Surface Hydration with an Ion Beam

Abstract

Systems and methods for controllably forming an analyte layer comprising amorphous ice and/or other frozen amorphous solids on a substrate. In an embodiment, the present invention provides simplified systems and methods for the preparation of cryo-EM samples, where the same particle beam, such as an ion beam, is used to deposit the desired analyte onto the substrate as well as to generate the amorphous ice or frozen solid layer on the substrate

Claims

1. A method for depositing an analyte on a substrate comprising the steps of: a) forming an analyte solution comprising analyte particles and a solvent; b) generating an analyte beam from the analyte solution, where the analyte beam comprises charged or uncharged analyte particles and molecules of the solvent; c) directing the analyte beam toward a substrate surface at atmospheric pressure or under a vacuum such that the charged or uncharged analyte particles and molecules of the solvent impinge on the substrate surface, wherein the substrate surface is at a temperature of 0° C. or less thereby forming an amorphous solid layer of the solvent on the substrate surface, wherein the amorphous solid layer has a thickness of 10 microns or less, and wherein the charged or uncharged analyte particles are embedded within the deposited amorphous solid layer.

2. The method of claim 1, wherein the substrate surface is at a temperature of −100° C. or less.

3. The method of claim 1, wherein the analyte beam is an ion beam formed using electrospray ionization (ESI) or laser desorption.

4. The method of claim 1, wherein the solvent comprises cyclohexanol, methanol, ethanol, isopentane, water, O.sub.2, Si, SiO.sub.2, S, C, Ge, Fe, Co, Bi, and combinations thereof.

5. The method of claim 1, wherein the solvent is water.

6. The method of claim 1, wherein the substrate is an electron microscopy (EM) grid comprising a continuous film or membrane positioned across a top or bottom surface of the EM grid.

7. The method of claim 1, wherein the analyte beam is directed toward the substrate surface under vacuum, wherein the charged or uncharged analyte particles and molecules of the solvent contact the substrate surface at a pressure equal to or less than 10.sup.−1 Torr.

8. The method of claim 7, wherein the charged or uncharged analyte particles and molecules of the solvent contact the substrate surface at a pressure equal to or less than 10.sup.−3 Torr.

9. The method of claim 1, wherein the amorphous solid layer has a thickness of 5 microns or less.

10. The method of claim 17, wherein the analyte beam is generated using a mass spectrometer device, wherein generating an analyte beam comprises performing mass spectrometry analysis on a mixture of particles, identifying desired analyte particles within the mixture, and isolating the desired analyte particles from the mixture based on the mass, size, mass-to-charge ratio, or combinations thereof, of the desired analyte particles.

11. The method of claim 10, wherein generating an analyte beam comprises isolating particles having a mass-to-charge-ratio within 2 m/z to the desired analyte particles.

12. The method of claim 10 further comprising enriching, reducing, or altering the solvent in the analyte solution to generate the analyte beam.

13. A sample preparation system comprising: a) a vacuum chamber or gas chamber; b) a substrate positioned with the vacuum chamber or gas chamber, wherein said substrate comprises a receiving surface; c) a temperature control means able to provide a temperature of 0° C. or less to the receiving surface of the substrate; and d) an analyte source in fluid communication with the vacuum chamber or gas chamber, wherein the analyte source is able to produce a controllable analyte beam comprising charged or uncharged analyte particles and molecules of a solvent, and direct said analyte beam to contact the receiving surface of the substrate.

14. The system of claim 13, wherein the analyte source is able to generate an ion beam using electrospray ionization (ESI) or laser desorption.

15. The system of claim 13, wherein the temperature control means is able to provide a temperature of −100° C. or less to the receiving surface of the substrate.

16. The system of claim 13, wherein the system is a cryo-electron microscopy (cryo-EM) system and the substrate is part of a cryo-EM probe.

17. The system of claim 13 further comprising a modified mass spectrometer device able to provide ions and molecules of the solvent to the analyte source.

18. The system of claim 17, wherein the modified mass spectrometer device is able to isolate particles in a mixture, wherein the isolate particles have a mass-to-charge-ratio within 2 m/z to preselected desired analyte particles, and wherein the modified mass spectrometer device is able to perform mass spectrometry analysis on the isolated particles.

19. The system of claim 13, wherein the vacuum chamber or gas chamber is a vacuum chamber able to provide a pressure equal to or less than 10.sup.−1 Torr.

20. The system of claim 13, wherein the vacuum chamber or gas chamber is a vacuum chamber able to provide a pressure equal to or less than 10.sup.−3 Torr.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 mass spectrum obtained by analyzing apoferritin ions landed onto a cryogenically cooled electron microscope grid using an ion beam containing residual water. A cluster of m/z peaks is present centered at 9,065 m/z. This corresponds to the ˜60 charge state of the apoferritin complex. The width of these m/z peaks suggests that some residual water molecules are likely remaining on the apoferritin ions.

[0028] FIG. 2 shows a mass spectrum using the same ion beam as FIG. 1, but after using a quadrupole mass filter to filter for ions having an m/z value between ˜9,000 and 10,000.

[0029] FIG. 3 shows an EM image of a grid hole of an electron microscope grid treated with the unfiltered beam utilized in FIG. 1. A layer of amorphous ice has formed, but no apoferritin can be visualized, likely because the ice is too thick to allow its observation.

[0030] FIG. 4 shows an EM image of a grid hole of an electron microscope grid treated with the filtered beam utilized in FIG. 2. Particles can be seen that are most likely one or two apoferritin subunits given that they are too small to be the intact complex.

[0031] FIG. 5 shows EM images of a carbon TEM grid cooled to −190° C. and treated with an ion beam containing GroEL proteins and residual water. The grid was removed from the chamber and negative stained. A layer of amorphous ice has formed, and GroEL particles can be seen gathered together in small pools or groups.

[0032] FIG. 6 shows a cryo-electron microscopy (cryo-EM) sample preparation system in an embodiment of the present invention.

[0033] FIG. 7 shows an ion analyte source used in an embodiment of the invention, where the analyte source comprises ions optics (such as and skimmers) to focus and direct an ion beam.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Ion beams are routinely generated for many applications including mass spectrometry. In mass spectrometry ion beams can be made in a variety of ways, perhaps the most prevalent being electrospray ionization (ESI). In ESI, analytes are diluted in solutions that typically contain some fraction of water or another solvent. This solution is then placed in a needle or capillary and an electric field applied. The application of an electric field and flow of the solution, either forced or induced by the field as in static nanospray, results in the formation of a plume of charged droplets. These charged droplets can be directed towards and into the inlet of a mass spectrometer. During their transfer and passage into the mass spectrometer inlet and into the vacuum system of the mass spectrometer, they continue to undergo desolvation and evaporation such that a beam containing charged analyte ions is created. These ions are then manipulated in various ways using the techniques of mass spectrometry.

[0035] The present study discovered that ion beams generated in this way also contain significant amounts of water or other solvents. The water or other solvent molecules sometimes remain attached to the analyte ion to form a solvent-analyte ion complex, such as a water-analyte ion complex. In conventional systems, techniques are typically used to ensure removal of the water so as to allow for the precise measurement of the analyte's mass. Water-containing ionic clusters that are devoid of analyte also exist. Often background signals are observed in mass spectra that might be attributed to such species, but, because their mass-to-charge values likely span a broad range on account of a highly varied distribution of sizes, these water-containing ionic clusters are often not observed in mass spectra. In certain embodiments, the optics of the mass spectrometry device may be adjusted to remove excess droplets and solvent clusters. Alternatively, such droplets and solvent clusters may be preserved.

Example 1—Surface Hydration with an Ion Beam

[0036] In an example, a modified mass spectrometer was used to land an ion beam onto a cryogenically cooled electron microscope grid. The ion beam was generated following electrospray of an aqueous solution of protein complex apoferritin. The mass spectrum obtained by analyzing the resultant ions in an Orbitrap mass spectrometer are shown in FIG. 1, where there is a cluster of m/z peaks centered at 9,065 m/z. This corresponds to the ˜60 charge state of the apoferritin complex. The width of these m/z peaks suggests that some residual water molecules are likely remaining on the apoferritin ions. The mass spectrum in FIG. 2 shows the same ion beam but after a quadrupole mass filter has been used to allow ions having an m/z value between 9,000 and 10,000 pass.

[0037] To understand the makeup of the ion beam that produced the spectra shown in FIGS. 1 and 2, the unfiltered beam was diverted to the cryo-cooled EM grid. The image on in FIG. 3 shows a close-up of one of the grid holes, where a layer of amorphous ice has formed. No apoferritin can be visualized—likely because the ice is too thick to allow its observation. FIG. 4 shows an image when the filtered beam (lower mass spectrum) is directed toward the surface. Here, particles can be seen that are most likely one or two apoferritin subunits given that they are too small to be the intact complex.

[0038] Exposure to radiation causes damage to the particles, which is expected behavior for proteins. Also notice the ring in the center of the image. This ring provides evidence that for a time liquid water was present prior to freezing. The ice thickness on the filtered ion beam is much thinner than that observed on the unfiltered ion beam.

[0039] These data provide direct evidence that a substantial component of the ion beam comprises water—in water-containing ionic clusters and/or attached to analyte ion clusters. This water contained in an ion beam, which has not been documented to such an extent before, can be used in the present invention to have practical purposes. In particular, the water or other solvent contained in the ion beam can be used to hydrate a surface, or otherwise apply the solvent to a surface. Such surface hydration can provide protection to radiation sensitive particles or samples that are also on the surface, including particles and samples derived from the same ion beam.

[0040] In a further example, a modified mass spectrometer was used to treat a cryogenically cooled electron microscope grid with an ion beam generated following the electrospray of an aqueous solution of GroEL, a bacterial chaperonin protein.

[0041] A carbon TEM grid was cooled to −190 degrees Celsius and the ion beam was used to soft land GroEL proteins onto the grid for a period of 30 minutes. While keeping the grid at −190, the pressure in the vacuum chamber was raised to atmospheric pressure. Once up to pressure, the grid was warmed to room temperature, 22 degrees Celsius (still in a helium environment). The warming time, with the assistance of a resistive thermal heater, was ten minutes. The grid was then removed from the chamber and negative stained. FIG. 5 shows two images which show that some of the GroEL survived and appeared to gather in small pools. Thus, these experiments illustrate that landing bioparticles on a cryogenic surface can preserve molecules using a single ion beam without the use of additional other sources of water or other solvents.

Example 2—Cryo-EM Sample Preparation Instrument

[0042] FIGS. 6 and 7 show an exemplary cryo-electron microscopy (cryo-EM) sample preparation systems 25 according to certain embodiments of the present invention where the analyte beam is used to deposit the analyte particles and amorphous solid layer. A cryo-EM probe 2 able to hold or contain a sample is inserted into vacuum chamber 1 (or gas chamber). The temperature of the system is maintained using a coolant, such as liquid nitrogen, which is stored in tank 8 and transferred through cold finger 5, while one or more turbo pumps 9 are used to maintain the vacuum.

[0043] Analyte particles and molecules of the solvent, typically in the form of a vapor, are collected in an analyte source 6 where they are focused into an analyte beam 13 (such as through electrospray ion deposition) and directed to contact the sample plate being held by cryo-EM probe 2. FIG. 7 shows one type of an analyte source 6 where analyte particles and solvent molecules are drawn into the analyte source 6 through capillary 16. One or more ion optic devices, such as skimmers 17, are used to focus the analyte ions and solvent molecules into a beam 13 and to control the release speed of the ions and molecules through exit aperture 18. An optical detection cell 11 can be used to monitor whether the deposited ice layer comprises vitreous ice or crystalline ice.

[0044] Having now fully described the present invention in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

[0045] When a group of materials, compositions, components or compounds is disclosed herein, it is understood that all individual members of those groups and all subgroups thereof are disclosed separately. Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. Additionally, the end points in a given range are to be included within the range. In the disclosure and the claims, “and/or” means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.

[0046] As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.

[0047] One of ordinary skill in the art will appreciate that starting materials, device elements, analytical methods, mixtures and combinations of components other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which 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. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Headings are used herein for convenience only.

[0048] All publications referred to herein are incorporated herein to the extent not inconsistent herewith. Some references provided herein are incorporated by reference to provide details of additional uses of the invention. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art.