Instant super-resolution imaging in live cells and embryos via analog image processing
TL;DR: An analog implementation of structured illumination microscopy is reported that enables three-dimensional (3D) super-resolution imaging with a lateral resolution of 145 nm and an axial resolution of 350 nm at acquisition speeds up to 100 Hz by using optical instead of digital image-processing operations.
Abstract: Existing super-resolution fluorescence microscopes compromise acquisition speed to provide subdiffractive sample information. We report an analog implementation of structured illumination microscopy that enables 3D super-resolution imaging with 145 nm lateral and 350 nm axial resolution, at acquisition speeds up to 100 Hz. By performing image processing operations optically instead of digitally, we removed the need to capture, store, and combine multiple camera exposures, increasing data acquisition rates 10–100x over other super-resolution microscopes and acquiring and displaying super-resolution images in real-time. Low excitation intensities allow imaging over hundreds of 2D sections, and combined physical and computational sectioning allow similar depth penetration to confocal microscopy. We demonstrate the capability of our system by imaging fine, rapidly moving structures including motor-driven organelles in human lung fibroblasts and the cytoskeleton of flowing blood cells within developing zebrafish embryos.
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TL;DR: In this Review,luorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity and the labelling of individual molecules to enable their visualization has emerged as a central challenge.
Abstract: Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge. One current direction for method improvement is the ambition to quantitatively account for each molecule under investigation and assess true molecular colocalization patterns via multi-colour analyses. In pursuing this goal, the labelling of individual molecules to enable their visualization has emerged as a central challenge. Extending nanoscale imaging into (sliced) tissue and whole-animal contexts is a further goal. In this Review we describe the successes to date and discuss current obstacles and possibilities for further development.
726 citations
TL;DR: An overview of current super-resolution microscopy techniques is given and guidance on how best to use them to foster biological discovery is provided.
Abstract: Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
665 citations
TL;DR: An overview of the important parameters involved in successful image reconstruction, a summary of the recent biological applications, and a brief outlook of the directions in which SR-SIM is headed in the future are provided.
Abstract: Super-resolved structured illumination microscopy (SR-SIM) is among the most rapidly growing fluorescence microscopy techniques that can surpass the optical diffraction limit. The strength of SR-SIM is that it can be readily applied to samples prepared for conventional fluorescence microscopy, requiring no sophisticated sample preparation protocols. As an extension of wide-field fluorescence microscopy, it is inherently capable of multicolor imaging and optical sectioning and, with sufficiently fast implementations, permits live cell imaging. Image reconstruction, however, currently relies on sophisticated computational procedures, susceptible to reconstruction artifacts, requiring trained users to recognize and avoid them. Here, we review the latest developments in SR-SIM research. Starting from a historical overview of the development of SR-SIM, we review how this method can be implemented in various experimental schemes, we provide an overview of the important parameters involved in successful image re...
309 citations
TL;DR: A deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM), which attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities.
Abstract: To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.
287 citations
TL;DR: It is proposed that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.
Abstract: While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.
DOI: http://dx.doi.org/10.7554/eLife.11553.001
231 citations
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...Tom20-mCherry was a gift from Andrew G. York (NIH, Bethesda, MD) and described in York et al. (2013)....
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References
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TL;DR: An iterative method of restoring degraded images was developed by treating images, point spread functions, and degraded images as probability-frequency functions and by applying Bayes’s theorem.
Abstract: An iterative method of restoring degraded images was developed by treating images, point spread functions, and degraded images as probability-frequency functions and by applying Bayes’s theorem. The method functions effectively in the presence of noise and is adaptable to computer operation.
3,869 citations
TL;DR: In this article, the authors consider the problem of determining the distribution of proper motions in a line-of-sight line of sight (LoSOS) image from a given number count.
Abstract: J q,•.=I:Pi,Vt;, i-I f \"'(~)d~=1 and \"'W~O of the variation of star density along a line-of-sight from the distribution of proper motions in that direction; (2) the determination of the space distribution of radio sources from number counts; (3) the determination of the radial variation of star density in a globular cluster from star counts; (4) the correction of radioastronomical and spectrographic observations for the effect of the instrumental profile; and (5) the determination of the temperature stratification in the solar atmosphere from limb-darkening data. These examples suffice to show that the problem under consideration arises in many branches of astronomy and that its solution is vital to the process of extracting useful information from observations.
3,670 citations
TL;DR: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide‐field fluorescence microscope with strikingly increased clarity compared to both conventional and confocal microscopes.
Abstract: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.
3,274 citations
Journal Article•
TL;DR: Python is an excellent "steering" language for scientific codes written in other languages, but with additional basic tools, it transforms into a high-level language suited for scientific and engineering code that's often fastenough to be immediately useful but also flexible enough to be sped up with additional extensions.
Abstract: Python is an excellent "steering" language for scientific codes written in other languages. However, with additional basic tools, Python transforms into a high-level language suited for scientific and engineering code that's often fast enough to be immediately useful but also flexible enough to be sped up with additional extensions.
2,841 citations
TL;DR: Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
Abstract: In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
2,730 citations