Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).
TL;DR: A high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores that can, in principle, reach molecular-scale resolution is developed.
Abstract: We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.
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TL;DR: In this article , a detailed statistical model of the temporal imaging process which relies on a hidden Markov model operating on two timescales is presented. But the model requires additional calibration measurements.
Abstract: With the advent of fluorescence superresolution microscopy, nano-sized structures can be imaged with a previously unprecedented accuracy. Therefore, it is rapidly gaining importance as an analytical tool in the life sciences and beyond. However, the images obtained so far lack an absolute scale in terms of fluorophore numbers. Here, we use, for the first time, a detailed statistical model of the temporal imaging process which relies on a hidden Markov model operating on two timescales. This allows us to extract this information from the raw data without additional calibration measurements. We show this on the basis of added data from experiments on single Alexa 647 molecules as well as GSDIM/dSTORM measurements on DNA origami structures with a known number of labeling positions.
TL;DR: Fluo as discussed by the authors is a domain-specific language (DSL) in Haskell for setting up fluorescence microscopy experiments that can be combined and nested freely, providing domain specific features such as stage loops and time lapses, and is modular in terms of hardware connections.
Abstract: AbstractFluorescence microscopy is a true workhorse in the domain of life sciences, essential for unraveling the inner workings of cells and tissue. It is not only used from day to day in industry, also academia push boundaries in research using and doing fluorescence microscopy. It is in the latter context that software that is sufficiently modular in terms of experiments and hardware is desirable. Existing solutions are too closely tailored to their accompanying hardware setup or too limited in terms of expressivity. We present \(\textsf {Fluo}\): a domain-specific language (DSL) in Haskell for setting up fluorescence microscopy experiments that can be combined and nested freely. It provides domain-specific features such as stage loops and time lapses, and is modular in terms of hardware connections. \(\textsf {Fluo}\) has been operational since 2015 at the Nanobiology Lab. It has not only improved researchers’ efficiency, but has also given rise to novel research results. For example, performing simultaneous Förster Resonant Energy Transfer (FRET) measurements, a mechanism for tracking energy transfer between a donor-acceptor pair, uses advanced time-lapse experiments and serves as an example use case in the paper. We reflect on the choice of Haskell as a host language and the usability of the DSL.
TL;DR: In this paper , the authors provide an overview of what is known and still to be discovered about organelle membrane protrusions in mammalian cells, focusing on the best-characterised examples of these membrane extensions arising from peroxisomes and mitochondria.
Abstract: Simple Summary Within cells, there are numerous compartments called ‘organelles’ that perform a range of specialised functions required to support life. Organelles are constantly adapting to their environment, changing shape and cooperating with each other depending on the cellular needs, which is essential for cell health as defects in these processes lead to human diseases. One example of organelle dynamic behaviour is the formation of thin tubules that extend and retract from the membranes that delimit the organelles. With a focus on two organelles (peroxisomes and mitochondria) that have roles in cell metabolism and protection, we examine how and why these membrane extensions form, and what their function is within the cell. This includes forming new organelles or organelle networks; increasing the organelle surface area to maximise uptake of molecules; mediating communication between different organelles. We propose that these membrane extensions allow organelles to ‘reach out’ and explore their surroundings more efficiently. Together, this review highlights the importance of organelle dynamics, and specifically membrane extension, in maintaining healthy cell function, as well as exploring the questions remaining to be answered to further our understanding of this essential aspect of cell biology. Abstract Organelles within eukaryotic cells are not isolated static compartments, instead being morphologically diverse and highly dynamic in order to respond to cellular needs and carry out their diverse and cooperative functions. One phenomenon exemplifying this plasticity, and increasingly gaining attention, is the extension and retraction of thin tubules from organelle membranes. While these protrusions have been observed in morphological studies for decades, their formation, properties and functions are only beginning to be understood. In this review, we provide an overview of what is known and still to be discovered about organelle membrane protrusions in mammalian cells, focusing on the best-characterised examples of these membrane extensions arising from peroxisomes (ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis) and mitochondria. We summarise the current knowledge on the diversity of peroxisomal/mitochondrial membrane extensions, as well as the molecular mechanisms by which they extend and retract, necessitating dynamic membrane remodelling, pulling forces and lipid flow. We also propose broad cellular functions for these membrane extensions in inter-organelle communication, organelle biogenesis, metabolism and protection, and finally present a mathematical model that suggests that extending protrusions is the most efficient way for an organelle to explore its surroundings.
01 Jan 2023
TL;DR: A mini review of the major modalities used to examine the plant cytoskeleton and the theory behind them can be found in this article , with a focus on light microscopy.
Abstract: The cytoskeleton is a dynamic and diverse subcellular filament network, and as such microscopy is an essential technology to enable researchers to study and characterize these systems. Microscopy has a long history of observing the plant world not least as the subject where Robert Hooke coined the term "cell" in his publication Micrographia. From early observations of plant morphology to today's advanced super-resolution technologies, light microscopy is the indispensable tool for the plant cell biologist. In this mini review, we will discuss some of the major modalities used to examine the plant cytoskeleton and the theory behind them.
TL;DR: Numerical simulated results demonstrate that the image, whose resolution exceeds the Rayleigh limit, can be stably reconstructed even if the detection signal-to-noise ratio (SNR) is less than 10 dB.
Abstract: We present an imaging approach via sparsity constraint and sparse speckle illumination which can dramatically enhance the optical system's imaging resolution When the object is illuminated by some sparse speckles and the sparse reconstruction algorithm is utilized to restore the blur image, numerical simulated results demonstrate that the image, whose resolution exceeds the Rayleigh limit, can be stably reconstructed even if the detection signal-to-noise ratio (SNR) is less than 10 dB Factors affecting the quality of the reconstructed image, such as the coded pattern's sparsity and the detection SNR, are also studied
References
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TL;DR: Multiphoton microscopy has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals and its use is now increasing exponentially.
Abstract: Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.
3,738 citations
TL;DR: A localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations), and results show good agreement with the derived precision equation.
2,390 citations
TL;DR: Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples, and a recently proposed method in which the nonlinearity arises from saturation of the excited state is experimentally demonstrated.
Abstract: Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arisesfromsaturationoftheexcitedstate.Thismethodcanbeused in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.
2,125 citations
TL;DR: The results strongly support a hand-over-hand model of motility, not an inchworm model, which moves processively on actin.
Abstract: Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of 1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 2x nm and 37 – 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model. Myosin V is a cargo-carrying processive motor
1,888 citations
TL;DR: A family of concepts has emerged that overcomes the diffraction barrier altogether and, relying on saturated optical transitions, these concepts are limited only by the attainable saturation level.
Abstract: For more than a century, the resolution of focusing light microscopy has been limited by diffraction to 180 nm in the focal plane and to 500 nm along the optic axis Recently, microscopes have been reported that provide three- to sevenfold improved axial resolution in live cells Moreover, a family of concepts has emerged that overcomes the diffraction barrier altogether Its first exponent, stimulated emission depletion microscopy, has so far displayed a resolution down to 28 nm Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever
983 citations