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Register a new userLocalization microscopy: a review of the progress in methods and applications
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Here, we report analysis and summary of research in the field of localization microscopy for optical imaging. We introduce the basic elements of super-resolved localization microscopy methods for PALM and STORM, commonly used both in vivo and in vitro, discussing the core essentials of background theory, instrumentation and computational algorithms. We discuss the resolution limit of light microscopy and the mathematical framework for localizing fluorescent dyes in space beyond this limit, including the precision obtainable as a function of the amount of light emitted from a dye, and how it leads to a fundamental compromise between spatial and temporal precision. The properties of a good dye are outlined, as are the features of PALM and STORM super-resolution microscopy and adaptations that may need to be made to experimental protocols to perform localization determination. We analyse briefly some of the methods of modern super-resolved optical imaging that work through reshaping point spread functions and how they utilize aspects of localization microscopy, such as stimulated depletion (STED) methods and MINFLUX, and summarize modern methods that push localization into 3D using non-Gaussian point spread functions. We report on current methods for analyzing localization data including determination of 2D and 3D diffusion constants, molecular stoichiometries, and performing cluster analysis with cutting-edge techniques, and finally discuss how these techniques may be used to enable important insight into a range of biological processes.
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Here, we discuss a collection of cutting-edge techniques and applications in use today by some of the leading experts in the field of correlative approaches in single-molecule biophysics. A key difference in emphasis, compared with traditional single-molecule biophysics approaches detailed previously, is on the emphasis of the development and use of complex methods which explicitly combine multiple approaches to increase biological insights at the single-molecule level. These so-called correlative single-molecule biophysics methods rely on multiple, orthogonal tools and analysis, as opposed to any one single driving technique. Importantly, they span both in vivo and in vitro biological systems as well as the interfaces between theory and experiment in often highly integrated ways, very different to earlier traditional non-integrative approaches. The first applications of correlative single-molecule methods involved adaption of a range of different experimental technologies to the same biological sample whose measurements were synchronised. However, now we find a greater flora of integrated methods emerging that include approaches applied to different samples at different times and yet still permit useful molecular-scale correlations to be performed. The resultant findings often enable far greater precision of length and time scales of measurements, and a more understanding of the interplay between different processes in the same cell. Many new correlative single-molecule biophysics techniques also include more complex, physiologically relevant approaches as well as increasing number that combine advanced computational methods and mathematical analysis with experimental tools. Here we review the motivation behind the development of correlative single-molecule microscopy methods, its history and recent progress in the field.
There are currently intense efforts being directed towards extending the range and energy of long distance nonlinear pulse propagation in the atmosphere by moving to longer infrared wavelengths, with the purpose of mitigating the effects of turbulence. In addition, picosecond and longer pulse durations are being used to increase the pulse energy. While both of these tacks promise improvements in applications, such as remote sensing and directed energy, they open up fundamental issues regarding the standard model used to calculate the nonlinear optical properties of dilute gases. Amongst these issues is that for longer wavelengths and longer pulse durations, exponential growth of the laser-generated electron density, the so-called avalanche ionization, can limit the propagation range via nonlinear absorption and plasma defocusing. It is therefore important for the continued development of the field to assess the theory and role of avalanche ionization in gases for longer wavelengths. Here, after an overview of the standard model, we present a microscopically motivated approach for the analysis of avalanche ionization in gases that extends beyond the standard model and we contend is key for deepening our understanding of long distance propagation at long infrared wavelengths. Our new approach involves the mean electron kinetic energy, the plasma temperature, and the free electron density as dynamic variables. The rate of avalanche ionization is shown to depend on the full time history of the pulsed excitation, as opposed to the standard model in which the rate is proportional to the instantaneous intensity.
We report experimental results on heterodyne holographic microscopy of subwavelength-sized gold particles. The apparatus uses continuous green laser illumination of the metal beads in a total internal reflection configuration for dark-field operation. Detection of the scattered light at the illumination wavelength on a charge-coupled device array detector enables 3D localization of brownian particles in water
The ribosome is one of the largest and most complex macromolecular machines in living cells. It polymerizes a protein in a step-by-step manner as directed by the corresponding nucleotide sequence on the template messenger RNA (mRNA) and this process is referred to as `translation of the genetic message encoded in the sequence of mRNA transcript. In each successful chemo-mechanical cycle during the (protein) elongation stage, the ribosome elongates the protein by a single subunit, called amino acid, and steps forward on the template mRNA by three nucleotides called a codon. Therefore, a ribosome is also regarded as a molecular motor for which the mRNA serves as the track, its step size is that of a codon and two molecules of GTP and one molecule of ATP hydrolyzed in that cycle serve as its fuel. What adds further complexity is the existence of competing pathways leading to distinct cycles, branched pathways in each cycle and futile consumption of fuel that leads neither to elongation of the nascent protein nor forward stepping of the ribosome on its track. We investigate a model formulated in terms of the network of discrete chemo-mechanical states of a ribosome during the elongation stage of translation. The model is analyzed using a combination of stochastic thermodynamic and kinetic analysis based on a graph-theoretic approach. We derive the exact solution of the corresponding master equations. We represent the steady state in terms of the cycles of the underlying network and discuss the energy transduction processes. We identify the various possible modes of operation of a ribosome in terms of its average velocity and mean rate of GTP hydrolysis. We also compute entropy production as functions of the rates of the interstate transitions and the thermodynamic cost for accuracy of the translation process.
Many polarisation techniques have been harnessed for decades in biological and clinical research, each based upon measurement of the vectorial properties of light or the vectorial transformations imposed on light by objects. Various advanced vector measurement/sensing techniques, physical interpretation methods, and approaches to analyse biomedically relevant information have been developed and harnessed. In this review, we focus mainly on summarizing methodologies and applications related to tissue polarimetry, with an emphasis on the adoption of the Stokes-Mueller formalism. Several recent breakthroughs, development trends, and potential multi-modal uses in conjunction with other techniques are also presented. The primary goal of the review is to give the reader a general overview in the use of vectorial information that can be obtained by polarisation optics for applications in biomedical and clinical research.
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