Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.     

Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.     

Near-isotropic 3D optical nanoscopy with photon-limited chromophores

Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution.     

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40?nm in 3D) over a large field of view (12?12?μm).     

Widely accessible method for superresolution fluorescence imaging of living systems

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.     

Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution

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 arises from saturation of the excited state. This method can be used 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.     

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.Fluorescence microscopy is an extremely powerful research tool in the life sciences. It combines highest sensitivity with molecular specificity and exceptional image contrast. However, as with all light-based microscopy techniques, its resolution is limited by the diffraction of light to a typical lateral resolution of ∼200 nm and an axial resolution of ∼500 nm (for 500-nm wavelength light). Only recently, this diffraction limit was broken by using the quantum, or nonlinear, character of fluorescence excitation and emission. The first of these superresolution methods was stimulated emission depletion (STED) microscopy (1). Later, methods based on single-molecule localization, such as photoactivated localization microscopy (PALM) (2) and stochastic optical reconstruction microscopy (STORM) (3), joined the field. These methods “break” the diffraction limit because they all use principles that operate beyond the diffraction of light.Although still bound to light diffraction, increased spatial resolution can be achieved in a class of advanced resolution methods that exploit a clever combination of excitation and detection modalities (4–7). Although these methods do not reach the resolution achievable with STED, PALM, STORM, and related techniques, they do not require any specialized labels or high excitation intensities, and they may be applied to any fluorescent sample at any excitation/emission wavelength. The most prominent example of this class is structured illumination microscopy (SIM) (5), in which one scans a sample with a structured illumination pattern while taking images with a wide-field imaging system. Meanwhile, several commercial instruments for SIM have become available. The disadvantages of SIM are its technical complexity, reflected in the rather large cost of the commercially available systems, and its sensitivity to optical imperfections and aberrations, which are unavoidable in biological samples.In a theoretical study in 1988, Sheppard (8) pointed out that it is possible to double the resolution of a scanning confocal microscope in a manner closely related to SIM. In SIM, one starts with a conventional wide-field imaging microscope, and by implementing a scanning structured illumination, one subsequently obtains, after appropriate deconvolution of the recorded images, an image with increased resolution. In image-scanning microscopy (ISM), as proposed by Sheppard, one starts with a conventional confocal microscope that uses a diffraction-limited laser focus for scanning a sample but replaces the point detector typically used for recording the excited fluorescence signal with an imaging detector. Also here, an image with enhanced resolution is obtained after applying an appropriate algorithm to the recorded images.We experimentally realized this idea first in 2010 (4), indeed demonstrating a substantial increase in resolution. The major drawback of this implementation was the slowness of the imaging. At each scan position of the laser focus, an image of the excited region had to be recorded, limiting the scan speed by the frame rate of the imaging camera used. For the small scan area of 2 µm × 2 µm shown with the original ISM setup, data acquisition took 25 s. In 2012, York et al. (6) demonstrated that this limitation may be overcome by using a multifocal excitation scheme. They generated an array of multiple excitation foci by implementing a digital micro-mirror device (DMD) into the excitation path of a wide-field microscope. Using this system, ISM images can be obtained with excellent speed, in two excitation/emission wavelengths (two-color imaging) and in three dimensions. However, this approach requires the incorporation of a DMD with all the necessity of perfect optical alignment.Here, we demonstrate that existing imaging detector-based confocal systems can be converted easily into a doubly resolving ISM system. This mainly includes two kinds of microscopes that are widely available in research laboratories: confocal spinning-disk (CSD) microscopes and rapid laser scanning confocal microscopes with an imaging camera as the detector. We present the results obtained with a CSD system.     

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.     

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.     

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.     

Quantitative biological imaging by ptychographic x-ray diffraction microscopy

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3·10⁵ Gy, and were shown to scale with photon fluence.     

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffraction-limited resolution of conventional light microscopy (∼200–300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.     

Biological imaging by soft x-ray diffraction microscopy

We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffraction microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.     

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.     

Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast

By introducing beam-scanning multifocal multiphoton 4Pi-confocal microscopy, we have attained fast fluorescence imaging of live cells with axial super resolution. Rapid scanning of up to 64 pairs of interfering high-angle fields and subsequent confocal detection enabled us to perform three to five times finer optical sectioning than confocal microscopy. In conjunction with nonlinear image restoration, we demonstrate, to our knowledge for the first time, three-dimensional imaging of live eukaryotic cells at an equilateral resolution of approximately 100 nm. This imaging mode allowed us to reveal the morphology and size of the green fluorescent protein-labeled mitochondrial compartment of live Saccharomyces cerevisiae (bakers' yeast) growing on different carbon sources. Our studies show that mitochondria of cells grown on medium containing glycerol as the only carbon source, as opposed to glucose-grown cells, exhibit a strongly branched tubular reticulum. We determine the average tubular diameter and find that it increases from 339 +/- 5 nm to 360 +/- 4 nm when changing from glucose to glycerol, that is, from a fermentable to a nonfermentable carbon source. Moreover, this change is associated with a 2.8-fold increase of the surface of the reticulum, resulting in an average increase in volume of the mitochondrial compartment by a factor of 3.0 +/- 0.2.     

High numerical aperture tabletop soft x-ray diffraction microscopy with 70-nm resolution

Light microscopy has greatly advanced our understanding of nature. The achievable resolution, however, is limited by optical wavelengths to approximately 200 nm. By using imaging and labeling technologies, resolutions beyond the diffraction limit can be achieved for specialized specimens with techniques such as near-field scanning optical microscopy, stimulated emission depletion microscopy, and photoactivated localization microscopy. Here, we report a versatile soft x-ray diffraction microscope with 70- to 90-nm resolution by using two different tabletop coherent soft x-ray sources-a soft x-ray laser and a high-harmonic source. We also use field curvature correction that allows high numerical aperture imaging and near-diffraction-limited resolution of 1.5lambda. A tabletop soft x-ray diffraction microscope should find broad applications in biology, nanoscience, and materials science because of its simple optical design, high resolution, large depth of field, 3D imaging capability, scalability to shorter wavelengths, and ultrafast temporal resolution.     

Macromolecular-scale resolution in biological fluorescence microscopy

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximately 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.     

Superresolution Imaging of Human Cytomegalovirus vMIA Localization in Sub-Mitochondrial Compartments

The human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, traffics to mitochondria-associated membranes (MAM), where the endoplasmic reticulum (ER) contacts the outer mitochondrial membrane (OMM). vMIA association with the MAM has not been visualized by imaging. Here, we have visualized this by using a combination of confocal and superresolution imaging. Deconvolution of confocal microscopy images shows vMIA localizes away from mitochondrial matrix at the Mitochondria-ER interface. By gated stimulated emission depletion (GSTED) imaging, we show that along this interface vMIA is distributed in clusters. Through multicolor, multifocal structured illumination microscopy (MSIM), we find vMIA clusters localize away from MitoTracker Red, indicating its OMM localization. GSTED and MSIM imaging show vMIA exists in clusters of ~100–150 nm, which is consistent with the cluster size determined by Photoactivated Localization Microscopy (PALM). With these diverse superresolution approaches, we have imaged the clustered distribution of vMIA at the OMM adjacent to the ER. Our findings directly compare the relative advantages of each of these superresolution imaging modalities for imaging components of the MAM and sub-mitochondrial compartments. These studies establish the ability of superresolution imaging to provide valuable insight into viral protein location, particularly in the sub-mitochondrial compartments, and into their clustered organization.     

Simultaneous atomic force microscope and fluorescence measurements of protein unfolding using a calibrated evanescent wave

Fluorescence techniques for monitoring single-molecule dynamics in the vertical dimension currently do not exist. Here we use an atomic force microscope to calibrate the distance-dependent intensity decay of an evanescent wave. The measured evanescent wave transfer function was then used to convert the vertical motions of a fluorescent particle into displacement (SD = < 1 nm). We demonstrate the use of the calibrated evanescent wave to resolve the 20.1 +/- 0.5-nm step increases in the length of the small protein ubiquitin during forced unfolding. The experiments that we report here make an important contribution to fluorescence microscopy by demonstrating the unambiguous optical tracking of a single molecule with a resolution comparable to that of an atomic force microscope.     

A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications

Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.