WO2013109405A1

WO2013109405A1 - Preparation of specimen arrays on an em grid

Info

Publication number: WO2013109405A1
Application number: PCT/US2013/000018
Authority: WO
Inventors: Bridget CARRAGHER; Clinton S. POTTER; Tilak Jain
Original assignee: The Scripps Research Institute
Priority date: 2012-01-17
Filing date: 2013-01-14
Publication date: 2013-07-25
Also published as: US20170025250A1; US9952128B2; US20150090899A1; EP2805144A1; US9594008B2; EP2805144A4

Abstract

The invention provides methods and compositions for preparation of complex specimen arrays for analysis by electron microscopy. These methods and compositions can permit high throughput screening of samples on single EM grid supports using sample volumes in the nanoliter and picoliter range.

Description

PREPARATION OF SPECIMEN ARRAYS ON AN EM GRID

BACKGROUND OF THE INVENTION

[0001 ] 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.

[0002] Screening of samples using transmission electron microscopy (EM) is often used in to characterize proteins such as antibodies used for therapeutics, viruses or virus like particles used in vaccines, drug delivery particles, or other formulations of nanoparticles. Screening may also be used to determine optimal conditions for 2D and 3D crystallization of proteins, optimizing preparation conditions for a novel protein or other macromolecular complexes, as well as formulation optimization.

[0003] EM applications such as those discussed above often require the analysis of a large number of conditions in parallel. EM sample preparation typically requires a

cumbersome procedure of obtaining several negative stained samples on EM grid supports for each condition. EM grids typically comprise of a 3 mm diameter copper mesh (-25 μιη thick) with open square windows (30 to 200 μιη wide) acting as the base substrate. A thin (typically 5-50 nm) carbon Film is layered on top this substrate, creating electron transparent ' (carbon Film) regions in the open windows. Other EM grid substrate materials are usually made of other metals or semiconductor materials such as silicon, with films made of Silicon Nitride, Silicon Dioxide or Silicon Carbide. By way of example, each condition being analyzed can require (i) plasma treating one or more grids to create a hydrophi lic specimen surface, (ii) pipetting 2-3 μL· of the appropriate sample in an appropriate buffer onto the specimen surface, (iii) blotting the grid using Filter paper to remove excess sample, (iv) pipetting 2-3 μΙ_, o stain immediately onto the specimen surface to avoid sample drying, (v) blotting the grid again using Filter paper to remove excess stain, and (vi) allowing the stain to dry. In many cases staining using this process requires optimization of several conditions, such as concentration (sample and stain), buffer constituents, pH, sample and stain application time. This results in a large number of grid trials, which entail loading individual grids into the electron microscope for analysis of each condition, and wastes large volumes of what are often very precious sample material. BRI EF SUMMARY OF THE INVENTION

[0004] It is an object of the invention to provide methods and compositions preparation of complex specimen arrays for analysis by electron microscopy. These methods and compositions can permit high throughput screening of samples on single EM grid supports using sample volumes in the nanoliter and picoliter range (required for rare and di fficult to obtain samples). Because picoliter scale volumes will not cover an entire EM sample grid surface under achievable contact angles, each EM sample grid may be processed to provide an array of specimen locations on a single grid.

[0005] In a first aspect, the present invention provides methods for preparing an electron microscopy sample on an EM sample grid. The methods comprise:

a. dispensing a plurality of discrete specimens onto an EM sample grid in an ordered array of specimen locations, each specimen of the plurality of discrete specimens being placed into an individual specimen location in the array of locations, and each individual specimen location in the array of locations having an area of between about 2000 μπι² to about 70,000 μιη² (for example, each location is a circular spot of between about 50 μιη and about 300 μηι in diameter); and

b. applying a discrete volume of a stain material suitable for contrast enhancement in an electron microscope to each individual specimen location in the array of locations that received a specimen.

[0006] In certain embodiments, a wash step precedes the application of stain to the specimen locations. In preferred embodiments, excess wash material is removed with a porous or bibulous material, or microstructures that induce local capillary effects, that "blots" or "wicks" material away from the specimen location. By way of example such a method may comprise:

a. dispensing a plurality of discrete specimens onto an EM sample grid in an ordered array of specimen locations, each specimen of the plurality of discrete specimens being placed into an individual specimen location in the array of locations, and each individual specimen location in the array of locations having an area of between about 2000 μ^ηι² to about 70,000 μηι² (for example, each location is a circular spot of between about 50 μ^ηι and about 300 μιη in diameter); b. applying a discrete volume of a wash solution to each individual specimen location in the array of locations that received a specimen, and removing excess wash solution by contact with a bibulous or porous material at the periphery of each ind ividual specimen location in the array of locations; and

c. applying a discrete volume of a stain material suitable for contrast enhancement in an electron microscope to each individual specimen location in the array of locations that received a specimen.

[0007] Suitable stains for use in the present methods include ammonium molybdate, uranyl acetate, uranyl formate, phosphotungstic acid, osmium tetroxide, osm ium ferricyanide and auroglucothionate, commercially avai lable stains such as NanoVan and NanoW as wel l as other stains that are described in the general l iterature.

[0008] Creation of the blotting regions on the grids can be performed by a variety of fabrication techniques. The material used as the blotting material can be patterned by microfabrication techniques on the grid. By way of example only, thin film blotting material (such as dried gels,adsorption papers and porous membranes) can be laser machined and then adhered to the surface of a grid. In another example, the blotting material in l iquid form can be printed using inkjet printing or stamped using soft-contact l ithography, and then desiccated. Other methods can include creating nano-wires and polymer-matrixes by first forming a patterned seed layer and subsequent deposition / polymerization. This list is not meant to be limiting.

[0009] In various embodiments, the sample vol me(s) are appl ied to subregions of the EM sample grid surface by spotting such as by the use of robotic m icropipetting techniques, or more preferably using "ink jet" printing technologies. I nk jet printing technologies known in the art include devices equipped with pins, or sample ejection elements that d ispense using thermal, sonic, or piezoelectric impulses. Among the methods mentioned above, the inkjet method is a preferred sample application method because of its ability to carry out high- density, precise spotting. As used herein, the term "picol iter vol umes" refers to a volume of liquid that is at least 1 pL and which is less than 1 nL; "nanoliter volumes" refers to a volume of liquid that is at least 1 nL and which is less than 1 iL; and "m icroliter volumes" refers to a volume of l iquid that is at least 1 μL· and which is less than 1 niL.

[0010] The inkjet method is a method in which a solution of interest is placed in an extra- fine nozzle, pressure or heat is instantaneously applied on a portion near the nozzle's lip to correctly eject an extremely low volume of aqueous material from the nozzle's tip and directed to the surface of the EM grid. For example, the inkjet head may be a bubble-jet head having a mechanism for discharging a solvent with the appl ication of thermal energy; a piezo-jet head that ejects a solution using a piezoelectric element; etc. Preferably, the drop dispenser comprises multiple nozzles that can be used to "print" sample volumes, wash solutions, stains, etc., onto the discretely addressable sample locations of the EM sample grid. When the aqueous solution is ejected from the inkjet head, each droplet forms a circu lar spot, the thickness and expansion of which is controlled by the structure of the EM grid surface. Connection with an adjacent spot can be effectively prevented even when the spots of sample solution are spotted in high density. On a standard ~3 mm diameter EM grid (2 mm diameter imaging area), when the dispensed spots are 50-300 μιη in diameter, transfer of the entire contents of the 12, 24, 48, 96 or 384 well-plate is possible.

[001 1 ] In a related aspect, the present invention relates to EM specimen grids that are configured for use in the present invention. The EM specimen grids comprise a plural ity of discrete specimen locations delimited from one another by peripheral regions comprising a bibulous or porous material, or microstructures that induce local capil lary effects, that "blots" or "wicks" material. These grids are referred to herein as "blotting m icrowell array^" or "BMA" grids.

[0012] In another related aspect, the present invention relates to a system for dispensing aqueous materials onto an EM sample grid at individual specimen locations in an ordered array of specimen locations, each individual specimen location in the array of locations having an area of between about 2000 μηη² to about 70,000 μηι². The systems comprise: a. a holder for reversibly receiving an EM sample grid;

b. a picol iter to nanoliter volume drop dispenser (e.g., an inkjet printing element) configured to dispense fluid from one or more dispensing elements onto each individual specimen location in the array of locations;

c. a drive mechanism to position the EM sample grid relative to the one or more dispensing elements; and

d. one or more reservoirs operably linked to the drop dispenser for holding one or more aqueous solutions to be dispensed onto each individual specimen location in the array of locations.

[00 1 3] The present invention is particularly appl icable to transm ission electron microscopy. Transm ission electron microscopy (TEM) is a m icroscopy techn ique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transm itted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic fi lm, or to be detected by a sensor such as a CCD camera. The image is in effect assumed to be a simple two-d imensional projection of the sample down the optical axis.

[0014] The methods and compositions of the present invention may be used to analyze particles selected from the group consisting of polymer beads, metal beads, proteins, protein- metal bead complexes, protein-polymer bead complexes, viruses, virus-l ike particles, liposomes and other nanoparticles. The methods may also be used to analyze sub micron aggregates of these particles.

[001 5] DETA I LED DESCRI PTION OF TH E IN V ENTION

[0016] Molecular microscopy is a non-invasive molecular imaging technology that uses advanced specimen preparation and imaging methods designed speci fically to visualize complex biological samples, under conditions close to their native state. For well-ordered samples such as viruses, and virus-antibody complexes, the achievable resolution can be <0.4 nm. 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 statistical ly signi ficant manner. Samples are preserved in solution by vitri fication (using an automated cryogenic robot) or by negative stain, and then imaged using a transm ission electron m icroscope (TEM) controlled by automated software that enables sampl ing of a sign i ficant portion of the specimen. Data is analyzed and stored in a secure database that tracks all aspects of sample preparation, imaging, and analysis to provide our current customers with a tightly control led system for biological imaging.

[001 7] In electron microscopy, stain ing is usually done with heavy metal salts common ly derived from molybdenum, uranium, or tungsten. Heavy ions are used since they wi l l readily interact with the electron beam and produce ampl itude contrast. A smal l drop of the sample is deposited on the carbon coated grid, allowed to settle for approximately one m inute, blotted dry if necessary, and then covered with a small drop of the stain (for example 2% ranyl acetate). After a few seconds, this drop is also blotted dry, and the sample is ready to be imaged in the TEM.

[001 8] The present invention here describes methods and compositions for conducting a high-throughput screen of samples on a single EM compatible grid . As shown in Fig. 1 , a standard wel l-plate (96 or 384 wells) contains the sample conditions to be tested (in lower throughput screens 12, 24 and 48 wel l-plates can also be accommodated). An inkjet head capable of delivering samples (picol iters to microl iters) transfers the sample condit ions from the stock plate onto a targeted area of a single EM grid. The dispensed samples are registered precisely for downstream identification and tracking during EM imaging at low and h igh magnification. Multiple inkjet heads can be used to fac il itate sample d ispensing onto the EM grid. On a 3 mm diameter grid (2 mm imaging area), when the d ispensed spots are 50-300 μιη in diameter, transfer of the entire contents of the 12, 24, 48, 96 or 384 well-plate is possible. This allows for complete mapping of sample conditions from the standard wel l- plate onto the grid. For screens requiring more thousands of sample cond itions, only a few EM grids will be required.

[0019] In one scenario of the invention, the samples are dispensed and dried on the grid prior to any staining. This scenario can be used i f the samples are relatively stable and the drying (accompanied by phenomenon such as salt crystal l ization) does not lead to particle destabilization or staining fai lure. In such situations, once inkjet sample transfer is complete, the grid can be washed and flooded with stain (3 μί). Alternatively, the stain can be dispensed onto the individual sample spots on the grid using a single inkjet head that precisely targets the registered areas. As shown in Figure 2, if multiple heads are used, the dispensing of stain can take place before the dispensed sample dries. In either case, dispensing of stain using an inkjet head al lows for much greater control of volume and uniformity of spreading across the grid, which is not possible with the standard blotting process. Additionally, multiple staining conditions (concentration and type of stain) can be tested on similar sample conditions. Multiplexing at the grid level al lows on ly a single grid (or a few, compared to hundreds to thousands) to be loaded in the electron m icroscope for the screen.

[0020] Sample constituents can include dissolvable materials such as sugars, gels and buffer salts that prevent the destabil ization of sensitive samples during the brief period of evaporation after the first droplet lands and spreads on the grid. As shown in Figure 2, diffusion of the stain particles occurs after the second droplet lands on the sample spot. A long with the spatial precision of droplet transfer, the time interval between the first and second droplet can also be accurately controlled within a few hundred m i l l iseconds to seconds. Mu ltiple dispense heads can al low for intermediate washes, bindi ngs and reactions, between the sample and stain droplet. The surface properties of the grid (flatness, wettabi lity and atomic roughness) govern the spreading of droplets given comparable environmental conditions. The grid surface can be made hydrophi lic (or super-hydrophilic) to ensure rapid spreading of the droplets and faster diffusion between the sample and stain.

[0021 ] To further control sample washing and staining without significant evaporation prior to drying, an array of blotting material can surround the targeted area on the grid. A fter sample spotting, the wash and stain steps with larger d ispense volume leads to local blotting in the surrounding material. In this manner the samples can be washed, without signi ficant buildup in the target area. Similarly, the subsequent dispensed stain wi l l be blotted local ly to create an even layer of negatively stained sample. As noted above, the material used as the blotting material can be patterned by m icrofabrication techniques on the grid. In one method, thin film blotting material (such as dried gels, adsorption papers or porous membranes) can be laser machined and then adhered to the surface of a grid. I n another method, the blotting material in liquid form can be printed using inkjet printing or stamped using soft-contact lithography, and then desiccated. Other methods can include creating nano-wires and polymer-matrixes by first forming a patterned seed layer and subsequent deposition / polymerization. Other methods can include creating m icrostructiircs, surround ing the targeted areas that induce local capi llary effects, such as an overhanging ledge or spiral with spaces of 0.5 to 10 μηι between hydrophilic walls. The B MA grids can be aligned accurately with the inkjet printer to dispense the droplets between the blotting areas. The blotting areas themselves can be used as physical markers for identi fying the registered samples and for downstream image recognition and processing.

[0022] While the invention has been described and exempl i fied in su fficient detai l for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as l imitations on the scope of the invention. Modi fications therein and other uses wi ll occur to those ski l led in the art. These mod i fications are encompassed within the spirit of the invention and are defined by the scope of the claims. [0023] It will be readily apparent to a person skil led in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0024] All patents and publications mentioned in the speci fication are indicative of the levels of those of ordinary skill in the art to which the invention pertains. Al l patents and publications are herein incorporated by reference to the same extent as i f each ind ividual publication was specifically and individual ly indicated to be incorporated by re ference.

[0025] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or lim itations that is not speci fical ly d isclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisti ng essential ly of and "consisting of may be replaced with either of the other two terms. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been speci fical ly disclosed by preferred embodiments and optional features, modi fication and variation of the concepts herein disclosed may be resorted to by those ski lled in the art, and that such modi fications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0026] Other embodiments are set forth within the fol lowing c laims.

Claims

We claim:

1 . A method for preparing an electron m icroscopy sample on an EM sample grid, comprising: dispensing a plurality of discrete specimens onto an EM sample grid in an ordered array of specimen locations, each specimen of the plural ity of discrete specimens being placed into an individual specimen location in the array of locations, and each ind ividual spec imen location in the array of locations having an area of between about 2000 μηΓ to about 70,000 μιη²; and applying a discrete volume of a stain material suitable for contrast enhancement in an electron microscope to each individual specimen location in the array of locations that received a specimen.

2. A method for preparing an electron m icroscopy sample on an EM sample grid, comprising:

dispensing a plurality of discrete specimens onto an EM sample grid in an ordered array of specimen locations, each specimen of the plural ity of discrete specimens being placed into an individual specimen location in the array of locations, and each ind ividual specimen location in the array of locations having an area of between about 2000 μητ to about 70,000 μιττ (for example, each location is a circular spot of between about 50 μιη and about 300 μηι in diameter);

applying a discrete volume of a wash solution to each individual spec imen location in the array of locations that received a specimen, and removing excess wash solution by contact with a bibulous or porous material at the periphery of each ind ividual specimen location in the array of locations; and

applying a discrete volume of a stain material suitable for contrast enhancement i n an electron microscope to each individual specimen location in the array of locations that received a specimen.

3. A method according to claim 1 or 2, wherein said d iscrete specimens are dispensed by applying more than material to each specimen location, wherein each specimen is formed by mixing the more than one material at each specimen location.

4. A method according to one of claims 1 -3, wherein the sta in materia l is selected from the group consisting of ammonium molybdale, uranyl acetate, uranyl formate, phosphotungstic acid, osm ium tetroxide, osm ium ferricyan ide and aurogl ucoth ionate, commercially available stains such as TManoW and NanoVan and other stains that have been described in the general literature.

5. A method according to one of claims 1 -3, wherein the bibulous or porous material is a microfabricated material applied to the surface of the EM sample grid, wherein the microfabricated material comprises a pattern of openings in the bibulous or porous material corresponding to the ordered array of specimen locations. .

6. A method according to one of claims 1 -3, wherein the bibulous or porous material comprises a dried gel material.

7. A method according to one of c laims 1 -3, wherein the bi bu lous or porous material comprises a fibrous material.

8. A method according to one of claims 1 -3, wherein the bibulous or porous material comprises a matrix forming one or more capi llary spaces.

9. An EM specimen grid, comprising:

a plurality of discrete specimen locations del imited from one another by peripheral regions comprising a bibulous or porous material, each individual specimen location in the array of locations having an area of between about 2000 μηι² to about 70,000 μηΓ.

10. A method according to claim 9, wherein the bibulous or porous material comprises a dried gel material.

1 1 . A method according to claim 9, wherein the bibulous or porous material comprises a fibrous material.

12. A method according to claim 9, wherein the bibulous or porous material comprises a matrix form ing one or more capi llary spaces.

13. A system for dispensing aqueous materials onto an EM sample grid at ind ividual specimen locations in an ordered array of specimen locations, each ind ividua l specimen location in the array of locations having an area of between about 2000 μιτι² to about 70,000 μιη². The systems comprise:

a holder for reversibly receiving an EM sample grid;

a volume drop dispenser configured to dispense fluid from one or more d ispensing elements onto each individual specimen location in the array of locations, at least one d ispensing element in the volume drop dispenser configured to d ispense picol iter volumes; a drive mechanism to position the EM sample grid relative to the one or more dispensing elements; and

one or more reservoirs operably l inked to the drop dispenser for holding one or more aqueous solutions to be dispensed onto each individual specimen location in the array of locations.