Reaching the information limit in cryo-EM of biological macromolecules: experimental aspects

Although cryo-electron microscopy (cryo-EM) of biological macromolecules has made important advances in the past few years, the level of current technical performance is still well below what the physics of electron scattering would allow. It should be possible, for example, to use cryo-EM to solve protein structures at atomic resolution for particle sizes well below 80 kDa, but currently this has been achieved only for particles at least 10 times larger than that. In this review, we first examine some of the reasons for this large gap in performance. We then give an overview of work that is currently in progress to 1), improve the signal/noise ratio for area detectors; 2), improve the signal transfer between the scattered electrons and the corresponding images; and 3), reduce the extent to which beam-induced movement causes a steep fall-off of signal at high resolution. In each case, there is substantial reason to think that cryo-EM can indeed be made to approach the estimated physical limits.     

CTF determination and correction for low dose tomographic tilt series

The resolution of cryo-electron tomography can be limited by the first zero of the microscope’s contrast transfer function (CTF). To achieve higher resolution, it is critical to determine the CTF and correct its phase inversions. However, the extremely low signal-to-noise ratio (SNR) and the defocus gradient in the projections of tilted specimens make this process challenging. Two programs, CTFPLOTTER and CTFPHASEFLIP, have been developed to address these issues. CTFPLOTTER obtains a 1D power spectrum by periodogram averaging and rotational averaging and it estimates the noise background with a novel approach, which uses images taken with no specimen. The background-subtracted 1D power spectra from image regions at different defocus values are then shifted to align their first zeros and averaged together. This averaging improves the SNR sufficiently that it becomes possible to determine the defocus for subsets of the tilt series rather than just the entire series. CTFPHASEFLIP corrects images line-by-line by inverting phases appropriately in thin strips of the image at nearly constant defocus. CTF correction by these methods is shown to improve the resolution of aligned, averaged particles extracted from tomograms. However, some restoration of Fourier amplitudes at high frequencies is important for seeing the benefits from CTF correction.     

Beam-induced motion of vitrified specimen on holey carbon film

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.     

A dose-rate effect in single-particle electron microscopy

A low beam intensity, low electron dose imaging method has been developed for single-particle electron cryo-microscopy (cryo-EM). Experiments indicate that the new technique can reduce beam-induced specimen movement and secondary radiolytic effects, such as "bubbling". The improvement in image quality, especially for multiple-exposure data collection, will help single-particle cryo-EM to reach higher resolution.     

TomoAlign: A novel approach to correcting sample motion and 3D CTF in CryoET

TomoAlign is a software package that integrates tools to mitigate two important resolution limiting factors in cryoET, namely the beam-induced sample motion and the contrast transfer function (CTF) of the microscope. The package is especially focused on cryoET of thick specimens where fiducial markers are required for accurate tilt-series alignment and sample motion estimation. TomoAlign models the beam-induced sample motion undergone during the tilt-series acquisition. The motion models are used to produce motion-corrected subtilt-series centered on the particles of interest. In addition, the defocus of each particle at each tilt image is determined and can be corrected, resulting in motion-corrected and CTF-corrected subtilt-series from which the subtomograms can be computed. Alternatively, the CTF information can be passed on so that CTF correction can be carried out entirely within external packages like Relion. TomoAlign serves as a versatile tool that can streamline the cryoET workflow from initial alignment of tilt-series to final subtomogram averaging during in situ structure determination.     

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.     

Electron cryo-microscopy of vitrified biological specimens: towards high spatial and temporal resolution

A decade after the development of electron cryo-microscopy for vitrified specimens, its advantages and limitations are analysed. Indeed, recent work carried out by different laboratories strengthens the idea that electron cryo-microscopy might soon be an alternative method to X-ray crystallography and NMR techniques for determining the structure of biological assemblies with both high spatial and temporal resolutions. High pressure freezing allows vitrification of larger volumes of biological suspensions. Thick vitrified objects can be cryosectioned. Electron cryo-microscopy of the sections gives images having a resolution better than 2 nm. Although the high resolution imaging mode under low dose conditions is not yet fully understood, microscopes are being developed to provide better and better images. Image averaging is being facilitated by the development of both crystallization and computer methods. Thus, we can expect that electron microscopy will soon become a potential technique for structural determination at atomic resolution. Finally, much effort is being devoted to improving the temporal resolution of electron cryo-microscopy. Soon, we may be able to observe molecules during their biological activity.     

Classification and 3D averaging with missing wedge correction in biological electron tomography

Strategies for the determination of 3D structures of biological macromolecules using electron crystallography and single-particle electron microscopy utilize powerful tools for the averaging of information obtained from 2D projection images of structurally homogeneous specimens. In contrast, electron tomographic approaches have often been used to study the 3D structures of heterogeneous, one-of-a-kind objects such as whole cells where image-averaging strategies are not applicable. Complex entities such as cells and viruses, nevertheless, contain multiple copies of numerous macromolecules that can individually be subjected to 3D averaging. Here we present a complete framework for alignment, classification, and averaging of volumes derived by electron tomography that is computationally efficient and effectively accounts for the missing wedge that is inherent to limited-angle electron tomography. Modeling the missing data as a multiplying mask in reciprocal space we show that the effect of the missing wedge can be accounted for seamlessly in all alignment and classification operations. We solve the alignment problem using the convolution theorem in harmonic analysis, thus eliminating the need for approaches that require exhaustive angular search, and adopt an iterative approach to alignment and classification that does not require the use of external references. We demonstrate that our method can be successfully applied for 3D classification and averaging of phantom volumes as well as experimentally obtained tomograms of GroEL where the outcomes of the analysis can be quantitatively compared against the expected results.     

In situ single-molecule imaging with attoliter detection using objective total internal reflection confocal microscopy

Confocal microscopy is widely used for acquiring high spatial resolution tissue sample images of interesting fluorescent molecules inside cells. The fluorescent molecules are often tagged proteins participating in a biological function. The high spatial resolution of confocal microscopy compared to wide field imaging comes from an ability to optically isolate and image exceedingly small volume elements made up of the lateral (focal plane) and depth dimensions. Confocal microscopy at the optical diffraction limit images volumes on the order of approximately 0.5 femtoliter (10(-15) L). Further resolution enhancement can be achieved with total internal reflection microscopy (TIRM). With TIRM, an exponentially decaying electromagnetic field (near-field) established on the surface of the sample defines a subdiffraction limit dimension that, when combined with conventional confocal microscopy, permits image formation from <7 attoL (10(-18) L) volumes [Borejdo et al. (2006) Biochim. Biophys. Acta, in press]. Demonstrated here is a new variation of TIRM, focused TIRM (fTIRM) that decreases the volume element to approximately 3 attoL. These estimates were verified experimentally by measuring characteristic times for Brownian motion of fluorescent nanospheres through the volume elements. A novel application for TIRM is in situ single-molecule fluorescence spectroscopy. Single-molecule studies of protein structure and function are well-known to avoid the ambiguities introduced by ensemble averaging. In situ, proteins are subjected to the native forces of the crowded environment in the cell that are not present in vitro. The attoL fluorescence detection volume of TIRM permits isolation of single proteins in situ. Muscle tissue contains myosin at a approximately 120 microM concentration. Evidence is provided that >75% of the bleachable fluorescence detected with fTIRM is emitted by five chromophore-labeled myosins in a muscle fiber.     

Direct observation of microtubules with the scanning tunneling microscope

To observe surface topography of microtubules, we have applied scanning tunneling microscopy (STM), which can image metal and semiconductive surfaces with atomic resolution. Isolated microtubules fixed in 0.1% glutaraldehyde in reassembly buffer containing 0.8 M glycerol were imaged in air on a graphite substrate. The presence of microtubules in solution was verified by electron microscopy. At atmospheric pressure and room temperature, STM probing of both freeze-dried and hydrated microtubules reveals structures approximately 25 nm in width, consisting of longitudinal filaments about 4 nm in width. These structures match electron microscopy images of microtubules and their component protofilaments. Microtubules imaged by STM frequently appear buckled and semiflattened. Top-view shaded scans show what appear to be individual tubulin subunits within protofilaments. We believe these results represent the first direct STM observation of protein assemblies in which components can be identified. Although the microtubule image resolution described here is no better than that presently obtainable by other techniques (e.g., electron microscopy with freeze-drying, shadowing, and/or negative staining), it is significant that suitably prepared biomolecules may be sufficiently conductive and stable for STM imaging, which is ultimately capable of atomic resolution. Further development of STM technology, computer-enhanced image processing, and elucidation of optimal STM sample preparation indicate that STM and related applications will offer unique opportunities for the study of biomolecular surfaces.     

The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals

Electron beam damage is the fundamental limit to resolution in electron cryomicroscopy (cryo-EM) of frozen, hydrated specimens. Radiation damage increases with the number of electrons used to obtain an image and affects information at higher spatial frequencies before low-resolution information. For the experimentalist, a balance exists between electron exposures sufficient to obtain a useful signal-to-noise ratio (SNR) in images and exposures that limit the damage to structural features. In single particle cryo-EM this balance is particularly delicate: low-resolution features must be imaged with a sufficient SNR to allow image alignment so that high-resolution features recorded below the noise level can be recovered by averaging independent images. By measuring the fading of Fourier components from images obtained at 200 kV of thin crystals of catalase embedded in ice, we have determined the electron exposures that will maximize the SNR at resolutions between 86 and 2.9 Å. These data allow for a rational choice of exposure for single particle cryo-EM. For example, for 20 Å resolution, the SNR is maximized at ～20 e^?/Ų, whereas for 3 Å resolution, it is maximized at ～10 e^?/Ų. We illustrate the effects of exposure in single particle cryo-EM with data collected at ～12–15 and ～24–30 e^?/Ų.     

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.     

Electronic light microscopy: present capabilities and future prospects

Electronic light microscopy involves the combination of microscopic techniques with electronic imaging and digital image processing, resulting in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images and a discussion of the sampling requirements for the accurate digital recording of optical images, I discuss the three most important imaging modalities in electronic light microscopy-video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning microscopy-considering their capabilities, their applications, and recent developments that will increase their potential. Video-enhanced contrast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloidal gold particles, an order of magnitude smaller than the resolution limit of the light microscope. It has revolutionised the study of cellular motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the technique of optical trapping (optical tweezers), it permits exquisitely sensitive force and distance measurements to be made on motor proteins. Digital fluorescence microscopy enables low-light-level imaging of fluorescently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particularly multiple bandpass dichroic mirrors, and developments in multiparameter imaging, which is becoming particularly important for in situ hybridisation studies and automated image cytometry, fluorescence ratio imaging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-focus blur deconvolution and image restoration are becoming increasingly important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical sectioning and 3D image acquisition, but suffers from a number of limitations. I discuss advances in confocal techniques that address the problems of temporal resolution, spherical and chromatic aberration, wavelength flexibility and cross-talk between fluorescent channels, and describe new optics to enhance axial resolution and the use of two-photon excitation to reduce photobleaching. Finally, I consider the desirability of establishing a digital image database, the BioImage database, which would permit the archival storage of, and public Internet access to, multidimensional image data from all forms of biological microscopy. Submission of images to the BioImage database would be made in coordination with the scientific publication of research results based upon these data. In the context of electronic light microscopy, this would be particularly useful for three-dimensional images of cellular structure and video sequences of dynamic cellular processes, which are otherwise hard to communicate. However, it has the wider significance of allowing correlative studies on data obtained from many different microscopies and from sequence and crystallographic investigations. It also opens the door to interactive hypermedia access to the multidimensional image data, and multimedia publishing ventures based upon this.Presented at the XXXVII Symposium of the Society for Histochemistry, 23 September 1995, Rigi Kaltbad, Switzerland     

Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.     

Combined use of freeze-fracture electron microscopy and X-ray diffraction for the structure determination of three-dimensionally ordered specimens

The cubic phases of lipid-water systems have been studied by freeze-fracture electron microscopy. The preservation of the sample structure following cryofixation was verified by low temperature X-ray diffraction. Different types of fracture planes were identified; all display highly ordered two-dimensional domains, each subdivided into sub-domains related to each other by displacements and rotations related to the symmetry of the space group. The images were filtered using cross-correlation averaging techniques and the filtered images were compared to the corresponding planar sections of the electron density maps. Several conclusions were drawn: 1) when properly cryofixed, as assessed by low temperature X-ray diffraction, the structure of the sample was well preserved in the replicas; 2) the symmetry of the space group was faithfully reflected in the electron microscope images; 3) the crystallographic orientations of the most frequently identified'fracture planes coincided with those of the most intense X-ray reflections indicating that the fracture propagates, preferentially, in regions where the electron density variations are the largest; 4) when different structural models are compatible with X-ray diffraction data, it is possible to determine the correct model by comparing the filtered images with sections of the corresponding electron density maps; and 5) this approach constitutes a new and powerful tool of general interest for the low resolution study of three-dimensionally ordered specimens.     

Three-dimensional imaging by deconvolution microscopy

Deconvolution is a computational method used to reduce out-of-focus fluorescence in three-dimensional (3D) microscope images. It can be applied in principle to any type of microscope image but has most often been used to improve images from conventional fluorescence microscopes. Compared to other forms of 3D light microscopy, like confocal microscopy, the advantage of deconvolution microscopy is that it can be accomplished at very low light levels, thus enabling multiple focal-plane imaging of light-sensitive living specimens over long time periods. Here we discuss the principles of deconvolution microscopy, describe different computational approaches for deconvolution, and discuss interpretation of deconvolved images with a particular emphasis on what artifacts may arise.     

Alignment error envelopes for single particle analysis

To determine the structure of a biological particle to high resolution by electron microscopy, image averaging is required to combine information from different views and to increase the signal-to-noise ratio. Starting from the number of noiseless views necessary to resolve features of a given size, four general factors are considered that increase the number of images actually needed: (1) the physics of electron scattering introduces shot noise, (2) thermal motion and particle inhomogeneity cause the scattered electrons to describe a mixture of structures, (3) the microscope system fails to usefully record all the information carried by the scattered electrons, and (4) image misalignment leads to information loss through incoherent averaging. The compound effect of factors 2-4 is approximated by the product of envelope functions. The problem of incoherent image averaging is developed in detail through derivation of five envelope functions that account for small errors in 11 "alignment" parameters describing particle location, orientation, defocus, magnification, and beam tilt. The analysis provides target error tolerances for single particle analysis to near-atomic (3.5 A) resolution, and this prospect is shown to depend critically on image quality, defocus determination, and microscope alignment.     

Real space refinement of acto-myosin structures from sectioned muscle

We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs.     

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

Fluorescence microscopy is an important and extensively utilised tool for imaging biological systems. However, the image resolution that can be obtained has a limit as defined through the laws of diffraction. Demand for improved resolution has stimulated research into developing methods to image beyond the diffraction limit based on far-field fluorescence microscopy techniques. Rapid progress is being made in this area of science with methods emerging that enable fluorescence imaging in the far-field to possess a resolution well beyond the diffraction limit. This review outlines developments in far-field fluorescence methods which enable ultrahigh resolution imaging and application of these techniques to biology. Future possible trends and directions in far-field fluorescence imaging with ultrahigh resolution are also outlined.