining and smear formation, including microfluidic devices; and optical and computational automated microscopy systems including smartphone-based devices. Image pre-processing and analysis methods for automated diagnosis are also briefly discussed. It concludes with a set of foreseeable research directions that could lead to affordable, integrated and accurate whole slide imaging systems.
Topics: Image Processing, Computer-Assisted; Microscopy; Pathology; Signal Processing, Computer-Assisted; Smartphone
PubMed: 34254690
DOI: 10.1111/jmi.13049 -
Applied Spectroscopy Sep 2020Microplastics (MPs) have been reported in various environmental compartments and their number is continuously increasing because of degradation into smaller fragments...
Microplastics (MPs) have been reported in various environmental compartments and their number is continuously increasing because of degradation into smaller fragments down to nanoplastics. Humans are exposed to these small-sized MPs through food and air with potential health consequences that still need to be determined. This requires, in the first place, efficient and detailed visualization, relocalization, and characterization of the same MPs with complementary analytical methods. Here, we show the first application of a correlative microscopy and spectroscopy workflow to MPs that meets these demands. For this purpose, standard MP particles on aluminum-coated polycarbonate membrane filters were investigated by an optical zoom microscope and a hyphenated scanning electron microscopy (SEM)-Raman system. By merging the obtained data in one software, it is possible to navigate on the entire filters' surface and correlate at identical locations MP morphology at the spatial resolutions of electron (1.6 nm at 1 kV for the used SEM, ∼100 nm minimum MP size in this study) and optical (∼1-10 µm) microscopies with chemical identification by micro-Raman spectroscopy. Moreover, we observed that low-voltage SEM works without a conductive coating of MPs, causes no detectable charging and structural changes, and provides high-resolution surface imaging of single and clustered MP particles, thus enabling subsequent Raman measurements. We believe that further work on the accurate identification and quantification of micro- and nanoplastics in real samples can potentially profit from this workflow.
Topics: Environmental Monitoring; Environmental Pollutants; Microplastics; Microscopy; Spectrum Analysis, Raman
PubMed: 32186214
DOI: 10.1177/0003702820916250 -
Current Opinion in Structural Biology Feb 2024
Topics: Cryoelectron Microscopy; Microscopy, Electron
PubMed: 38237366
DOI: 10.1016/j.sbi.2023.102772