Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.@en

Recent advances in electron microscopy techniques have led to a significant scale up in volumetric imaging of biological tissue. The throughput of electron microscopes, however, remains a limiting factor for the volume that can be imaged in high resolution within reasonable time. Faster detection methods will improve throughput. Here, we have characterized and benchmarked a novel detection technique for scanning electron microscopy: optical scanning transmission electron microscopy (OSTEM). A qualitative and quantitative comparison was performed between OSTEM, secondary and backscattered electron detection and annular dark field detection in scanning transmission electron microscopy. Our analysis shows that OSTEM produces images similar to backscattered electron detection in terms of contrast, resolution and signal-to-noise ratio. OSTEM can complement large scale imaging with (scanning) transmission electron microscopy and has the potential to speed up imaging in single-beam scanning electron microscope.

@en

Multi-modal imaging techniques have become essential for better understanding fundamental questions in cell biology such as disease progression. While individual microscopy methods have rapidly advanced in recent years, the information content of any one imaging technique is limited to the type of contrast that particular technique is sensitive to. By tagging particular biomolecules with a fluorescent protein, fluorescence microscopy (FM), for example, can relay dynamic information about the distribution of these biomolecules in their cellular environment. It struggles, however, to convey information regarding the structure of the organelles that might contain these biomolecules or the surroundings of their cellular environment. Electron microscopy (EM), on the other hand, can provide detailed layouts of cellular structure by staining membranes with heavy metals. Thus, by correlating these modalities (correlative light and electron microscopy, CLEM), a more holistic understanding of the relationship between structure and function at the (sub-)cellular level can be achieved. Array tomography (AT) is a technique combining FM and EM for volumetric imaging, first introduced in 2007 for studying brain tissue. The technique has since expanded, but the approach has largely remained the same. Biological material is cut into a series of ultrathin (∼100 nm) sections (an array) and prepared for sequential FM and EM imaging by applying a series of immunofluorescence and heavy metal stains. Correlative images of the serial sections are then computationally aligned to reconstruct the 3D structure (tomography). Compared to other volumetric imaging techniques in the life sciences, AT offers the ability to correlate structure and function at high resolution across large fields of view. Moreover, it enables high axial resolutionfor both EM and FM as determined by the section thickness...@en

Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D image acquisition and reconstruction pipeline that overcomes both of these limitations by using a widefield fluorescence microscope integrated inside of a scanning electron microscope. The workflow consists of acquiring large field of view fluorescence microscopy (FM) images, which guide to regions of interest for successive EM (integrated correlative light and electron microscopy). High precision EM-FM overlay is achieved using cathodoluminescent markers. We conduct a proof-of-concept of our integrated workflow on immunolabelled serial sections of tissues. Acquisitions are limited to regions containing biological targets, expediting total acquisition times and reducing the burden of excess data by tens or hundreds of GBs.

@en

Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server-based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management.

@en

Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging. However, optimization of negative bias potential for application in thin section imaging will be needed prior to routine use and application in large-scale EM. Here, we present negative bias potential optimized through a combination of simulations and empirical measurements. We find that the use of a negative bias potential generally results in improvement of image quality and signal-to-noise ratio (SNR). The extent of these improvements depends on the presence and strength of a magnetic immersion field. Maintaining other imaging conditions and aiming for the same image quality and SNR, the use of a negative stage bias can allow for a 20-fold decrease in dwell time, thus reducing the time for a week long acquisition to less than 8 h. We further show that negative bias potential can be applied in an integrated correlative light electron microscopy (CLEM) application, allowing fast acquisition of a high precision overlaid LM-EM dataset. Application of negative stage bias potential will thus help to solve the current bottleneck of image acquisition of large fields of view at high resolution in large-scale microscopy.

@en

The authors present the application of a retarding field between the electron objective lens and sample in an integrated fluorescence and electron microscope. The retarding field enhances signal collection and signal strength in the electron microscope. This is beneficial for samples prepared for integrated fluorescence and electron microscopy as the amount of staining material added to enhance electron microscopy signal is typically lower compared to conventional samples in order to preserve fluorescence. We demonstrate signal enhancement through the applied retarding field for both 80-nm post-embedding immunolabeled sections and 100-nm in-resin preserved fluorescence sections. Moreover, we show that tuning the electron landing energy particularly improves imaging conditions for ultra-thin (50 nm) sections, where optimization of both retarding field and interaction volume contribute to the signal improvement. Finally, we show that our integrated retarding field setup allows landing energies down to a few electron volts with 0.3 eV dispersion, which opens new prospects for assessing electron beam induced damage by in situ quantification of the observed bleaching of the fluorescence following irradiation.

@en

In the past decades, correlative light and electron microscopy (CLEM) methods have evolved from being mostly used by a few pioneering, specialist labs to a collection of techniques and workflows practiced by a broad group of researchers in structural biology. In most cases, CLEM involves a distinct set of sequentially used specimen preparation and labeling techniques, followed by diverse types of light and electron microscopy techniques. This chapter focuses on those areas in present-day CLEM that are faced with challenges for which these advantages of integrated microscopes may well be key for further advancement. These areas are large-scale and high-throughput correlated (volume) microscopy, super-resolution localization in resin or cryo-frozen sections, fluorescence-guided focused ion beam milling for cryo-electron tomography, and the integration of sample preparation and transfer. Ultimately this should lead to the development of specific integrated CLEM systems with complete and fully automated workflows, leading to high-throughput and high-yield systems.@en