Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200 keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200 keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30 K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70 K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.     

A new setup for in vivo fluorescence imaging of photosynthetic activity

Here, we describe a new imaging setup able to assess in vivo photosynthetic activity. The system specifically measures time-resolved chlorophyll fluorescence in response to light. It is composed of a fast digital camera equipped with a wide-angle lens for the analysis of samples up to 10 × 10 cm, i.e. entire plants or petri dishes. In the choice of CCD, we have opted for a 12-bits high frame rate [150 fps (frames per second)] at the expense of definition (640 × 480 pixels). Although the choice of digital camera is always a compromise between these two related features, we have designed a flexible system allowing the fast sampling of images (down to 100 μs) with a maximum spatial resolution. This image readout system, synchronized with actinic light and saturating pulses, allows a precise determination of F ₀ and F _M, which is required to monitor PSII activity. This new imaging system, together with image processing techniques, is useful to investigate the heterogeneity of photosynthetic activity within leaves or to screen large numbers of unicellular algal mutant colonies to identify those with subtle changes in photosynthetic electron flow.     

Achievable resolution from images of biological specimens acquired from a 4k x 4k CCD camera in a 300-kV electron cryomicroscope

Bacteriorhodopsin and ε 15 bacteriophage were used as biological test specimens to evaluate the potential structural resolution with images captured from a 4k × 4k charge-coupled device (CCD) camera in a 300-kV electron cryomicroscope. The phase residuals computed from the bacteriorhodopsin CCD images taken at 84,000× effective magnification averaged 15.7° out to 5.8-Å resolution relative to Henderson’s published values. Using a single-particle reconstruction technique, we obtained an 8.2-Å icosahedral structure of ε 15 bacteriophage with the CCD images collected at an effective magnification of 56,000×. These results demonstrate that it is feasible to retrieve biological structures to a resolution close to 2/3 of the Nyquist frequency from the CCD images recorded in a 300-kV electron cryomicroscope at a moderately high but practically acceptable microscope magnification.     

Detection of bioluminescence from individual bacterial cells: a comparison of two different low-light imaging systems

Detection of very low light levels arising from individual cells of the naturally bioluminescent bacterium Vibrio fischeri as well as from a luminescence-marked Pseudomonas putida strain was achieved by the aid of two different camera systems. Using a liquid nitrogen-cooled slow-scan CCD (charge-coupled device) camera we were able to detect single-cell bioluminescence within 1 min, and the pictures obtained were of good resolution. In contrast, employing a photon-counting video camera we were able to detect bioluminescent cells within 10 seconds, but at the expense of spatial resolution. This study demonstrates the feasibility of microscopic single cell analysis employing bioluminescence as reporter system. © 1997 John Wiley & Sons, Ltd.     

Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells

Cryo-electron tomography (cryo-ET) allows for the visualization of biological material in a close-to-native state, in three dimensions and with nanometer scale resolution. However, due to the low signal-to-noise ratio inherent to imaging of the radiation-sensitive frozen-hydrated samples, it appears oftentimes impossible to localize structures within heterogeneous samples. Because a major potential for cryo-ET is thereby left unused, we set out to combine cryo-ET with cryo-fluorescence microscopy (cryo-FM), in order to facilitate the search for structures of interest. We describe a cryo-FM setup and workflow for correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) that can be easily implemented. Cells are grown on finder grids, vitally labeled with one or two fluorescent dyes, and vitrified. After a structure is located by cryo-FM (with 0.4 μm resolution), its image coordinates are translated to cryo-ET stage coordinates via a home-built software routine. We tested our workflow on whole mount primary human umbilical vein endothelial cells. The correlative routine enabled us to investigate mitochondrial ultrastructure for the first time on intact human mitochondria, and led us to find mitochondrial cristae that were connected to the intermembrane space via large slits, which challenges the current view that such connections are established exclusively via small circular pores. Taken together, this study emphasizes that cryo-CLEM can be a routinely used technique that opens up exciting new possibilities for cryo-ET.     

Advantages of CCD detectors for de novo three-dimensional structure determination in single-particle electron microscopy

For three-dimensional (3D) structure determination of large macromolecular complexes, single-particle electron cryomicroscopy is considered the method of choice. Within this field, structure determination de novo, as opposed to refinement of known structures, still presents a major challenge, especially for macromolecules without point-group symmetry. This is primarily because of technical issues: one of these is poor image contrast, and another is the often low particle concentration and sample heterogeneity imposed by the practical limits of biochemical purification. In this work, we tested a state-of-the art 4 k x 4 k charge-coupled device (CCD) detector (TVIPS TemCam-F415) to see whether or not it can contribute to improving the image features that are especially important for structure determination de novo. The present study is therefore focused on a comparison of film and CCD detector in the acquisition of images in the low-to-medium ( approximately 10-25 A) resolution range using a 200 kV electron microscope equipped with field emission gun. For comparison, biological specimens and radiation-insensitive carbon layers were imaged under various conditions to test the image phase transmission, spatial signal-to-noise ratio, visual image quality and power-spectral signal decay for the complete image-processing chain. At all settings of the camera, the phase transmission and spectral signal-to-noise ratio were significantly better on CCD than on film in the low-to-medium resolution range. Thus, the number of particle images needed for initial structure determination is reduced and the overall quality of the initial computed 3D models is improved. However, at high resolution, film is still significantly better than the CCD camera: without binning of the CCD camera and at a magnification of 70 kx, film is better beyond 21 A resolution. With 4-fold binning of the CCD camera and at very high magnification (> 300 kx) film is still superior beyond 7 A resolution.     

High‐resolution photo‐mosaic time‐series imagery for monitoring human use of an artificial reef

Successful marine management relies on understanding patterns of human use. However, obtaining data can be difficult and expensive given the widespread and variable nature of activities conducted. Remote camera systems are increasingly used to overcome cost limitations of conventional labour‐intensive methods. Still, most systems face trade‐offs between the spatial extent and resolution over which data are obtained, limiting their application. We trialed a novel methodology, CSIRO Ruggedized Autonomous Gigapixel System (CRAGS), for time series of high‐resolution photo‐mosaic (HRPM) imagery to estimate fine‐scale metrics of human activity at an artificial reef located 1.3 km from shore. We compared estimates obtained using the novel system to those produced with a web camera that concurrently monitored the site. We evaluated the effect of day type (weekday/weekend) and time of day on each of the systems and compared to estimates obtained from binocular observations. In general, both systems delivered similar estimates for the number of boats observed and to those obtained by binocular counts; these results were also unaffected by the type of day (weekend vs. weekday). CRAGS was able to determine additional information about the user type and party size that was not possible with the lower resolution webcam system. However, there was an effect of time of day as CRAGS suffered from poor image quality in early morning conditions as a result of fixed camera settings. Our field study provides proof of concept of use of this new cost‐effective monitoring tool for the remote collection of high‐resolution large‐extent data on patterns of human use at high temporal frequency.     

Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4–6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.     

Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for detailed observation, scenarios that make it challenging to observe with conventional imaging systems. One of these is the drop impact of liquids, which usually happens within one tenth of millisecond. To tackle this challenge, a fast imaging technique is introduced that combines a high-speed camera (capable of up to one million frames per second) with a macro lens with long working distance to bring the spatial resolution of the image down to 10 µm/pixel. The imaging technique enables precise measurement of relevant fluid dynamic quantities, such as the flow field, the spreading distance and the splashing speed, from analysis of the recorded video. To demonstrate the capabilities of this visualization system, the impact dynamics when droplets of non-Newtonian fluids impinge on a flat hard surface are characterized. Two situations are considered: for oxidized liquid metal droplets we focus on the spreading behavior, and for densely packed suspensions we determine the onset of splashing. More generally, the combination of high temporal and spatial imaging resolution introduced here offers advantages for studying fast dynamics across a wide range of microscale phenomena.     

Methods for aligning and for averaging 3D volumes with missing data

The visibility and resolution of a tomographic reconstruction containing multiple copies of discrete particles can be enhanced by averaging subtomograms after they are corrected aligned. However, the “missing wedge” in electron tomography can easily lead to erroneous alignment. We have explored a Fourier space cross-correlation method with a proper weighting scheme to align and average different sets of volumetric data, each of which has different missing data due to the limited specimen tilts. This approach depends neither on a preexisting template, nor an exact knowledge of the geometry, orientation, or amount of the missing data. This paper introduces a procedure where the missing data might be gradually “filled in” by consecutively aligning and averaging volumes with different orientations of their missing data. We have validated these techniques by a set of simulated data with various symmetries and extent of missing data. We have also successfully applied these procedures to experimental cryo-electron tomographic data [Chang, J.T., Schmid, M.F., Rixon, F.J., and Chiu, W., 2007. Electron cryotomography reveals the portal in the herpesvirus capsid. J. Virol. 81, 2065–2068; Schmid, M.F., Paredes, A.M., Khant, H.A., Soyer, F., Aldrich, H.C., Chiu, W., and Shively, J.M., 2006. Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography. J. Mol. Biol. 364, 526–535].     

Modular Scanning Confocal Microscope with Digital Image Processing

In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength.     

A Microscope for Two-Dimensional Measurements of In Vivo Chlorophyll Fluorescence Kinetics Using Pulsed Measuring Radiation,Continuous Actinic Radiation,and Saturating Flashes

Transients of chlorophyll fluorescence in photosynthetic objects are often measured using short pulses of exciting radiation, which has recently been employed to capture kinetic images of fluorescence at the macroscopic level. Here we describe an instrument introducing this principle to recording of two dimensional fluorescence transients in microscopic objects. A modified fluorescence microscope is equipped with a CCD camera intensified by a micro-channel plate image amplifier. The microscopic field is irradiated simultaneously by three types of radiation: actinic radiation, saturating flashes, and pulsed measuring radiation. The measuring pulses are generated by a light-emitting diode and their duration is between 10 to 250 µs. The detection of fluorescence images (300×400 pixels, 8 bit) has a maximum time resolution of 40 ms and is gated in synchrony with the exciting pulses. This allows measuring on a background of a continuous actinic radiation up to irradiance that can elicit the maximal fluorescence yield (F_M). On the other hand, the integral irradiance of the objects by the measuring radiation is very low, e.g., 0.08 µmol m^–2 s^–1 at 0.05 µm spatial resolution and 0.006 µmol m^–2 s^–1 at 4 µm spatial resolution. This allows a reliable recording of F₀ even in very short time intervals (e.g., 5×80 ms). The software yields fluorescence kinetic curves for objects in user-selected areas as well as complete false-colour maps of the essential fluorescence kinetics parameters (F_M, F_O, F_V, F_V/F_M, etc.) showing a two-dimensional distribution of their values. Several examples demonstrate that records of fluorescence kinetics can be obtained with a reasonable signal-to-noise ratio with all standard microscope objectives and with object sizes reaching from segments of leaf tissue to individual algal cells or chloroplasts.     

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy

Gammaherpesviruses, including the human pathogens Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral–host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.     

Active microwave tomographic imaging of isolated, perfused animal organs

The relative transparency of biological materials to high-frequency electromagnetic waves has encouraged the development of new systems for imaging. This report describes experiments of microwave tomography conducted on a prototype. The object to be analyzed is submerged in water and is illuminated by a plane wave. The total electric field is analyzed by a microwave camera. The recorded data are then processed numerically in order to reconstruct the image that corresponds to the distribution of equivalent currents in a defined plane of a section. Experiments have been conducted on isolated kidneys with and without perfusion. The influence of the perfusing solution temperature has also been studied. These experiments show the potential of this system, especially through the correlation between microwave images and the biological structures. They also confirm previous results concerning spatial resolution and depth of exploration. Finally, the results demonstrate the influence of temperature and support the applicability of this imaging system in non-invasive thermometry, especially for clinical hyperthermia.     

Imaging system for morphometric assessment of absorption or fluorescence in stained cells

An image acquisition and processing system has been developed for quantitative microscopy of absorption or fluorescence in stained cells. Three different light transducers are used in the system to exploit the best characteristics of these sensors for different biological measurements. A digital scanner, in the form of a linear array charge-coupled device (CCD), acquires data with high spatial and photometric resolution. A color (RGB) camera is employed when spectral information is required for the segmentation of cellular subcomponents. An image-intensified charged-injection device (CID) camera provides for very low light intensity measurements, primarily for fluorescence-labeled cells. Properties of these transducers, such as contrast transfer function, linearity, and photo-response nonuniformity, have been measured. Two dedicated image processing units were incorporated into the system. The front-end processor, based on a digital signal processor, provides functions such as object detection, raw image calibration, compression, artifact removal, and filtering. The second image processor is associated with the frame memory and includes a histogram processor, a dedicated arithmetic logic unit for image processing functions, and a graphics module for one-bit overlay functions. An interactive program was developed to acquire cell images and to experiment with a range of segmentation algorithms, feature extractions, and other image processing functions. The results of any image operation are displayed on the video monitor. Once a desired processing sequence is determined, the sequence may be stored to become part of a command library and can be executed thereafter as a single instruction.     

CdTe compact gamma camera for coded aperture imaging in radioguided surgery

The aim of this work was to assess the performance of a prototype compact gamma camera (MediPROBE) based on a CdTe semiconductor hybrid pixel detector, for coded aperture imaging. This probe can be adopted for various tasks in nuclear medicine such as preoperative sentinel lymph node localization, breast imaging with ^99mTc radiotracers and thyroid imaging, and in general in radioguided surgery tasks. The hybrid detector is an assembly of a 1-mm thick CdTe semiconductor detector bump-bonded to a photon-counting CMOS readout circuit of the Medipix2 series or energy-sensitive Timepix detector. MediPROBE was equipped with a set of two coded aperture masks with 0.07-mm or 0.08-mm diameter holes. We performed laboratory measurements of field of view, system spatial resolution, and signal-difference-to-noise ratio, by using gamma-emitting radioactive sources (¹⁰⁹Cd, ¹²⁵I, ²⁴¹Am, ^99mTc). The system spatial resolution in the lateral direction was 0.56 mm FWHM (coded aperture mask with holes of 0.08 mm and a 60 keV source) at a source-collimator distance of 50 mm and a field of view of 40 mm by side. Correspondingly, the longitudinal resolution in 3D source localization tasks was about 3 mm. MediPROBE showed a significant improvement in terms of spatial resolution when equipped with the high-resolution coded apertures, with respect to the performance previously reported with 1–2 mm pinhole apertures as well as with respect to adopting a 0.35 mm pinhole aperture.     

Performance validation of an improved Xenon-arc lamp-based CCD camera system for multispectral imaging in proteomics

Advances in gel-based nonradioactive protein expression and PTM detection using fluorophores has served as the impetus for developing analytical instrumentation with improved imaging capabilities. We describe a CCD camera-based imaging instrument, equipped with both a high-pressure Xenon arc lamp and a UV transilluminator, which provides broad-band wavelength coverage (380-700 nm and UV). With six-position filter wheels, both excitation and emission wavelengths may be selected, providing optimal measurement and quantitation of virtually any dye and allowing excellent spectral resolution among different fluorophores. While spatial resolution of conventional fixed CCD camera imaging systems is typically inferior to laser scanners, this problem is circumvented with the new instrument by mechanically scanning the CCD camera over the sample and collecting multiple images that are subsequently automatically reconstructed into a complete high-resolution image. By acquiring images in succession, as many as four different fluorophores may be evaluated from a gel. The imaging platform is suitable for analysis of the wide range of dyes and tags commonly encountered in proteomics investigations. The instrument is unique in its capabilities of scanning large areas at high resolution and providing accurate selectable illumination over the UV/visible spectral range, thus maximizing the efficiency of dye multiplexing protocols.     

Resolution-recovery-embedded image reconstruction for a high-resolution animal SPECT system

The small-animal High-Resolution SPECT (HiReSPECT) is a dedicated dual-head gamma camera recently designed and developed in our laboratory for imaging of murine models. Each detector is composed of an array of 1.2 × 1.2 mm² (pitch) pixelated CsI(Na) crystals. Two position-sensitive photomultiplier tubes (H8500) are coupled to each head's crystal. In this paper, we report on a resolution-recovery-embedded image reconstruction code applicable to the system and present the experimental results achieved using different phantoms and mouse scans. Collimator-detector response functions (CDRFs) were measured via a pixel-driven method using capillary sources at finite distances from the head within the field of view (FOV). CDRFs were then fitted by independent Gaussian functions. Thereafter, linear interpolations were applied to the standard deviation (σ) values of the fitted Gaussians, yielding a continuous map of CDRF at varying distances from the head. A rotation-based maximum-likelihood expectation maximization (MLEM) method was used for reconstruction. A fast rotation algorithm was developed to rotate the image matrix according to the desired angle by means of pre-generated rotation maps. The experiments demonstrated improved resolution utilizing our resolution-recovery-embedded image reconstruction. While the full-width at half-maximum (FWHM) radial and tangential resolution measurements of the system were over 2 mm in nearly all positions within the FOV without resolution recovery, reaching around 2.5 mm in some locations, they fell below 1.8 mm everywhere within the FOV using the resolution-recovery algorithm. The noise performance of the system was also acceptable; the standard deviation of the average counts per voxel in the reconstructed images was 6.6% and 8.3% without and with resolution recovery, respectively.     

CZT gamma camera for scintimammography

A high performance prototype gamma camera based on the semiconductor radiation detector Cd(Zn)Te is described. The camera features high spatial resolution, high-energy resolution, a reduced dead space on the edge of the field of view, and a compact format. The camera performance was first examined by comparison of small field of view examinations with those from an Elscint SP6HR standard clinical gamma camera. The new camera was found to give equal or improved image quality. The camera was then used for a systematic phantom study of small lesions in a background as would be found in breast cancer imaging. In this study the camera was able to systematically detect smaller, deeper, and fainter lesions. The camera is presently being used in a clinical trial aimed to assess its value in scintimammography where previous limitations of image quality and detector size have restricted the use of the functional imaging techniques. Preliminary results from 40 patients show high sensitivity and specificity with respect to X-ray mammography and surgery.     

99mTc thyroid imaging system using multiple imaging plates

A system for taking static thyroid ^99mTc images was devised by using multiple imaging plates (IPs) and a low-energy high resolution collimator. System spatial resolution of the IP systems and the gamma camera was determined by referring to standards set by the National Electrical Manufacturers Association. Sensitivity was represented by using lower detection limits (LDLs). The sensitivity and resolution of IP systems using 16 IP probes connecting two collimators and 9 IPs were determined by using a 20 ml thyroid phantom, and compared with the sensitivity of gamma cameras. The sensitivity of the IP systems increased in proportion to the number of IPs. The sensitivity and resolution of a probe using 6 IPs and a high resolution collimator were equivalent to or superior to the gamma camera for taking static thyroid ^99mTc images. IP systems can be applied clinically as mobile static nuclear imaging devices. The performance of IP systems should be thoroughly investigated for combinations of various collimators and the number of IPs in order to verify their efficacy for imaging all organs.