Automated sample mounting and alignment system for biological crystallography at a synchrotron source

High-throughput data collection for macromolecular crystallography requires an automated sample mounting and alignment system for cryo-protected crystals that functions reliably when integrated into protein-crystallography beamlines at synchrotrons. Rapid mounting and dismounting of the samples increases the efficiency of the crystal screening and data collection processes, where many crystals can be tested for the quality of diffraction. The sample-mounting subsystem has random access to 112 samples, stored under liquid nitrogen. Results of extensive tests regarding the performance and reliability of the system are presented. To further increase throughput, we have also developed a sample transport/storage system based on "puck-shaped" cassettes, which can hold sixteen samples each. Seven cassettes fit into a standard dry shipping Dewar. The capabilities of a robotic crystal mounting and alignment system with instrumentation control software and a relational database allows for automated screening and data collection to be developed.     

High-vacuum optical platform for cryo-CLEM (HOPE): A new solution for non-integrated multiscale correlative light and electron microscopy

Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique (high-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The ‘touch-free’ specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope.     

A method for determining the crystal orientation for any tilt position when using a transmission electron microscope double-tilt specimen holder

A method is described in this short communiction which provides a rapid and precise determination of the crystal specimen orientation in any tilt position when using a doule-tilt holder in the transmission electron microscope (TEM). It is necessary to establish three pairs of tilt angles (α_i, β_i) (i = 1, 2, 3) where three recognized directions of the crystal specimen are aligned to the electron beam direction respectively, in order to determine the crystal specimen orientation for any tilt position in a double-tilt specimen holder. In addition, the tilt position (α, β), where any orientation [ u v w] is aligned to the beam direction, can be determined with the aid of this method.A software system for computer applications of the above method has beed developed.     

Development of a periodontium-approximated specimen holder for attrition studies of telescopic crowns]

To enable attritional studies on telescopic crowns, a specimen holder has been constructed that is intended to simulate, in zero approximation, the biomechanical properties of abutments bearing telescopic crowns. The specimen holder consists essentially of a system with two metal "concertinas" with different elasticity constants reflecting the different biomechanical parameters for periodontium and bone. The biomechanical processes in the periodontium are too complex to be simulated accurately in vitro. Nevertheless, the specimen holder described here enables the forces acting during insertion and removal of a partial denture to be accommodated elastically in the angular and axial directions, to simulate the possible degrees of freedom of the abutments in situ. In this way, the unavoidable errors of specimen fixation are rendered tolerable, so that local preferential wear is decisively reduced.     

Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes

This protocol details the steps used for visualizing the frozen-hydrated grids as prepared following the accompanying protocol entitled 'Preparation of macromolecular complexes for visualization using cryo-electron microscopy.' This protocol describes how to transfer the grid to the microscope using a standard cryo-transfer holder or, alternatively, using a cryo-cartridge loading system, and how to collect low-dose data using an FEI Tecnai transmission electron microscope. This protocol also summarizes and compares the various options that are available in data collection for three-dimensional (3D) single-particle reconstruction. These options include microscope settings, choice of detectors and data collection strategies both in situations where a 3D reference is available and in the absence of such a reference (random-conical and common lines).     

An effective tool for homogeneous assays using a simple robot arm with Stackers-TRISTAN

The Twister & RapidPlate Integrated System by TANabe, or TRISTAN, consists of a 96-channel dispenser (RapidPlate 96), a plate reader (V-MAX), and a simple robot arm (Twister). We developed TRISTAN for effectively conducting a homogeneous assay. Although this system accommodates fewer than 20 microplates, it has several advantages over conventional robotic systems for high-throughput screening in the following aspects: parameter setting, running time, hardware errors, manpower, and cost-effectiveness. The system proved to be effective and efficient for homogeneous assays.     

Automated 100-position specimen loader and image acquisition system for transmission electron microscopy

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the "Gatling", permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 h. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science, and nanotechnology that require rapid screening and image analysis of multiple specimens.     

An Alternative to the Flat Substrate Method of Preparing Electron Microscope Autoradiographs

Difficulty with flat substrate methods of preparing electron microscope autoradiographs has prompted reconsideration and refinement of a technique in which an electron microscope grid is placed beneath the specimen prior to dipping. This technique avoids the problems commonly associated with the direct application of emulsions to specimen grids, and should be considered as an alternative to flat substrate techniques when difficulty with these methods is encountered.     

The HOME microscope workstation. A new tool for cervical cancer screening.

The Highly Optimized Microscope Environment (HOME) is a computerized microscope design for assisting pathologists and cytotechnologists in routine clinical tasks. The prototype system consists of an IBM PC-compatible computer and a light microscope in which a built-in high-resolution computer display image is super-imposed on the optical image of the specimen. Also, an encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on the stage to be uniquely defined. Software, written in C language and running under the MS-DOS/MS-Windows environment, is controlled by means of a mouse-driven cursor. A specific application has been developed for cervical cancer screening, taking into account the needs and constraints of microscopists performing this task. Informatics tools offered by the HOME system provide them with precise flagging and relocation of objects on the slide, control of the scanning pathway, and ability to write and print the report directly through the microscope. The computer files generated by microscopic examination are stored and contain information available for quality control assessment and laboratory management.     

Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM

Over the last three decades, Cryo-TEM has developed into a powerful technique for high-resolution imaging of biological macromolecules in their native vitrified state. However, the method for vitrifying specimens onto EM grids is essentially unchanged - application of ～3 μL sample to a grid, followed by blotting and rapid plunge freezing into liquid ethane. Several trials are often required to obtain suitable thin (few hundred nanometers or less) vitrified layers amenable for cryo-TEM imaging, which results in waste of precious sample and resources. While commercially available instruments provide some level of automation to control the vitrification process in an effort to increase quality and reproducibility, obtaining satisfactory vitrified specimens remains a bottleneck in the Cryo-TEM pipeline. We describe here a completely novel method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A first prototype system (Spotiton v0.5) demonstrates feasibility of this new approach for specimen vitrification. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process. Using TEM imaging and biochemical assays we demonstrate that the piezo-electric inkjet mechanism does not disrupt the structural or functional integrity of macromolecules. These preliminary studies provide insight into the factors and components that will need further development to enable a robust and repeatable technique for specimen vitrification using this novel approach.     

Robotic nanolitre protein crystallisation at the MRC Laboratory of Molecular Biology

We have set up high-throughput robotic systems to screen and optimise crystallisation conditions of biological macromolecules with the aim to make difficult structural biology projects easier. The initial screening involves two robots. A Tecan Genesis liquid handler is used to transfer commercially available crystallisation reagents from 15 ml test tubes into the reservoirs of 96-well crystallisation plates. This step is fully automated and includes a carousel for intermediate plate storage, a Beckman plate sealer and a robotic arm, which transfers plates in between steps. For adding the sample, we use a second robot, a 17-tip Cartesian Technologies PixSys 4200 SynQuad liquid handler, which uses a syringe/solenoid valve combination to dispense small quantities of liquid (typically 100 nl) without touching the surface of the plate. Sixteen of the tips are used to transfer the reservoir solution to the crystallisation wells, while the 17th tip is used to dispense the protein. The screening of our standard set of 1440 conditions takes about 3 h and requires 300 microl of protein solution. Once crystallisation conditions have been found, they are optimised using a second Tecan Genesis liquid handler, which is programmed to pipette gradients from four different corner solutions into a wide range of crystallisation plate formats. For 96-well plates, the Cartesian robot can be used to add the sample. The methods described are now used almost exclusively for obtaining diffraction quality crystals in our laboratory with a throughput of several thousand plates per year. Our set-up has been copied in many institutions worldwide.     

High-throughput film-densitometry: an efficient approach to generate large data sets

A film-handling machine (robot) has been built which can, in conjunction with a commercially available film densitometer, exchange and digitize over 300 electron micrographs per day. Implementation of robotic film handling effectively eliminates the delay and tedium associated with digitizing images when data are initially recorded on photographic film. The modulation transfer function (MTF) of the commercially available densitometer is significantly worse than that of a high-end, scientific microdensitometer. Nevertheless, its signal-to-noise ratio (S/N) is quite excellent, allowing substantial restoration of the output to "near-to-perfect" performance. Due to the large area of the standard electron microscope film that can be digitized by the commercial densitometer (up to 10,000 x 13,680 pixels with an appropriately coded holder), automated film digitization offers a fast and inexpensive alternative to high-end CCD cameras as a means of acquiring large amounts of image data in electron microscopy.     

Staining and Washing Ultrathin Sections; a Device for Handling Various Materials Simultaneously

To eliminate individual manipulation, as many as 10 grids, each held firmly by a small notched bar of polyethylene plastic, are simultaneously stained, then washed. If the stain used is reactive with atmospheric CO₂ it can be forced through a Millipore filter into a small chamber made of glass tubing which contains the grid holder. The stain, cleared of any solid particles, has very little contact with air and remains free of lead carbonate contamination. Washing is carried out by submerging the chamber and removing the grid holder under water (Feldman, D. G., J. Cell Biol., 15: 592-5, 1962). Washing is minimized because there is not the risk of contaminating grids and wash water with stain trapped between the points of forceps. The polyethylene is nonadherent to the wash water, and the grids can therefore be dried quickly on the holder. With this method, the relative stainability of different materials may be observed because each grid within a batch receives identical treatment.     

High-throughput screening of chromatographic separations: IV. Ion-exchange

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions.     

Review of different platforms to perform rapid onsite evaluation via telecytology

There is increased utilisation of cytopathology to provide a rapid onsite evaluation (ROSE) of fine needle aspiration and touch preparations of small biopsies. A well‐executed ROSE procedure can significantly impact the diagnostic quality and appropriate specimen triage of procured biopsy materials. To accommodate the demand for ROSE, telecytology has been increasingly implemented to facilitate ROSE occurring remotely. Telecytology can be categorised based on camera systems including eyepiece system, camera port system and robotic microscope/whole slide image scanner system. Image sharing methods include static images, broadcast only live video streaming, teleconferencing and whole slide image management system. In this review, we will discuss the advantages and disadvantages of each of these systems and deployment considerations.     

Microscale Fluid Behavior during Cryo-EM Sample Blotting

Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy for over three decades. This technique removes the excess solution from a transmission electron microscope grid by pressing absorbent filter paper against the specimen before vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood. Here, high-speed interference contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during the removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topographical features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses.     

Production and Cleaning of Freeze-Etch Replicas which Show Complementary Surfaces of Fractured Fungus Spores and Hyphae

London finder grids (H-2) and 3 mm copper discs were flared on opposite sides by use of a die and punch. The grids were registered on the discs and attached with vaseline. The specimen was squeezed through the grid apertures by the 2 copper discs. The complete sandwich was frozen in freon 22 and placed in a hinged specimen holder which fractured the layer of the specimen between the 2 finder grids. Platinum-carbon and carbon replicas were made immediately after fracturing. After drying the replicas, the vaseline was removed by chloroform, and replicas were cleaned with 70% H₂SO₄ for 1-18 hr at 52 C, followed by 6% commercial sodium hypochlorite (Chlorox) for 1-2 hr at 23-25 C.     

A fully automated plasma protein precipitation sample preparation method for LC-MS/MS bioanalysis

This report describes the development and validation of a robust robotic system that fully integrates all peripheral devices needed for the automated preparation of plasma samples by protein precipitation. The liquid handling system consisted of a Tecan Freedom EVO 200 liquid handling platform equipped with an 8-channel liquid handling arm, two robotic plate-handling arms, and two plate shakers. Important additional components integrated into the platform were a robotic temperature-controlled centrifuge, a plate sealer, and a plate seal piercing station. These enabled unattended operation starting from a stock solution of the test compound, a set of test plasma samples and associated reagents. The stock solution of the test compound was used to prepare plasma calibration and quality control samples. Once calibration and quality control samples were prepared, precipitation of plasma proteins was achieved by addition of three volumes of acetonitrile. Integration of the peripheral devices allowed automated sequential completion of the centrifugation, plate sealing, piercing and supernatant transferral steps. The method produced a sealed, injection-ready 96-well plate of plasma extracts. Accuracy and precision of the automated system were satisfactory for the intended use: intra-day and the inter-day precision were excellent (C.V.<5%), while the intra-day and inter-day accuracies were acceptable (relative error<8%). The flexibility of the platform was sufficient to accommodate pharmacokinetic studies of different numbers of animals and time points. To the best of our knowledge, this represents the first complete automation of the protein precipitation method for plasma sample analysis.     

Development of a technology for automation and miniaturization of protein crystallization

The usage of standard 96 well microplates for the screening of crystallization conditions of recombinant proteins offers several advantages when compared to commonly used crystallization plate formats. The adoption of robotic technology for plate and glass slide preparation within a "hanging drop" vapour diffusion crystallization experiment enables to work with an increased throughput at reduced costs. In addition to commercial pipetting devices with a 96-channel aspirator/dispenser, solenoid ink-jet technology was applied to form 250 nl droplets with a diameter of approximately 1 mm. This allows miniaturization of crystallization screening set-ups with an estimated ten-fold cost reduction when compared to commonly used 24 well plates.