Apparatus and method for producing specimens for electron microscopy

#The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

#Molecular microscopy is a non-invasive electron imaging technology that uses advanced specimen preparation and imaging methods designed specifically to visualize complex biological samples, under conditions close to their native state. Samples are preserved in a very thin film of solution on an EM grid, by vitrification (using an automated cryogenic robot) or by negative stain, and then imaged using a transmission electron microscope (TEM) controlled by automated software that enables sampling of a significant portion of the specimen. High-throughput molecular microscopy combines robotic instruments, automated data collection and processing software, and a relational database into a pipeline to prepare, image, and analyze samples in a reproducible manner and with throughputs capable of addressing biopharmaceutical characterization needs in a statistically significant manner. For well-ordered samples such as viruses, and virus-antibody complexes, the achievable resolution can be <0.4 nm.

#In preparing EM grids for molecular microscopy, preparation of the sample grid is often the least controlled step in the process. Typically, a few microliters of sample are applied to a specimen grid. Excess sample is removed by contact blotting with a piece of absorbent paper to thin the film of solution. Finally, vitrification, achieved by plunging the thin sample into a liquid coolant (typically liquid ethane). The ultimate thickness of the sample is related to, among other things, the relative hydrophilicity of the grid surface, the relative humidity and temperature of the environment, and the length of blotting time. In addition, once the solution is applied to the grid it has an extremely high surface to volume ratio and its behavior is dominated by the heat and mass transfer that result from evaporation of the aqueous sample. The result of combining these events is that grid surfaces are not reproducible, either within the same grid or within grids. Portions of the specimen grid will be too thick for effective penetration by the electron beam, while others will be too thin for proper specimen preservation, while still others will suffer from increases in solute concentrations due to evaporation of water.

#Over thirty years ago, Dubochet and colleagues first demonstrated that biological specimens in their native hydrated state could be embedded in vitrified buffer and imaged in the transmission electron microscope (TEM) without loss of structural preservation. This paved the way for the development of cryo-TEM as a field, and the technique has been used to study a vast array of structures over the past 3 decades. Specimens for cryo-EM are normally prepared by embedding them in a thin layer of vitrified ice suspended over an EM grid substrate that may be either continuous or fenestrated. Ideally the vitrified layer should be just slightly thicker than the diameter of the particle (50-500 nm), providing a well distributed and well separated single layer of particles in random orientations.

#FIG. 1 is a schematic illustration of traditional blotting method and inkjet dispensing method disclosed herein. (A) Traditional blotting method. (B) New method utilizes inkjet dispensing. (C) piezo-electric based inkjet head.

#FIG. 2 shows important time points starting from droplet launch out of the dispense head to the desired time of vitrification.

#Over thirty years ago, Dubochet and colleagues first demonstrated that biological specimens in their native hydrated state could be embedded in vitrified buffer and imaged in the transmission electron microscope (TEM) without loss of structural preservation. This paved the way for the development of cryo-TEM as a field, and the technique has been used to study a vast away of structures over the past 3 decades. Specimens for cryo-EM are normally prepared by embedding them in a thin layer of vitrified ice suspended over an EM grid substrate that may be either continuous or fenestrated. Ideally the vitrified layer should be just slightly thicker than the diameter of the particle (50-500 nm), providing a well distributed and well separated single layer of particles in random orientations.

#Current methods of preparing the vitrified sample typically start with applying a relatively large volume (˜3 μl) of sample to the surface of an EM grid, then reducing the sample to a thin layer by applying filter paper to one or both sides of the grid to blot away most of the volume, followed by quick plunging into liquid ethane FIG. 1A. A variety of devices have been developed to try to optimize this method, including both home-made guillotine plungers and more automated commercial devices. All of these devices, however, rely on using macroscopically large filter paper surfaces to try to produce a layer of liquid some 100's of nm thick. These methods allow very limited control of the sample layer on the substrate surface and, not surprisingly, the outcome of these methods is not optimal, normally resulting in large areas of the grid surface area that have vitrified ice that is either too thick or too thin and particles are also sometimes distributed unevenly as a result of varying ice thickness. In addition to the traditional method of specimen pipetting and blotting, variant methods have been described that involve creating a dense spray of specimen (1-20 um droplets) using nebulizers, micro-nozzles and electrospray mechanisms, where the grid is plunged through the sprayed region on its way to vitrification. Droplets landing on the grid surface do not need further blotting and spread out on contact due to surface hydrophilicity. However, there is little control over individual droplet sizes and the timing of when those droplets impact the grid surface. Moreover, the liquid-handling devices used for these processes often entail large dead-volumes and thus specimen wastage, probably a primary reason why this particular method has not seen more mainstream usage.

#The quality and thickness of the vitrified layer will depend on a number of factors including the properties of the specimen itself (e.g. viscosity, particle concentration), the nature of the substrate surface (e.g. hydrophobicity, flatness), the blotting paper (e.g. angle of contact, surface roughness, absorbency, blot time), and the plunging parameters (e.g. dwell time after blotting, speed of plunge, temperature of the cryogen). Thus, even highly skilled technicians may need to spend considerable effort optimizing this process for a new specimen; precious specimen is consumed by each trial, and time is required both in the laboratory and on the microscope to evaluate the prepared grids.