Showing papers on "Photoactivated localization microscopy published in 2014"

Precisely and accurately localizing single emitters in fluorescence microscopy

Abstract: Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing number of researchers using single-particle tracking and super-resolution localization microscopy.

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Abstract: Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.

Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography.

Abstract: Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.

Eight years of single-molecule localization microscopy

Abstract: Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.

The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging.

Abstract: Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.

Multicolor two-photon light-sheet microscopy.

Abstract: Two-photon microscopy is the most effective approach for deep-tissue fluorescence cellular imaging; however, its application to high-throughput or high-content imaging is often hampered by low pixel rates, challenging multicolor excitation and potential cumulative photodamage. To overcome these limitations, we extended our prior work and combined two-photon scanned light-sheet...

Superresolution imaging of biological systems using photoactivated localization microscopy.

Abstract: A progressive reduction of the spatial scale accessible by microscopes has catalyzed our increasing understanding of cells and their constituents. Light microscopy has been at the forefront of this journey, beginning in the 1600s with the first observation of cells and bacteria using a simple lens. With the advent of phase contrast and fluorescence microscopy, a second phase of visualization into the micron-scale world began, with researchers now able to discern membrane-bounded organelles and cytoskeletal elements. It was not until the advent of genetically encoded fluorescent proteins (FPs),1-4 however, that biologists could begin imaging the constituents of these structures. This marked a third phase of light-based, biological visualization. Still, because cellular structures closer together than ∼200 nm could not be resolved using these techniques due to the diffraction limit of light,5 light microscopy could not yet cross into the nanoscopic world. Now, this frontier is finally being crossed. Recently developed light-based superresolution (SR) techniques are allowing imaging of biological structures with spatial resolutions more than an order of magnitude finer than conventional optical microscopes. This is being achieved in two ways: by spatially modulating the excitation radiation, as used by stimulated emission depletion (STED) microscopy 6 and structured illumination microscopy (SIM),7,8 and by temporally modulating the emission of individual fluorescent molecules, as used in photoactivated localization microscopy (PALM),9,10 stochastic optical reconstruction microscopy (STORM),11 and related point-localization SR imaging approaches.12,13 In this review, we discuss the second of these SR imaging techniques, focusing particularly on PALM along with some illustrative applications. PALM relies on accurate localization of single FPs based on temporal isolation of single molecule emission, combining this precise positional information to reconstruct superresolution images. Because PALM employs genetically-encoded, photo-controllable FPs to localize single molecules 14-17, it has broad applicability for investigating the spatial organization and motion of diverse types of proteins associated with various structures and environments inside cells and tissues. Recent applications of PALM, with other point-localization SR techniques, are stimulating new testable hypothesis, refining the prevailing conceptual frameworks, and extending our understanding of mechanistic principles in biology at the nanoscale.

Light sheet microscopy.

Abstract: This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation. Based on our experience over the last 10 years, we summarize the key concepts in light sheet microscopy, typical implementations, and successful applications. In particular, sample mounting for long time-lapse imaging and the resulting challenges in data processing are discussed in detail.

Progress in quantitative single-molecule localization microscopy

Abstract: With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM).

Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) for nanoscale imaging of protein-protein interactions in cells.

Abstract: Bimolecular fluorescence complementation (BiFC) has been widely used to visualize protein-protein interactions (PPIs) in cells. Until now, however, the resolution of BiFC has been limited by the diffraction of light to ∼250 nm, much larger than the nanometer scale at which PPIs occur or are regulated. Cellular imaging at the nanometer scale has recently been realized with single molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM). Here we have combined BiFC with PALM to visualize PPIs inside cells with nanometer spatial resolution and single molecule sensitivity. We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments. PAmCherry1 BiFC exhibits high specificity and high efficiency even at 37°C in detecting PPIs with virtually no background from spontaneous reconstitution. Moreover, the reconstituted protein maintains the fast photoconversion, high contrast ratio, and single molecule brightness of the parent PAmCherry1, which enables selective PALM localization of PPIs with ∼18 nm spatial precision. With BiFC-PALM, we studied the interactions between the small GTPase Ras and its downstream effector Raf, and clearly observed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD (Ras-binding domain) complexes on the cell membrane. These observations provided novel insights into the regulation of Ras/Raf interaction at the molecular scale, which would be difficult with other techniques such as conventional BiFC, fluorescence co-localization or FRET.

Increasing the Brightness of Cyanine Fluorophores for Single‐Molecule and Superresolution Imaging

Abstract: In spite of their relatively low fluorescence quantum yield, cyanine dyes such as Cy3, Cy5, or Cy7 are widely used in single-molecule fluorescence applications due to their high extinction coefficients and excellent photon yields. We show that the fluorescence quantum yield and lifetime of red-emitting cyanine dyes can be substantially increased in heavy water (D2 O) compared with water (H2 O). We find that the magnitude of the quantum yield increase in D2 O scales with the emission wavelength, reaching a particularly high value of 2.6-fold for the most red-emitting dye investigated, Cy7. We further demonstrate a higher photon yield in single-molecule superresolution experiments in D2 O compared to H2 O, which leads to an improved localization precision and hence better spatial resolution. This finding is especially beneficial for biological applications of fluorescence microscopy, which are typically carried out in aqueous media and which greatly profit from the red spectral range due to reduced cellular auto-fluorescence.

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant

Abstract: We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slow-switching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.

Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging.

Abstract: Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.

3D superresolution microscopy by supercritical angle detection

Abstract: We present a fundamentally new approach to 3D superresolution microscopy based on the principle of surface-generated fluorescence. This near-field fluorescence is strongly dependent on the distance of fluorophores from the coverslip and can therefore be used to estimate their axial positions. We established a robust and simple implementation of supercritical angle fluorescence detection for single-molecule localization microscopy, calibrated it using fluorescent bead samples, validated the method with DNA origami tetrahedra, and present proof-of-principle data on biological samples.

Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.

Abstract: Many biomolecules in cells can be visualized with high sensitivity and specificity by fluorescence microscopy. However, the resolution of conventional light microscopy is limited by diffraction to ~200-250 nm laterally and >500 nm axially. Here, we describe superresolution methods based on single-molecule localization analysis of photoswitchable fluorophores (PALM: photoactivated localization microscopy) as well as our recent three-dimensional (3D) method (iPALM: interferometric PALM) that allows imaging with a resolution better than 20 nm in all three dimensions. Considerations for their implementations, applications to multicolor imaging, and a recent development that extend the imaging depth of iPALM to ~750 nm are discussed. As the spatial resolution of superresolution fluorescence microscopy converges with that of electron microscopy (EM), direct imaging of the same specimen using both approaches becomes feasible. This could be particularly useful for cross validation of experiments, and thus, we also describe recent methods that were developed for correlative superresolution fluorescence and EM.

Super-resolution Microscopy: A Comparison of Commercially Available Options

Abstract: Although fluorescence microscopy has had a major impact on biomedical research, the resolution barrier inherent in light microscopy restricts the ability to differentiate between objects closer together than ∼250 nm and prevents the true sizing of structures smaller than this limit. Recent innovations have led to the development of three main commercially available options for super-resolution microscopy that effectively break this diffraction limit: structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM)/photoactivation localization microscopy (PALM), and stimulated emission depletion microscopy (STED). The goal of this chapter is to describe the physical basis for these techniques as well as practical information for each, to provide the potential user with a basis for comparison and determination of the optimal choice for specific applications. Finally, innovative variations of these techniques for particular biological studies, as well as descriptions of new alternative techniques are presented.

A Blueprint for Cost‐Efficient Localization Microscopy

Abstract: Crystal clear: The authors introduce a miniaturized localization microscopy setup based on cost-effective components. They demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.

A Single-Photon Avalanche Camera for Fluorescence Lifetime Imaging Microscopy and Correlation Spectroscopy

Abstract: Confocal laser scanning microscopy (CLSM) is commonly used to observe molecules of biological relevance in their native environment, the live cell, and study their spatial distribution and interactions nondestructively. CLSM can be easily extended to measure the lifetime of the excited state, the concentration and the diffusion properties of fluorescently labeled molecules, using fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), respectively, in order to provide information about the local environment and the kinetics of molecular interaction in live cells. However, these parameters cannot be measured simultaneously using conventional CLSM due to damaging effects that are associated with strong illumination, including phototoxicity, photobleaching, and saturation of the fluorescence signal. To overcome these limitations, we have developed a new camera consisting of 1024 single-photon avalanche diodes that is optimized for multifocal microscopy, FLIM and FCS. We show proof-of-principle measurements of fluorescence intensity distribution and lifetime of the enhanced green fluorescent protein expressed in live cells and measurement of quantum dot diffusion in solution by FCS using the same detector.

Time-Resolved Emission Imaging Microscopy Using Phosphorescent Metal Complexes: Taking FLIM and PLIM to New Lengths

Abstract: Luminescent metal complexes are increasingly being investigated as emissive probes and sensors for cell imaging using what is traditionally termed fluorescence microscopy. The nature of the emission in the case of second- and third-row metal complexes is phosphorescence rather than fluorescence, as it emanates from triplet rather than singlet excited states, but the usual terminology overlooks the distinction between the quantum mechanical origins of the processes. In steady-state imaging, such metal complexes may be alternatives to widely used fluorescent organic molecules, used in exactly the same way but offering advantages such as ease of synthesis and colour tuning. However, there is a striking difference compared to fluorescent organic molecules, namely the much longer lifetime of phosphorescence compared to fluorescence. Phosphorescence lifetimes of metal complexes are typically around a microsecond compared to the nanosecond values found for fluorescence of organic molecules. In this contribution, we will discuss how these long lifetimes can be put to practical use. Applications such as time-gated imaging allow discrimination from background fluorescence in cells and tissues, while increased sensitivity to quenchers provides a means of designing more responsive probes, for example, for oxygen. We also describe how the technique of fluorescence lifetime imaging microscopy (FLIM) – which provides images based on lifetimes at different points in the image – can be extended from the usual nanosecond range to microseconds. Key developments in instrumentation as well as the properties of complexes suitable for the purpose are discussed, including the use of two-photon excitation methods. A number of different research groups have made pioneering contributions to the instrumental set-ups, but the terminology and acronyms have not developed in a systematic way. We review the distinction between time-gating (to eliminate background emission) and true time-resolved imaging (whereby decay kinetics at each point in an image are monitored). For instance, terms such as PLIM (phosphorescence lifetime imaging microscopy) and TRLM (time-resolved luminescence microscopy) refer essentially to the same technique, whilst TREM (time-resolved emission imaging microscopy) embraces these long timescale methods as well as the more well-established technique of FLIM.

Bending the Rules: Widefield Microscopy and the Abbe Limit of Resolution

Abstract: One of the most fundamental concepts of microscopy is that of resolution-the ability to clearly distinguish two objects as separate. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. These techniques, however, are not always feasible or optimal for live cell imaging. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope-deconvolution, model convolution, and Gaussian fitting. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy.

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Abstract: Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Fluorescence imaging of nanoscale domains in polymer blends using stochastic optical reconstruction microscopy (STORM).

Abstract: High-resolution fluorescence techniques that provide spatial resolution below the diffraction limit are attractive new methods for structural characterization of nanostructured materials. For the first time, we apply the super-resolution technique of Stochastic Optical Reconstruction Microscopy (STORM), to characterize nanoscale structures within polymer blend films. The STORM technique involves temporally separating the fluorescence signals from individual labeled polymers, allowing their positions to be localized with high accuracy, yielding a high-resolution composite image of the material. Here, we describe the application of the technique to demixed blend films of polystyrene (PS) and poly(methyl methacrylate) (PMMA), and find that STORM provides comparable structural characteristics as those determined by Atomic Force Microscopy (AFM) and scanning electron microscopy (SEM), but with all of the advantages of a far-field optical technique.

Axial nanoscale localization by normalized total internal reflection fluorescence microscopy.

Abstract: We present a simple modification of a standard total internal reflection fluorescence microscope to achieve nanometric axial resolution, typically ≈10 nm. The technique is based on a normalization of total internal reflection images by conventional epi-illumination images. We demonstrate the potential of our method to study the adhesion of phopholipid giant unilamellar vesicles.

Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM).

Abstract: Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Nanoprobes for super-resolution fluorescence imaging at the nanoscale

Abstract: Compared with other imaging techniques, fluorescence microscopy has become an essential tool to study cell biology due to its high compatibility with living cells. Owing to the resolution limit set by the diffraction of light, fluorescence microscopy could not resolve the nanostructures in the range of < 200 nm. Recently, many techniques have been emerged to overcome the diffraction barrier, providing nanometer spatial resolution. In the course of development, the progress in fluorescent probes has helped to promote the development of the high-resolution fluorescence nanoscopy. Here, we describe the contributions of the fluorescent probes to far-field super resolution imaging, focusing on concepts of the existing super-resolution nanoscopy based on the photophysics of fluorescent nanoprobes, like photoswitching, bleaching and blinking. Fluorescent probe technology is crucial in the design and implementation of super-resolution imaging methods.