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An Introduction to Live-Cell Super-Resolution Imaging
Published in Margarida M. Barroso, Xavier Intes, In Vivo, 2020
Siân Culley, Pedro Matos Pereira, Romain F. Laine, Ricardo Henriques
Fluorescence microscopy has been a crucial tool in the advancement of modern cell biology because of its noninvasive nature, compatibility with imaging live samples, and molecule-specific labeling tools. However, the resolving power of conventional fluorescence microscopy is limited to ~250–300 nm. To resolve cellular structures on a smaller size scale than this, researchers have typically relied on electron microscopy, which can provide insight into structures on a nanometer scale. While electron microscopy continues to be a valuable tool for investigating fine intracellular structures, incompatibility with live samples and its limited labeling capabilities remain obstacles to studying dynamic phenomena with high confidence in molecular identities. This has led to the development of super-resolution microscopy methods, which were developed in the early 2000s. Super-resolution microscopy bridges the resolution gap between conventional fluorescence microscopy and electron microscopy while retaining the advantages associated with light microscopy. This chapter provides a brief overview of commonly used super-resolution microscopy techniques, their applications to live-cell imaging, and future directions for this family of techniques.
Polarized Nano-Optics
Published in Klaus D. Sattler, st Century Nanoscience – A Handbook, 2020
Today, fluorescence microscopy is the most widely used tool for in vivo imaging in cells and tissues to address current questions in biology and biomedical optics. Fluorescence is based on the emission of light using molecules used as labels (fluorophores), in which the interesting property is the ability to specifically report the behavior of chosen proteins. Using the significant potential of genetics today, and benefiting from its ability to image in vivo situations, in real time and with a submicrometric resolution, fluorescence imaging is still able to address open questions in cell biology, developmental biology, neurosciences, or in pathologies. Since fluorescence provides a sensitivity down the single molecule level, it is also able to tackle problems related to proteinprotein interactions at specific locations in cells, bringing important elements to the understanding of fundamental biological questions. Yet, while the imaging capabilities of fluorescence give invaluable information, researchers turn to other methods to learn on the way the molecules are organized, e.g., in a more or less ordered way with respect to their orientations. These methods, typically electron microscopy or X-ray diffraction, are however incompatible with in vivo measurements or in-depth in tissues. In this chapter, we describe how the control of light polarization, in the excitation or detection path of a fluorescence microscope, is able to provide significant information on the way molecules are aligned.
Methods of Identifying Microbiological Hazards in Indoor Environments
Published in Rafał L. Górny, Microbiological Corrosion of Buildings, 2020
Particles in samples can be observed in transmitted light (for transparent specimens) or reflected light (for opaque specimens), in light field or dark field or in polarised light to enhance the contract and precisely visualise the details of observed elements. Specimens can be stained using e.g. methylene blue, crystal violet, safranin, fuchsine or other differentiating stains in order to classify microbiological particles into appropriate groups. On the other hand, when the observed particles are almost invisible and observation of the specimen in another medium is impossible or unacceptable, a phase-contrast microscope is used. Fluorescence microscopy is a variant of light microscopy; it uses an ultraviolet or near-ultraviolet light source which causes particles with fluorescent properties to emit light.
Blind structured illumination as excitation for super-resolution photothermal radiometry
Published in Quantitative InfraRed Thermography Journal, 2020
Peter Burgholzer, Thomas Berer, Mathias Ziegler, Erik Thiel, Samim Ahmadi, Jürgen Gruber, Günther Mayr, Günther Hendorfer
Our method for increasing the spatial resolution was inspired by recent work in fluorescence microscopy, where super-resolution imaging was demonstrated using multiple unknown speckle illumination patterns [8,9]. We extend this concept to thermographic imaging, with several illumination patterns, similar to what we have done for photoacoustic imaging [10]. Figure 1 illustrates one of different illumination patters to illuminate the sample. The illumination patterns and the absorption pattern are represented by discrete vectors , where is the number of acquired camera pixels and the components denote values at equidistant points in the imaging domain. According to Equation (1) the reconstructed thermal signal, measured by the infrared camera is
Comparison of STED, confocal and optical microscopy of ultra-short pitch cholesterics
Published in Liquid Crystals, 2020
J. Pišljar, G. Posnjak, S. Pajk, A. Godec, R. Podlipec, B. Kokot, I. Muševič
In polarised light microscopy, the acquired image is a result of phase and amplitude contrast of birefringent sample under observation. Although LC director fields are most often observed using polarised light microscopy, fluorescent imaging methods often prove as beneficial because of their different principle of operation. In contrast to ordinary light microscopy, fluorescence microscopy acquires image of the sample by exciting fluorescent molecules in the sample to radiate fluorescent light. In this case, fluorescent dye molecules which are designed to align their radiative moment along (or perpendicular) to the local director, are added in small amounts to the LC. Since excitation of the anisotropic dye molecule is dependent on the polarisation of the excitation beam, the fluorescent emission intensity distribution is anisotropic and the amount of acquired fluorescent light depends on the local alignment of the radiative dipole moment [2]. This contrast in fluorescent light is used to acquire an image of the sample in fluorescent microscopy and it is quite clear that the image contrast depends on the degree of ordering of dye molecules with respect to the director.
A review of microfluidic concepts and applications for atmospheric aerosol science
Published in Aerosol Science and Technology, 2018
Andrew R. Metcalf, Shweta Narayan, Cari S. Dutcher
By far, the majority of microfluidic experiments are performed on a microscope where the most basic on-chip measurement available is visual microscopy; that is, taking an image of the device and fluid flow with an attached camera. Brightfield and darkfield microscopy images rely on a refractive index contrast between immiscible liquid phases or between suspended particles and the surrounding liquid. Brightfield imaging is used to determine interfacial boundaries for physical property measurements. Fluorescence microscopy, on the other hand, typically excites the sample with a pulse of light at one (range of) wavelength(s) and then images at a shorter (range of) wavelength(s) to measure the excitation from the sample. Fluorescence is typically used in biological agent detection; however, a recent study used fluorescence microscopy to image phase-separated atmospheric aerosol (You et al. 2012). In either imaging scenario, high-speed cameras, with frame rates in excess of 10,000 images per second, allow capture of fast, dynamic phenomena, such as interfacial deformation and relaxation.