Diffraction Limit Of Light Microscopy Research Articles

Correlative Stimulated Emission Depletion and Scanning Ion Conductance Microscopy.

Correlation microscopy combining fluorescence and scanning probe or electron microscopy is limited to fixed samples due to the sample preparation and nonphysiological imaging conditions required by most probe or electron microscopy techniques. Among the few scanning probe techniques that allow imaging of living cells under physiological conditions, scanning ion conductance microscopy (SICM) has been shown to be the technique that minimizes the impact on the investigated sample. However, combinations of SICM and fluorescence microscopy suffered from the mismatch in resolution due to the limited resolution of conventional light microscopy. In the last years, the diffraction limit of light microscopy has been circumvented by various techniques, one of which is stimulated emission depletion (STED) microscopy. Here, we aimed at demonstrating the combination of STED and SICM. We show that both methods allow recording a living cellular specimen and provide a SICM and STED image of the same sample, which allowed us to correlate the membrane surface topography and the distribution of the cytoskeletal protein actin. Our proof-of-concept study exemplifies the benefit of correlating SICM with a subdiffraction fluorescence method and might form the basis for the development of a combined instrument that would allow the simultaneous recording of subdiffraction fluorescence and topography information.

Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules.

Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.

SRpHi ratiometric pH biosensors for super-resolution microscopy

Fluorescence-based biosensors have become essential tools for modern biology, allowing real-time monitoring of biological processes within living cells. Intracellular fluorescent pH probes comprise one of the most widely used families of biosensors in microscopy. One key application of pH probes has been to monitor the acidification of vesicles during endocytosis, an essential function that aids in cargo sorting and degradation. Prior to the development of super-resolution fluorescence microscopy (nanoscopy), investigation of endosomal dynamics in live cells remained difficult as these structures lie at or below the ~250 nm diffraction limit of light microscopy. Therefore, to aid in investigations of pH dynamics during endocytosis at the nanoscale, we have specifically designed a family of ratiometric endosomal pH probes for use in live-cell STED nanoscopy.

Land-locked mammalian Golgi reveals cargo transport between stable cisternae

The Golgi is composed of a stack of cis, medial, trans cisternae that are biochemically distinct. The stable compartments model postulates that permanent cisternae communicate through bi-directional vesicles, while the cisternal maturation model postulates that transient cisternae biochemically mature to ensure anterograde transport. Testing either model has been constrained by the diffraction limit of light microscopy, as the cisternae are only 10–20 nm thick and closely stacked in mammalian cells. We previously described the unstacking of Golgi by the ectopic adhesion of Golgi cisternae to mitochondria. Here, we show that cargo processing and transport continue—even when individual Golgi cisternae are separated and “land-locked” between mitochondria. With the increased spatial separation of cisternae, we show using three-dimensional live imaging that cis-Golgi and trans-Golgi remain stable in their composition and size. Hence, we provide new evidence in support of the stable compartments model in mammalian cells.

Four-Channel Super-Resolution Imaging by 3-D Structured Illumination.

Multichannel imaging is used as a readout of relative localization of two or more components and is often the first step in investigating functional ensembles in cells. However, the localization volume of diffraction-limited light microscopy (approx. 200nm by 500nm) can accommodate hundred of proteins, calling for increased resolution for these types of analyses. Here, we present a protocol for 4-channel imaging using structured illumination microscopy (SIM), which increases resolution by a factor of two. We use adherent, fixed cells to identify the localization of adhesion proteins using immunofluorescence and fluorescent proteins. We discuss how labeling with the necessary brightness is achieved and how data has to be processed for colocalization analysis.

Optical and force nanoscopy in microbiology.

Microbial cells have developed sophisticated multicomponent structures and machineries to govern basic cellular processes, such as chromosome segregation, gene expression, cell division, mechanosensing, cell adhesion and biofilm formation. Because of the small cell sizes, subcellular structures have long been difficult to visualize using diffraction-limited light microscopy. During the last three decades, optical and force nanoscopy techniques have been developed to probe intracellular and extracellular structures with unprecedented resolutions, enabling researchers to study their organization, dynamics and interactions in individual cells, at the single-molecule level, from the inside out, and all the way up to cell-cell interactions in microbial communities. In this Review, we discuss the principles, advantages and limitations of the main optical and force nanoscopy techniques available in microbiology, and we highlight some outstanding questions that these new tools may help to answer.

Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles.

Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)-pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.

Correlative Light and Electron Microscopy II, Volume 124 (Methods in Cell Biology) Thomas Müller-Reichert and Paul Verkade (Eds.). Academic Press. Cambridge, MA, 2014, 452 pp. ISBN: 978-0128010754.

A picture is worth a thousand words. Correlative light and electron microscopy (CLEM) is a valuable imaging tool as it bridges the resolution gap between the two modalities. Fluorescent markers are applied routinely to capture a broad field of view in two-dimensional space (2D), 3D, or 4D. In diffraction-limited light microscopy (LM), resolution approaches 200 nm. Alternatively, the physical properties of an electron beam allow an improvement in resolution approaching 1 nm for the visualization of ultrastructural features, including the surrounding architecture of some feature of interest. It follows that CLEM is especially powerful. Correlative Light and Electron Microscopy II, Volume 124 (Methods in Cell Biology) expands on techniques and applications introduced in Correlative Light and Electron Microscopy, Volume 111 (Methods in Cell Biology). The use of CLEM has wide applicability and can benefit many studies. For example, it can be used to directly identify a small organelle, thus bridging the resolution gap between LM and electron microscopy (EM). It can also be used to identify rare events or a region of interest for subsequent ultrastructural analysis, such as the dynamics of a vesicle being trafficked throughout the cell.

Super-resolved insights into human immunodeficiency virus biology.

The recent development of fluorescence microscopy approaches overcoming the diffraction limit of light microscopy opened possibilities for studying small-scale cellular processes. The spatial resolution achieved by these novel techniques, together with the possibility to perform live-cell and multicolor imaging, make them ideally suited for visualization of native viruses and subviral structures within the complex environment of a host cell or organ, thus providing fundamentally new possibilities for investigating virus-cell interactions. Here, we review the use of super-resolution microscopy approaches to study virus-cell interactions, and discuss recent insights into human immunodeficiency virus biology obtained by exploiting these novel techniques.

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope.

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.

Protein clustering and spatial organization in T-cells.

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.

Applying superresolution localization-based microscopy to neurons.

Proper brain function requires the precise localization of proteins and signaling molecules on a nanometer scale. The examination of molecular organization at this scale has been difficult in part because it is beyond the reach of conventional, diffraction-limited light microscopy. The recently developed method of superresolution, localization-based fluorescent microscopy (LBM), such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), has demonstrated a resolving power at a 10 nm scale and is poised to become a vital tool in modern neuroscience research. Indeed, LBM has revealed previously unknown cellular architectures and organizational principles in neurons. Here, we discuss the principles of LBM, its current applications in neuroscience, and the challenges that must be met before its full potential is achieved. We also present the unpublished results of our own experiments to establish a sample preparation procedure for applying LBM to study brain tissue.

Correlative multicolor 3D SIM and STORM microscopy.

Within the last decade, super-resolution methods that surpass the diffraction limit of light microscopy have provided invaluable insight into a variety of biological questions. Each of these approaches has inherent advantages and limitations, such that their combination is a powerful means to transform them into versatile tools for the life sciences. Here, we report the development of a combined SIM and STORM setup that maintains the optimal resolution of both methods and which is coupled to image registration to localize biological structures in 3D using multicolor labeling. We utilized this workflow to determine the localization of Bld12p/CrSAS-6 in purified basal bodies of Chlamydomonas reinhardtii with utmost precision, demonstrating its usefulness for accurate molecular mapping in 3D.

Photocontrollable Fluorescent Proteins for Superresolution Imaging

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization-based and nonlinear ensemble-based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine.

The Pre/Post LTP Debate

The pre/post debate involves the question of whether long-term potentiation (LTP) is mediated by enhancement of release, enhancement of postsynaptic receptors, or both. Recent papers have presented evidence for purely postsynaptic or purely presynaptic changes, and a paper by Ahmed and Siegelbam (in this issue of Neuron) suggests a mechanism by which release is enhanced. This debate is increasingly constrained by technical advances that allow central synapses to be studied with increasing precision. A possible of way of reconciling conflicting evidence is suggested.

Properties of coherence-gated wavefront sensing

Coherence-gated wavefront sensing (CGWS) allows the determination of wavefront aberrations in strongly scattering tissue and their correction by adaptive optics. This allows, e.g., the restoration of the diffraction limit in light microscopy. Here, we develop a model, based on ray tracing of ballistic light scattered from a set of discrete scatterers, to characterize CGWS performance as it depends on coherence length, scatterer density, coherence-gate position, and polarization. The model is evaluated by using Monte Carlo simulation and verified against experimental measurements. We show, in particular, that all aberrations needed for adaptive wavefront restoration are correctly sensed if circularly polarized light is used.

Heterogeneous Molecular Distribution in Supported Multicomponent Lipid Bilayers

Membrane domains contribute important structural and functional attributes to biological membranes. We describe the heterogeneous nanoscale distribution of lipid molecules within microscale membrane domains in multicomponent lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). The lipids were labeled with the fluorescent lipid analogues Bodipy-PC and DiI-C20:0 to identify the distribution of individual membrane components. We used a near-field scanning optical microscope (NSOM) at room temperature to identify the nanoscale structures in the membrane. Simultaneous multicolor NSOM imaging at the emission maxima of the fluorescent analogues revealed a patchy distribution of Bodipy-PC and DiI-C20:0 indicative of phase separations in the bilayer. In a cholesterol-free system (DPPC/DLPC = 1:1), NSOM images proved that the two phosphatidylcholine molecules can coexist in domains at the micrometer level but form nanoscopic patches within the domains; DPPC occurs at the edge of the domains, whereas DLPC is present throughout the domains. In the presence of cholesterol (DPPC/DLPC = 7:3, chol = 18.9%), the two lipid molecules were more miscible but incomplete phase separations also occurred. The average domain sizes were 140-200 nm, well below the resolution capabilities of diffraction-limited light microscopy techniques; the domains were unresolvable by confocal microscopy. Our high-resolution NSOM studies of membrane domain behavior provide a better understanding of complex membrane phase phenomena in multicomponent biological membranes.

FRET: A Spectral Ruler for Interacting Molecules Involved in Apoptosis

Abstract Fluorescence Resonance Energy Transfer (FRET) is a process by which a fluorophore (the donor) in the excited state transfers its energy to a neighboring fluorophore (the acceptor) non-radiatively through dipole-dipole interactions. Since the efficiency of energy transfer varies as the inverse of the sixth power of the distance separating the donor and acceptor chromophores, for FRET to occur the distance between the two molecules cannot exceed 10 to 100 angstroms (1 to l0nm). The combination of FRET and optical microscopy allows examination and quantitation of dynamic molecular interactions between cellular constituents at resolutions beyond the Abbe diffraction limit of light microscopy. Through the microscope one may detect FRET by an overall decrease in fluorescence emission of the donor with a concomitant increase in fluorescence emission of the acceptor.