Apparatus and method for depositing ions on a surface - Patents.com

Abstract

An apparatus and method for preparing samples for examination by cryo-electron microscopy is described. Molecules of interest, such as protein molecules in solution, are electrosprayed and converted into singly charged gas-phase ions. A specific conformation or conformations of the protein ion are selected as a means of ion mobility filtering and deposited on a cryo-EM grid with sub-eV kinetic energies with the goal of preserving the higher-order structure of the molecule.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 288,478, filed December 10, 2021, which is incorporated herein by reference in its entirety. This application also claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 291,441, filed December 19, 2021, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT N / A

[0003] Notice of copyrighted material Portions of the material in this patent document may be subject to copyright protection under the copyright laws of the United States and other countries. The copyright owner has no objection to the reproduction by any person of either the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office publicly available files or records, but otherwise reserves all copyright rights. The copyright owner does not waive any rights to have this patent document maintained in confidence, including, but not limited to, the rights pursuant to 37 C.FR § 1.14.

[0004] A. Technical Field

[0005] The techniques of this disclosure relate generally to preparing molecular and particulate samples on surfaces, and more specifically to preparing samples for examination by cryo-EM. [Background technology]

[0006] B. References

[0007] The following publications are incorporated herein by reference in their entireties:

[0008] 1. Badu-Tawiah, AK, C Wu, and RG Cooks, Analytical Chemistry, No. 83, 2011, pp. 2648-2654.

[0009] 2. Baker ES, BH Clowers, F Li, K Tang, AV Tolmachev, DC Prior, ME Belov, Oyo RD Smith, Journal of the American Society for Mass Analytical Science, 2007, No. 18(7), pp. 1176-1187.

[0010] 3. Belov ME, BH Clowers, DC Prior, WF Danielson III, AV Liyu, BO Petritis, およびRD Smith, Analytical Chemistry, No. 80(15), 2008, pp. 5873~5883.

[0011] 4.Benesch JLP, BT Ruotolo, DA Simmons, NP Barrera, N Morgner, L Wang, HR Saibil, and Robinson CV, 2010, Journal of Structural Biology, No. 172, pp. 161~168.

[0012] 5. Blake TA, Z Ouyang, JM Wiseman, Z Taka'ts, AJ Guymon, S Kothari, Oyobi Cooks RG, 2004, Analytical Chemistry, 76, 6293-6305.

[0013] 6. Chaudhary A, FH van Amerom, RT Short, Scientific Machinery, No. 85(10), 2014, page 105101.

[0014] 7.Clowers BH, YM Ibrahim, DC Prior, WF Danielson III, ME Belov, およびRD Smith, Analytical Chemistry, No. 80(3), 2008, pp. 612~623.

[0015] 8. R. G. Cooks, D. Snyder, A. S. Basu, and W. P. Peng, 2021, U.S. Patent and Trademark Office No. 11,037,777.

[0016] 9. Deng ZT, N Thontasen, N Malinowski, G Rinke, L Harnau, S Rauschenbach, and K Kern, Nano Letters 2012, 12, 2452-2458.

[0017] 10. Earl et al., Current Opinion in Structural Biology, 2017, No. 46, pp. 71-78.

[0018] 11. Fernandez de la Mora, J., S Ude, and BA Thompson, Journal of Biotechnology, 2006, No. 1, pp. 988-997.

[0019] 12. Gologan B, JM Wiseman, and RG Cooks, 2006. In Laskin J and C Lifshitz C, eds., Principles of mass spectrometry applied to biomolecules, Hoboken, NJ: John Wiley & Sons, Inc.

[0020] 13. Hogan, Jr., CJ, EM Kettleson, B Ramaswami, DR Chen, and P Biswas, Analytical Chemistry, 2006, 78, 844-852.

[0021] 14. Johnson, G. E., D Gunarantne, and J Laskin, Mass Spectrometry Reviews, 2016, No. 35, pp. 439-479.

[0022] 15. Julian RR, SR Mabbett, Jarrold MF, Journal of the American Society for Mass Spectrometry, No. 16(10), 2005, pp. 1708-1712.

[0023] 16. Kaufman, S. L., J. W. Skogen, F. Dorman, F. Zarrin, and K. C. Lewis, Analytical Chemistry, 1996, No. 68, pp. 1895-1904.

[0024] 17. Kim T, AV Tolmachev, R Harkewicz, DC Prior, G Anderson, HR Udseth, and RD Smith, Analytical Chemistry, 2000, No. 72(10), pp. 2247-2255.

[0025] 18. Mikhailov VA, TH Mize, JLP Benesch, and Robinson CV, 2014, Analytical Chemistry No. 86, pp. 8321-8328.

[0026] 19. Ouyang Z, Z Takats, TA Blake, B Gologan, AJ Guymon, JM Wiseman, JC Oliver, VJ Davisson, and RG Cooks, 2003, Science 301, 1351-1354.

[0027] 20. Page JS, AV Tolmachev, K Tang, and RD Smith, Journal of the American Society for Mass Spectrometry, 2006, No. 17(4), pp. 586-592.

[0028] 22. Rinke, G., S Rauschenbach, L Harnau, A Albarghash, M Peuly, and K Kern, NanoLetters, DOI 10.1021 / nI502122j.

[0029] 22. Tang K, AA Shvartsburg, HN Lee, DC Prior, MA Buschbach, F Li, AV Tolmachev, GA Anderson, and RD Smith, Analytical Chemistry, 2005, No. 77(10), pp. 3330-3339.

[0030] 23. Tata, A., C Salvitti, and F Pepi, International Journal of Mass Spectrometry, 2020, No. 450, pp. 116309.

[0031] 24. Wang P and J Laskin, 2008, Applied Chemistry International, Vol. 47, pp. 6678-6680.

[0032] 25. Volny M, WT Elam, BD Ratner, and F Turecek, 2005, Analytical Chemistry No. 77, pp. 4846-4853.

[0033] 26. Walton, B. L., W. D. Hoffmann, and G. F. Verbeck, International Journal of Mass Spectrometry, 2014, 370, 66-74.

[0034] 27. Wyttenbach T, PR Kemper, and MT Bowers, International Journal of Mass Spectrometry, 2001, 212(1-3), pp. 13-23.

[0035] C. Background Considerations

[0036] In recent years, cryogenic electron microscopy (cryo-EM) has become a more accepted method for investigating the structure of single molecules, especially proteins. The increased acceptance is due in large part to improvements in hardware and software. The resolution of cryo-EM is beginning to rival that of X-ray crystallography, without the need to generate protein crystals as required for X-ray crystallography. Improvements in methods for preparing samples for examination by cryo-EM have lagged behind the development of cryo-electron microscopy, and have remained largely unchanged since the methods first developed in the 1980s. A typical method for preparing a sample for cryo-EM investigation follows several manual steps. First, the protein is purified and transferred into an appropriate aqueous buffer. Next, a small amount of the sample-containing buffer is pipetted onto a cryo-EM grid, after which most of the liquid is removed by absorbing the liquid onto a piece of blotting paper, so that the protein molecules adhere to the grid surface and are covered by a thin film of water. Finally, the wet grid is rapidly pressed into the cryogen in order to freeze the water film fast enough so that vitreous ice is formed rather than crystalline ice. The vitreous ice immobilizes molecules of interest for examination by cryo-EM. The indentation step is the most difficult to perform and, if not performed correctly, is the most frequent cause of sample preparation failure. Improvements are needed to prepare samples more efficiently and more reproducibly (Earl). Summary of the Invention [Problem to be solved by the invention]

[0037] The apparatus and methods described in this disclosure provide a new method for depositing ions and charged particles onto surfaces for subsequent analysis. The techniques described in this disclosure provide a means for depositing ions and charged particles onto surfaces in a process known as soft landing.

[0038] Soft landing refers to a condition that allows the ion or charged particle to land softly enough on the surface without the molecular conformation of the ion or charged particle being changed during the landing process and without the landing process destroying weakly associated molecular complexes or agglomerated particles. The concept of soft landing means that the kinetic energy of the ion or particle at the moment of impact is of an insufficient amount of energy to cause damage to the molecule or particle. [Means for solving the problem]

[0039] The major difference between the technique described in this disclosure and previous approaches for soft deposition of ions and particles on surfaces concerns the technique performed at atmospheric pressure. This new technique provides a means to land ions on fragile membranes, such as thin carbon or graphene films, which are easily damaged by high-velocity bombardment by ions or charged particles. The soft landing approach provides an alternative to the common technique of suspending the analyte molecules in frozen vitreous water ice, as is commonly done to prepare cryo-EM samples.

[0040] Further aspects of the technology described herein will be set forth in the remainder of the specification, the detailed description being intended to fully disclose preferred embodiments of the technology without imposing limitations.

[0041] The techniques described herein may be more fully understood by reference to the following drawings, which are for illustrative purposes only. [Brief description of the drawings]

[0042] [Figure 1] FIG. 1 is a diagram of the main components that make up a device for depositing mobility-selected ions or charged particles onto a collection surface. The inset shows the operation of the nDMA in more detail. [Diagram 2] FIG. 1 is a detailed view of the components used to deposit mobility selected ions or charged particles onto a collection surface. [Diagram 3] An ion detector is used to collect ions and charged particles for the purpose of detecting and recording a current of ions or charged particles. [Figure 4] FIG. 1 illustrates the design of a humidifier. [Diagram 5] FIG. 1 illustrates the design of an ion focusing funnel. [Figure 6] FIG. 13 is a diagram of a circular base and angled arms useful for securing the circular wiring that makes up the ion-focusing electrode. [Figure 7] Further details of the design of the ion-focusing funnel. The device of Figure 7 is inside a cylindrical cage (center). An end view shows the structure of the arms of Figure 6. [Figure 8A] FIG. 7 is a more detailed view of the device of FIG. 6, showing a means for fixing the wiring with the arm from the device of FIG. 6. [Figure 8B] 8B is a further detail of the device of FIG. 7 and FIG. 8A. [Figure 9] FIG. 2 is a diagram of a fixture for holding a substrate onto which ions or charged particles are deposited. [Figure 10A] FIG. 1 is a diagram of a means for cooling a substrate while measuring the temperature of the substrate. [Figure 10B] FIG. 1 is a diagram of a method and device for generating LN2 blow-off gas and a means for directing the blow-off gas to a substrate holder. [Figure 10C] FIG. 1 is an electrical schematic for providing heat to liquid nitrogen for the purpose of producing liquid LN2 blow-off gas. [Figure 11] FIG. 8 is a further view of the device of FIG. 7 showing the location of the ion or charged particle collector. [Figure 12] FIG. 1 is a diagram of a two-part cylindrical chamber useful for protecting ions or charged particles deposited on a collecting substrate. [Figure 13] 1 is a diagram of the connection between the outlet of the nDMA and the inlet to the ion-focusing funnel. The curves represent the diverging flows of gas. [Figure 14]A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 15] A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 16] A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 17] A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 18] A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 19]A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 20] A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 21] A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 22] A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Diagram 23] A diagram of ion trajectories (solid lines) within the ion-focusing funnel. The dotted lines represent the positions of the ions at different times. The small squares represent the positions of the circular wires that make up the electrodes within the ion-focusing funnel. The gas velocity, voltages applied to the electrode rings (listed from largest to smallest rings), and ion molecular weight and charge conditions are listed with the diagram. [Figure 24]2A-2C are two ion mobility spectra of tocilizumab ions recorded while the indicated voltages were applied to the exit ring of the ion-focusing funnel. [Diagram 25] 2A-2C are two ion mobility spectra of tocilizumab ions recorded while the indicated voltages were applied to the exit ring of the ion-focusing funnel. [Figure 26] 2A-2C are two ion mobility spectra of tocilizumab ions recorded while the indicated voltages were applied to the exit ring of the ion-focusing funnel. [Figure 27] 2A-2C are two ion mobility spectra of tocilizumab ions recorded while the indicated voltages were applied to the exit ring of the ion-focusing funnel. [Figure 28] 2A-2C are two ion mobility spectra of tocilizumab ions recorded while the indicated voltages were applied to the exit ring of the ion-focusing funnel. [Figure 29] 1 is a plot showing the mobility peak height of the M+, M2+ and 2M+ peaks as a function of voltage applied to the ion focusing funnel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] A. Introduction

[0044] This disclosure describes an apparatus and method for depositing ions and particles onto a surface using the following steps, all performed at atmospheric pressure: 1) converting a solution of analytes, such as a mixture of protein molecules or nanoparticles, into gas-phase ions or charged particles by means of electrospray; 2) selecting ions and particles having a predetermined electrical mobility from the mixture of ions and particles by a differential mobility analyzer (DMA); 3) exposing the mobility-selected ions and particles to a humidity-controlled gas to control hydration; 4) directing the ions and charged particles through an ion-focusing chamber where they are focused along the centerline of the chamber and further directed to land on a substrate at a low collision velocity; 5) changing the conformation of the ions and the shape of the particles by exposure to water vapor, heat or cooling; and 6) maintaining the conformation of the ions and the shape of the particles by freezing.

[0045] The techniques described in this disclosure provide improvements over previous designs. Compared to the generation of ion beams generated in a vacuum, a major improvement is achieved by operating the disclosed apparatus at atmospheric pressure, eliminating the need for a vacuum chamber and deceleration lenses to control the ion energy as the ions hit the collector. Selecting ions or particles of specific electrical mobility from a mixture of ions or particles by DMA provides a means to analyze ions and charged nanoparticles with specific conformations or shapes. Previous reports have used a system based on mass spectrometry rather than DMA to select ions or particles according to their molecular mass. One application of the disclosed techniques is the preparation of samples for subsequent examination by cryo-electron microscopy. A requirement of cryo-EM is to collect and analyze images of protein molecules that have the same conformation but are randomly oriented on the cryo-EM grid surface. Thus, the ability to select and deposit a large number of molecules with nearly identical conformations on a collecting surface such as a cryo-EM grid improves the quality of the cryo-EM images and reduces the number of molecular images that need to be processed to reveal high-quality molecular structures. The use of ion focusing in the present device provides a means to deposit the majority of mobility-selected ions onto the cryo-EM grid, thus minimizing the time required to deposit the required number of molecules onto the cryo-EM grid. The low energy deposition process provides a means to land undistorted proteins onto the cryo-EM grid, thus preventing collision damage. The combination of these advantages conceptualizes the soft landing device described herein. When particles are directed to strike a surface, they may bounce off the collection surface and be lost. Applying the disclosed soft landing technique to the process of collecting particles allows for better control of the deposition process. In this disclosure, the term "ion" or "ions" also refers to and encompasses the concepts of "particle," "articles," "nanoparticle," and "nanoparticles."

[0046] Ions land softly when they strike a surface at low velocity. Surface-induced dissociation is the opposite approach to current techniques, a process that induces ions to strike a surface at high velocity such that the kinetic energy of the ions is sufficient to fragment the ions when they strike the surface. A review of soft landing methodology and applications by Johnson describes previous attempts to deposit ions at low energies, typically kinetic energies below 200 eV. A series of reported developments reveal the exploration of lower kinetic energy collisions. Deposition of proteins using soft landing techniques based on the vacuum environment of a mass spectrometer has been reported by Gologan et al. and Wang and Laskin. Electrospray multiply charged protein ions that were deposited and then recovered intact were shown to retain biological activity (Volnay, 2005; Ouyang et al., 2003; Blake et al., 2004). Deng reported that the charge state of the deposited protein ions affects the structure of the deposited ions. They observed small cytochrome C structures when low charge states were deposited and spaghetti-like strings when more highly charged cytochrome C ions were deposited. Benesch et al. extended soft landing to large macroions of GroEL and reported that, overall, the shape of the GroEL complex survived the collision. However, Michailov et al. noted that depositing proteins on surfaces in the vacuum of a mass spectrometer is difficult, requiring low-temperature collision velocities below 100 eV (Rinke et al.). Walton et al. described deposition of ions in the sub-eV energy regime. Only a few publications have reported ion depositions performed at atmospheric pressure, and when such depositions are described, they do not include conditions for ion focusing (Tata, Badu-Tawiah et al.). The technique described in this disclosure induces ions, carried by a gas with controlled conditions of humidity and temperature, to strike a collecting surface at much lower energies than previously reported, such that the tertiary and quaternary structures of the protein are not damaged during deposition.The techniques described in this disclosure provide a means to deposit ions onto cryo-EM grids at deposition rates corresponding to ion energies of less than a few meV.

[0047] B. Exemplary Embodiments

[0048] 1. Gas-phase molecular ion generation

[0049] One mode for operating the apparatus described in this disclosure begins with selecting proteins for analysis by cryo-EM. Antibody molecules are an exemplary material processed by the disclosed apparatus. The charge-reduced form of electrospray ionization used in the techniques described in this disclosure can convert electrosprayed antibodies into singly charged gas-phase antibody ions. The charge-reduced electrospray process presents different sample preparation requirements than those typically performed by those skilled in the art of electrospray mass spectrometry. The charge-reduced process reduces the charge carried by the electrospray droplets to a low level. The low level of charge prevents droplet fission, i.e., the droplets are electrostatically split into progressively smaller droplets, ultimately forming nearly anhydrous protein ions. When non-volatile buffer salts are present in the electrospray droplets, the non-volatile salts form a thin coating on the surface of the analyte molecules, causing the size of the analyte ions to be larger than expected. In the present invention, the salt coating is minimized by using a molecular weight spin filter to efficiently remove non-volatile salts from the sample and exchanging the sample into a volatile buffer, such as an ammonium acetate solution. Spin filtration using molecular weight cut-off spin filters, for example passing the sample through an Amicon spin filter (PN UFC510096) several times, sufficiently reduces the salt content in the sample to acceptable levels, such as reducing the salt content to below micromolar concentrations. Spin filtering along with the use of a volatile buffer, such as 25-200 mM aqueous ammonium acetate, provides a means of producing uncontaminated, nearly anhydrous, single-charged phase ions.

[0050] Preparing samples for charge reduction electrospray further requires the use of low analyte concentrations, such as 1-50ug / mL. Just as the presence of non-volatile salts contaminates ions with a salt crust, the presence of two or more analyte molecules in a droplet will cause the analyte molecules to coalesce as the droplet evaporates, forming clusters such as dimers and trimers of analyte molecules. Dimers, trimers and any multimers that are produced by the use of relatively high analyte concentrations are called artificial aggregates. These are called artificial because they are not present in the sample solution before the sample solution is electrosprayed. Detection of artificial multimers is problematic because their presence can lead to the erroneous conclusion that the antibody molecules were already aggregated in solution before the droplets evaporated. For this reason, relatively low concentrations of antibody must be analyzed to prevent the formation of artificial dimers, etc.

[0051] The size of the primary electrospray droplet also affects the formation of artificial dimers, since as the droplet size increases, the probability of two or more antibody molecules being trapped within the droplet increases. Electrospray droplet size is related to viscosity, conductivity, surface tension, and sample delivery rate. The viscosity and surface tension of dilute ammonium acetate solutions can be modified by adding organic liquids, such as isopropyl alcohol or acetonitrile, to the sample. Addition of organic solvents is undesirable as the organic solvents may distort the conformation of the exemplary antibody. The conductivity of the sample increases with the concentration of ammonium acetate. A 200 mM ammonium acetate solution produces smaller droplets when electrosprayed than a 25 mM ammonium acetate solution, thus allowing higher antibody concentrations to be analyzed without the formation of artificial dimers, etc. Lower sample flow rates also produce smaller droplets than higher flow rates, but the electrospray is not stable at very low or very high flow rates, and therefore all conditions must be adjusted to produce a stable electrospray. Electrospraying a 100 mM ammonium acetate solution at 300 nL / min produces droplets with an average diameter of 250 nm, allowing analysis of analyte concentrations as high as 1 micromolar while minimizing the formation of artificial aggregates.

[0052] A schematic diagram of the apparatus described in this disclosure is shown in FIG. 1. A sample solution 102, such as an antibody dissolved in a dilute solution of ammonium acetate, is electrosprayed using an electrospray ion source 104. A preferred electrospray ion source is a charge-reducing source that produces singly charged ions. The ions are directed to a nano-differential mobility analyzer (nDMA) 106. The nDMA, labeled nDMA, shown in detail in inset 106a, provides a means for directing ions of a certain mobility through a humidifier and then introducing the ions into an ion-focusing device. It should be understood that many different types of humidifiers and ion-focusing devices have been described above. Often herein, humidifiers are referred to for use in controlling the water vapor content and temperature of ion-containing or particle-containing gas streams. An example of an alternative embodiment for humidifying a gas stream is a bubbler. An example of an alternative embodiment for a means for controlling the temperature of a gas stream is a cold tube of a hot tube. It can be an ion-focusing funnel, but any type of ion-focusing device may be used in place of the funnel-like device. As described in this disclosure, the ion focusing funnel focuses ions toward the centerline of the focusing funnel as they pass through it, with the goal of efficiently directing the ions toward a collecting surface, such as a cryo-EM grid located at the exit end of the focusing funnel. Collecting surfaces other than cryo-EM grids may be used and benefit from the use of ion focusing. Frequent references to cryo-EM or cryo-EM grids are used to illustrate exemplary applications for analyzing samples or exemplary subsequent analytical methods. One example of an alternative embodiment for providing ion focusing is an electrically resistive cylinder or tube, such as resistive glass from Photonis Inc, or a cylinder or tube made of antistatic plastic, such as polyurethane.

[0053] The illustrations in this disclosure use solid arrows to indicate fluid flow and flow direction. Dashed arrows serve to identify components in the figures. Referring again to FIG. 1, the liquid sample is directed to the electrospray ion source by a capillary tube, such as a 50 μm ID×220 μm OD fused silica capillary (Trajan Scientific, PN062444). The charge-reduced electrospray ions generated in the electrospray ion source are directed to the nDMA. The mobility-selected ions or charged particles are further directed through a temperature and humidity control device 108 to an ion or particle focusing funnel 110, where they are deposited on a collection surface 112 supported by a holder 114. The inset 106a in FIG. 1 shows the operation of the nDMA. A mixture of ions or charged particles 116 is directed into an annular space along the inside of the outer wall of the nDMA. The ions or charged particles are merged with a stream of clean sheath gas 118 and are then subjected to an electric field provided by a voltage supplied to a cylindrical inner member 120. The inset shows the result of a fixed voltage applied to the inner member. Ions of constant mobility 122 are induced to pass through an exit tube 124.

[0054] FIG. 2 details one embodiment of the technique described in this disclosure. A capillary tube 1 delivers a liquid sample to an electrospray ion source 2. The liquid sample contains the analyte molecules. An exemplary sample is an antibody dissolved in 100 nm ammonium acetate. A preferred capillary is a polyimide-coated fused silica capillary with an outer diameter of 220 μm and an inner diameter of 50 μm, less than 30 cm long, such as PN062462 from Trajan Scientific (Australia). At its inlet, the capillary tube 1 is provided with a pressurized sample source, such as a syringe pump or autosampler connected to a microfluidic pump that applies pressure to move the sample through the capillary tube 1. The capillary tube 1 is also provided with an electrical connection 1a connecting a voltage from a power supply 1b to the liquid sample flowing through 1a for the purpose of generating an electrospray. In a preferred embodiment, the electrical connection is a stainless steel microfluidic union (e.g., from IDEX Healthcare). Sometimes, it is necessary to replace the electrospray capillary 1 with a new capillary after the capillary becomes clogged. Because high voltage connection 1a is supplied with a high voltage, such as 2 kV, it is a safety requirement to remove the high voltage when changing the capillary. In the unlikely event that high voltage is not removed from 1a while the capillary is being changed, a current-limiting resistor and a bleed-down resistor are installed in the high-voltage wire leading from high-voltage source 1b to high-voltage connection 1a. The combination current-limiting resistor and bleed-down resistor 1c is shown in an enlarged view of 1c in Figure 2. Each electrical resistor in 1c has a resistance of 5 kV, 1 M ohms. 1c reduces, but does not eliminate, the possibility of exposure to high voltage if 1a is touched by an operator while high voltage is supplied to 1a.

[0055] Those skilled in the art of electrospray manufacturing will know that a tip on the exit end of capillary 1 allows for the formation of a stable electrospray. The tip on capillary 1 is held in a central position within electrospray ion source 2. Electrospray ion source 2 contains a small sealed source of radioactive polonium 210, such as a Model P-2042 NucleoSpot manufactured by NRD LLC, Grand Island, NY. Alpha particles emitted by the decay of polonium 210 atoms generate a cloud of bipolar air ions in the vicinity of the electrospray droplets. The bipolar air ions generated by the polonium 210 alpha particles interact with the charge carried by the electrospray droplets, reducing the charge on the droplets to a low level, such that a known percentage of the droplets carry a single charge as predicted by the Boltzmann charge distribution. The importance of generating singly charged ions is discussed below. The design of electrospray ion source 2 follows previous ion sources designed to reduce the charge on electrospray droplets and ions, such as designs described by Kaufman et al., Fernandez et al., and Hogan et al. The techniques described in this disclosure provide improvements over the work of Kaufman et al., Fernandez et al., and Hogan et al. In a preferred embodiment of the techniques described in this disclosure, a mass flow controller 3a provided with a pressurized source of dry air supplies 1.5 Lpm of dry air to the ion source 2. A second mass flow controller 3b provided with a supply of pressurized CO2 supplies 0.2 Lpm of CO2 to the ion source 2. Introducing a combined flow of air and CO2 to the electrospray ion source 2 provides a means of carrying the electrospray droplets emitted from the tip of the capillary 1 through the body of the electrospray ion source 2, where the droplets interact with the gas and air ions as they travel through the electrospray ion source 2. The use of mass flow controllers achieves better control of the gas supplied to the electrospray ion source 2 than previously described by Kaufman et al., Fernandez et al., and Hogan et al.Mass flow controllers provide more stable control of these gas flow rates than the previously described rotameters, especially when the supply of compressed air is provided by an air compressor, because the pressure delivered by the compressor is not constant over time, but rises and falls during the on-off cycles of the compressor pump.

[0056] With continued reference to FIG. 2, a video camera, such as a Dino-Lite model AD4113TL, www.dinolite.com, is attached to the ion source 2 and focused on the tip of the end of the electrospray capillary tube 1 as a means of inspecting the Taylor cone while the sample is being electrosprayed. Those skilled in the art of electrospray ion formation will appreciate the usefulness of the ability to inspect the stability of the Taylor cone. The Taylor cone represents the pointed shape of liquid emerging from the tip of the electrospray capillary. A live-in-time video image of the Taylor cone allows an operator to inspect the Taylor cone and adjust the voltage supplied to the sample as a means of producing a stable Taylor cone, i.e., a Taylor cone that does not flicker for the purpose of producing a stable ion current. The video camera implementation of the technology described in this disclosure is an improvement over the apparatus described by Kaufman et al., Fernandez et al., and Hogan et al., because the image of the Taylor cone can be inspected at high magnification with the video camera. In a preferred embodiment of the technique described in this disclosure, a Dinolite (www.microscopes.com) model AD4113TL camera is used to visualize the Taylor cone in the electrospray ion source 2.

[0057] 2. Selection of ions with a given electrical mobility

[0058] Electrospray droplets generated in ion source 2 evaporate as they travel through the electrospray ion source 2. The charge carried by each droplet is transferred to the analyte molecules carried by the electrospray droplets as the droplets evaporate. Evaporation results in the formation of gas phase analyte ions. The gas phase analyte ions are conducted out of the electrospray ion source 2 by exit tube 4. Exit tube 4 directs the ions to nano-DMA 5 where the analyte ions are separated according to their electrical mobility, as shown in the inset of FIG. 1.

[0059] In a preferred embodiment, the nDMA 5 is a nanoDMA (TSI Inc, Model 3085). The nDMA 5 operates with a sheath flow of air supplied by a 25 psig dry air source and a mass flow controller 6 (e.g., Sierra Instruments, Monterey, Calif.) configured to supply 20 Lpm of air to a port labeled "sheath flow" on the nDMA 5. The nDMA can optionally operate with a sheath flow other than 20 Lpm, such as a sheath flow ranging from 5 to 40 Lpm, to select a range of ion or charged particle sizes in which ions or charged particles of a given size can be induced through the nDMA. A sheath flow of 5 Lpm provides a range of selectable particle and ion sizes that is smaller than the range of particles and ions induced through the nDMA when the nDMA is operating with a sheath flow of 40 Lpm.

[0060] Those skilled in the practice of differential mobility analyzers will appreciate the concept of operating a differential mobility analyzer as a filter for ions and charged particles. In the techniques described in this disclosure, the Model 3085 provides a means of selecting ions having a narrow range of sizes from a population of ions having a much wider range of sizes. The operating conditions of the Model 3085 provide a means of selecting a subset of ions having diameters between about 2 nm and 200 nm. The term "selecting a subset of ions" refers to the state of the sheath gas flow and the voltage applied to the inner member of the nDMA 5. For example, when a sheath flow of 20 Lpm is used and 180 volts is applied to the inner member of the nDMA, singly charged ions with a diameter of approximately 7 nm will transmit through the nDMA. The use of a sheath gas flow of 20 Lpm and the application of 180 volts to the inner member of the nDMA are operating conditions that allow singly charged albumin ions to pass through the nDMA. The term "mobility selected ions" is an expression that describes ions that are directed through the nDMA for the purpose of selecting a subset of ions.

[0061] The exact range of particle or ion sizes that can be selected by the nDMA5 is provided by the manufacturer of the nDMA5. In addition to the sheath flow, a flow of ion-containing gas is supplied to the nDMA5. The electrospray ion source 2 provides a flow of ion-containing gas that is directed from the electrospray source 2 to the nDMA5 by tube 4. The gas flow carries the mobility selected ions out of the nDMA5.

[0062] Mobility selected ions are generated in the nDMA 5 of the device using two different techniques. In the first technique, the voltage applied to the inner member of the nDMA 5 is a ramping voltage. An example of a ramping voltage is a voltage applied to the inner member of the nDMA 5 that starts at 0 volts and ramps up to 10 kV over a period of several minutes. During ramping, as the voltage applied to the inner member of the nDMA 5 increases, ions with increasingly lower mobilities are guided through the nDMA 5. As shown in Figures 24-28, the ramping voltage is used to generate a mobility spectrum. The ramping voltage is also useful for determining the voltage required to guide ions with a certain mobility to a collection surface such as a cryo-EM grid. By noting the location of the peak in the mobility spectrum relative to the ramping time or ramping voltage, it is possible to estimate the voltage applied to the inner member of the nDMA 5 while the peak was recorded. In the second technique, the voltage applied to the inner member of the nDMA 5 is a fixed voltage. A fixed voltage is useful for directing ions with a certain mobility to a collection surface over an extended period of time. Fixed and ramping voltages are provided to the nDMA5 by customized software driving a National Instruments USB-611 module. The 611 module is connected via analog signals to a high voltage power supply such as a Bertan Model 255. The output from the Model 255 is connected to the inner components of the nDMA5.

[0063] It is preferred to balance the flow rates of the inlet sheath flow fed to the nDMA5 and the outlet sheath flow exiting the nDMA5. The flow balancing is accomplished by valve 7. Valve 7 provides a flow restriction to the flow of gas carrying mobility selected ions from the nDMA5. In a preferred embodiment, the valve is a Swagelok model SS-1RS4-A valve. Adjustment valve 7 provides a means to balance the inlet and outlet flows of sheath gas through the nDMA5, while the nDMA5 operates with a mass flow controller to establish the sheath flow through the nDMA5. Valve 7 is adjusted by observing the gas flow rate indicated by the flow meter. After the flow rate indicated by the flow meter equals the flow rate established by mass flow controller 6, it can be seen that the flow rates are evenly balanced. Mass flow controller 6 is easily adjusted by custom software available from IonDX, Inc., Monterey, Calif., in comparison to the recirculation flow provided by a TSI Inc. model 3080, which cannot be controlled by third party software.

[0064] The manufacturer of the nDMA provides a means of balancing the inlet and outlet sheath gas flows based on a closed circuit recirculating blower (Model 3080). The use of the recirculating blower introduces a constant volumetric flow rate of sheath gas into the nDMA and then withdraws an equal volumetric flow rate of gas from the nDMA. The recirculating flow of sheath gas provided by the Model 3080 is insufficient to provide a sheath flow as large as 40 Lpm. The recirculating sheath flow provided by the Model 3080 recirculates room air through the nDMA. During the course of experiments conducted using the Model 3080 and the recirculating blower, partial mixing of the sheath gas flow with the gas flow provided by the electrospray ion source results in the accumulation of water vapor in the sheath flow. The technique described in this disclosure establishes a sheath flow condition where the inlet sheath flow is continuously supplied by dry gas provided to the mass flow controller 6. The advantage of this design over the Model 3080 is better control of the population of ions selected by the nDMA 5. In the technique described in this disclosure, all selected ions are passed through a sheath gas stream of known and controlled water vapor content. Thus, this design provides more precise control of the ions that are directed to land on the collection surface, since the water vapor content of the sheath gas affects the electrical mobility of the ions. Also, ions passing through a humid gas will have a few water molecules attached to them, so they will be slightly larger than ions passing through a dry gas. As will be appreciated by those performing ion mobility analysis, when considering selecting ions or particles with a certain electrical mobility, it should be understood that the technique described in this disclosure provides more precise control over the ions that are directed through the nDMA5. Later in this description, we will describe the provision of the technique described in this disclosure in which the water vapor content of the sheath gas stream is an optionally adjustable parameter, a capability not provided by the Model 3080.

[0065] In one embodiment of the technique described in this disclosure, mobility selected analyte ions exit the nano-DMA 5 through valve 7 and are directed only to the ion detector 19 via tube 8, as shown in FIG. 2. The ion detector 19 is described in more detail in FIG. 3. The ion detector 19 acts as a particle and ion filter. The ion-containing gas directed through tube 8 is directed through a 20 mm long plug of fine steel wool 300 in a 50 mm long 5 mm by 4 mm inner diameter glass tube 302. After the gas flow passes through the steel wool, the resulting particle-free and ion-free gas exits the distal end of the glass tube 302, as indicated by the curved arrow 304 representing the gas flow streamline. The steel wool is electrically connected to a picoammeter 306, such as a Keithley model 6400, via a BNC connector 308 fixed to the wall of the ion detector 310 or 19, as shown in FIG. 2. The charge carried by the charged particles and ions after being filtered by the steel wool is transferred to the steel wool and further detected as an electric current by the picoammeter 306. The 10 mm long inlet of the glass tube is enclosed inside a 5 mm inner diameter by 60 mm long brass tube 312. The brass tube 312 is secured by a plastic grommet 314 in a hole in the wall of a 4.5 in. by 3.5 in. by 2 in. metal enclosure 310. The metal enclosure is connected to an electrical ground 316 to provide an electrical shield around the steel wool so that the small currents can be measured without interference from environmental electrical noise.

[0066] As described above, a ramping voltage or a fixed voltage can be applied to the inner member in the nDMA 5. The ion detector 19 can be used to record ion mobility spectra while the voltage applied to the inner member of the nDMA 5 is ramped. The ion current is detected by the ion detector 19 while ions of selected mobility are directed to the ion detector 19. The ion detector 19 can also be used to monitor the ion current while a fixed voltage is applied to the inner member of the nDMA 5. Monitoring the ion current while a fixed voltage is applied to the inner member of the nDMA 5 provides a reference signal of the magnitude of the ion current before the ion flow has passed too far through a component of the technology described in this disclosure, and is useful for determining the extent to which ions are lost during passage through the component. The percentage of ions lost during passage through a component of the system is determined by calculating the ratio of the ion current exiting the component to the ion current measured at the outlet of the nDMA 5.

[0067] 3. Exposure of mobility-selected ions to controlled humidity

[0068] Referring again to FIG. 2, in one embodiment of the technique described in this disclosure, mobility selected analyte ions exit the nano-DMA 5 through valve 7 and are directed through tube 8 to humidifier 12a. The humidifier provides a means of controlling the temperature and water vapor content of the ion-containing gas stream. Temperature control module 10a heats the water in humidifier 12a and, when set to a particular temperature, produces a predetermined level of humidity in the ion-containing gas. The humidity controlled ion-containing gas is directed by 8 to a dew point sensor 13a, such as an Easydew OnLine monitor, model 0422, Mitchell Instruments, Hauppauge, NY, and then to the ion focusing chamber 9. A second humidifier 12b provides a means of controlling the dew point of the sheath gas exiting the nano-DMA 5. The sheath gas exits the nano-DMA 5 through side port 11. The sheath gas is directed to humidifier 12b. Humidifier 12b operates similarly to humidifier 12a. The humidity controller also controls the temperature of the gas stream. In a preferred embodiment of the humidifier, the humidity controlled sheath gas is directed from 14 into the dew point sensor 13b and then into the ion focusing chamber 9 for use as a sheath flow in the ion focusing chamber 9. A preferred embodiment of the humidifier is shown below in FIG. 4. It should also be understood that the humidifier 12a and / or humidifier 12b can be operated powered off so as not to provide humidification. Additionally, the humidifier can be operated to reduce the temperature of the gas stream as a means of reducing the humidity of the gas stream. In another embodiment, the humidifier can be operated to cool the gas stream and condense water vapor onto the ions or particles passing through the humidifier. This mode of operation provides a means of depositing ion-containing droplets on a collection surface. In another embodiment, the humidifier can be operated to freeze the condensed droplets as they pass through the humidifier. This mode of operation provides a means of depositing the ions or particles on a surface in small ice balls.

[0069] The flow of humidity control gas derived from the dew point sensors 13a and 13b is introduced into the ion focusing chamber 9 where the ions enter the ion funnel 14 for the purpose of focusing the ions towards the centerline of the funnel 14 for the purpose of focusing the ions and / or particles for landing on a collecting surface such as a cryo-EM grid. The design of the focusing chamber 9 provides a means of focusing the ions into the humidity control gas. In the techniques described in this disclosure, control of the humidity of the gas directing the ions through the funnel is a key parameter. Humidity affects the degree to which the ions or particles are hydrated. Higher humidity produces ions with higher levels of hydration. The techniques described in this disclosure provide a means of depositing ions with a specific hydration level onto a landing surface such as a cryo-EM grid. By controlling the humidity of the ion-containing gas, a specific hydration level is established. It is particularly important to generate a humidity level that hydrates the ions while maintaining a low humidity level to prevent condensation of water vapor onto the collecting surface while the ions are being deposited onto the cooled collecting surface. In one use of the disclosed apparatus, it is desirable to prevent condensation on the collecting surface of the cryo-EM grid while it is attached to the cold finger. In another type of study performed with the disclosed apparatus, it is desirable to study chemical reactions in humidified particles to create reaction conditions found in outdoor environments, such as fog or clouds. Upper cylinder 17 and lower cylinder 18 form the main body of focusing chamber 9. Cylinders 17 and 18 can be separated to provide access to end 15 of rod 16 for the purpose of loading or unloading a collecting surface, such as a cryo-EM grid, from end 15 of rod 16.

[0070] A preferred embodiment of the humidifiers 12a and 12b is shown in more detail in FIG. 4 while the humidifiers are operating to add water vapor to a gas stream. The humidifiers 12a and 12b each include a wetted tubular wick 19 housed within a metal tube 19A. The tube 19A is surrounded by an electrical resistance tube heater 21 controlled by the temperature control module 10a. Air passing through the tubular wick 19 picks up water vapor evaporated from the wick 19, thus humidifying the air stream. An example of a tubular wick 19 is a 1 / 4 inch diameter, three-ply thick roll of filtration membrane, such as DVPP from Millipore-Sigma. The tubular wick 19 is held in place by inlet 22 and outlet 23 tubes. The outlet end 23 of the wick 19 is located within water 25. Water 25 is drawn into the wick 19 by capillary action to keep the wick wet. This design of wick is similar to that described by Herring, Spielman, and Lewis in U.S. Pat. No. 11,029,240, and operation of the wet wick provides the conditions for generating ion- or particle-containing droplets.

[0071] 4. Focusing the ions to strike the cryo-EM grid

[0072] The realization of ion focusing in the ion focusing chamber 9 is based on prior art described in the literature. Baker et al., Belov et al., Chaudhary et al., Clowers et al., Ibrahim et al., Julian et al., Kelley et al., Kim et al., Page et al., Tang et al., and Wyttenbach et al. have described the design and operation of ion funnels as a means to improve the performance of electrospray mass spectrometers. Ion funnels described in the prior art are generally used in the field of mass spectrometry to capture plumes of electrospray ions and efficiently guide them onto a collection surface in the vacuum chamber of the mass spectrometer. Ion funnels are also used in ion mobility mass spectrometers, but again for the purpose of allowing efficient transport of ions into the vacuum chamber. The use of ion funnels in the present disclosure to focus and then deposit slow ions onto a target such as a cryo-EM grid is not an obvious extension of the prior art description of ion funnels. A commercially available device called the Nanometer Aerosol Sampler Model 3089 by TSI Inc. was developed for the purpose of depositing charged particles onto a collection surface held in an atmospheric pressure gas such as room air. It uses a voltage of opposite polarity to that of the charge carried by the particle of interest as a means of attracting the electrostatically charged particles onto the deposition surface. In this device, a voltage of less than + / - 10 kV is applied to the collection surface for the purpose of electrostatically attracting the charged particles to land on the collection surface located inside the nanoparticle sampler. The use of the nanometer aerosol sampler is not described as a soft landing device due to the high voltage applied to the collection surface attached to the collector rod inside the nanometer aerosol sampler.

[0073] Referring to FIG. 5, the ion focusing funnel 14 provides a flow of gas and an electric field surrounding the analyte ions that move the analyte ions toward the longitudinal centerline of the ion funnel, where they are concentrated, or focused, near the centerline of the funnel. The focused ions are directed toward the end 15 of the rod 16 by the gas flowing through the funnel. The rod 16 is designed to fit into a sliding seal at the bottom of the chamber 18. The sliding seal, such as an O-ring seal, allows the rod to move in and out of the chamber 18. The flow of gas that directs the ions or particles toward the end 15 of the rod 16 pushes the ions or particles near the end 15 of the rod 16, thus allowing some of the ions to be deposited on the end 15 of the rod 16 by diffusion. In one embodiment of the apparatus described in this disclosure, a cryo-EM grid is attached to the end 15 of the rod 16, and diffusion acts to deposit the ions on the cryo-EM grid. In another embodiment of the technique described in this disclosure, the rod is a cold finger. Those skilled in the art of refrigeration will appreciate that the cold finger provides a means for cooling the end of the rod 16 and the collection surface attached thereto as a means of preserving material such as proteins deposited on the collection surface. In another embodiment of the ion focusing chamber, the rod 16 is connected to a voltage source and energized with a voltage that attracts ions to move towards the rod, such as application of a negative voltage to improve the efficiency of collecting positive ions. It will be appreciated that collection is not limited to positive ions, and negative ions or particles can be collected after the polarity of the ions and collection surface are reversed.

[0074] With continued reference to FIG. 5, further details of the design and operation of the ion focusing chamber 9 and ion focusing funnel 14 show the means by which ions are directed through the extension of tube 8 to the inlet of the ion focusing funnel 14. The humidity control gas directed through the extension of tube 11 passes through a diffuser 26. The diffuser 26 provides resistance to the flow of the humidity control gas, causing it to spread across the inlet of the diffuser 26, as indicated by gas diffusion 27. The diffuser 26 thus provides a uniform flow of gas across the diameter of the tubular column 17. The resulting uniformly diffused sheath gas flow surrounds the ion-containing gas flow introduced by tube 8. It will be appreciated that the uniform flow surrounding the ion-containing flow from tube 8 is the sheath flow within the ion focusing funnel 14. The sheath flow continues to surround the ion-containing gas flow as these gases flow through the ion focusing funnel 14. A preferred embodiment of the technique described in this disclosure introduces 2.0 Lpm of ion-containing gas and 20 Lpm of sheath gas into the ion focusing funnel 14.

[0075] With continued reference to FIG. 5, five circular wire rings make up the electrostatic focusing elements of the focusing funnel 14. This design is not limited to wire rings and can include electrodes made using a stack of circular plates with a hole in the center. Ring 28 is an exemplary ring. Ions enter the funnel through the widest diameter ring and exit the funnel 14 through the smallest diameter ring. FIG. 5 shows an exemplary funnel assembled with a band and five rings supported by four ribs. 29 is an exemplary ring support post. The preferred embodiment of the ion focusing funnel 14 is a stack of one circular band and five circular focusing rings. The band is 58 mm in diameter and the rings are circular rings of wire with diameters of 50, 41, 32, 24 and 15 mm made of stainless steel wire with a thickness (diameter) of 1 mm. The five rings are supported by six ribs. The rib assembly was 3D printed using ABS plastic. The overall length of the ring assembly is 70 mm. Five electrical resistors 30 connected in series between a voltage source 31 and electrical ground 32 constitute a voltage divider used to supply voltage to each ring. In a preferred embodiment of the technique described in this disclosure, the electrical connections between the rings and the resistors in voltage divider 30 supply an exemplary voltage of 1 kV to the smallest ring and 0.8, 0.6, 0.4, 0.2 and 0 kV to each of the successively larger diameter rings, respectively. Operation of focusing funnel 14 is not limited to 1, 0.8, 0.6, 0.4, 0.2 and 0 volts, but may also be powered with RF voltages.

[0076] A further illustration of an ion focusing funnel 14 is shown in FIG. 6. A cylinder (600) having an inlet section 602, an outlet section 604, and a middle section 606 is constructed from components of a 3D printed part. A band of sheet metal (0.015 inches thick), such as stainless steel, is wrapped around the entire width and circumference of the middle section 606, and the ends of the wrapped sheet metal are secured together to form a cylinder. This metal cylinder is referred to as the band, as described above. Six ribs 608 extend from the inner diameter of 600 toward the center of the longitudinal axis of 600. The ribs 608 are attached to the inner surface of the middle section 606 and angled toward the longitudinal centerline of 600. The angle of the ribs relative to the longitudinal centerline of 600 is shown by angle 610. The ribs have a cross section with the longer dimension of the cross section aligned with the radius of 806 in FIG. 8A. Five holes, 1 mm in diameter, are evenly spaced along the length of the ribs. Holes 612 in the ribs provide support for rings of 1 mm thick copper wire.

[0077] An end view of the entrance to the ion focusing funnel 14 is shown at 700 in FIG. 7. Six ribs are evenly distributed around the circumference of the entrance section 600, extending from the inner diameter of 600 towards the longitudinal centerline of 600. 7022 indicates that component 700 is a feature of a larger assembly, shown by an isometric view of the mechanism that holds the focusing funnel 14 in communication with the exit assembly 704. Six ribs extend from the inner diameter of 704 towards the center of 704, where they are attached to a central 1 / 4 inch cylinder 706. Cylinder 706 is a fixture for securing the ion collecting pedestal to the center of the exit of the funnel.

[0078] A further view of one embodiment of the ion focusing funnel 14 is shown in Figure 8A, where diagram 800 shows an end view of the entrance to the ion focusing funnel 14. The lines drawn along the radius of 800 are ribs 608 that hold the wires. In 800, the wires 802 are represented as circles. Diagram 804 shows the attachment of the wires 802 to the ribs of the focusing funnel 14. It can be understood that references to wires are synonymous with wiring.

[0079] A further view of the funnel 14 is shown in Fig. 8B. 806 is a side view of the 3D printed part holding the metal bands 808 and ribs 810. Figs. 700 and 704 show end views of the funnel 14.

[0080] End view 700 is a projection onto the inlet of the funnel and shows the ribs 608 of the funnel 14 extending from the inlet ring 808 towards the smaller end of the funnel 14. The inclination of the ribs relative to the centerline of the part is represented by the angle 610. In the preferred embodiment, the angle 610 is 42 degrees. End view 704 shows a configuration that looks like spokes in a wheel. The spokes, which are not to be confused with ribs in end view 700, extend from the outer periphery of 604 towards the center of where they meet to provide circular support to maintain the pedestal, not shown, while being inserted through the circular support in a central position. The center panel of FIG. 8B provides an isometric view of the converging funnel 14 and shows the location of the ribs and spokes.

[0081] FIG. 9 shows a fixture 900 for holding a collection surface on the end 15 of a rod 16 or on the end of a tube 41. A short tubular section 38 attached to the end 15 of the rod 16 secures a cryo-EM grid 39 to the end 15 of the rod 16. In a preferred embodiment, the rod and tubular section have an outer diameter of 0.25 inches. The height of the tubular section is 0.031 inches, the inner diameter of the tubular section is 0.25 inches, and the diameter of the cryo-EM grid is 0.118 inches (3 mm grid). The outer tube 41 and inner tube 40, along with a device for securing the cryo-EM grid, are collectively referred to as the pedestal 900. Fixture 902 further shows another means for securing the cryo-EM grid to the end of the ion collection pedestal. The widest diameter of 902 is 1 / 4 inch and is attached to the end of the rod or tube, i.e., the pedestal, by adhesive or a portion of a threaded rod such as 4-40 set screws 1 / 4 inch long. The fixture 902 has an upper portion adapted for mounting a cryo-EM grid and a tapered transition adapted for attachment to a larger diameter rod. The upper feature is a countersunk chamber designed to hold a cryo-EM grid. A slot provides tweezers access to a collection surface mounted within the chamber. The tapering of fixture 902 from a 0.25 inch diameter to a 0.15 inch diameter helps reduce the cross section of the fixture and ensures that the cross section interacts minimally with the ion-laden air flow stream, thus providing a guide for the ions to impinge on the cryo-EM grid without introducing turbulence into the ion-laden gas flow that would reduce deposition efficiency. Those skilled in the art of gas dynamics practice and design and gas flow around objects will appreciate that by minimizing the cross section of the pedestal, redirection of the gas flow streamlines is minimized in that the diffusing streamlines are not beneficial for guiding ions onto the collection surface as they guide ions away from the cryo-EM grid.

[0082] In another embodiment, the rod 16 or tube 41 is fabricated from an electrically insulating ¼ inch outer diameter tube, such as white Delrin. The Delrin tube provides a means of electrically insulating a collection surface, such as a fixture 902, attached to the end of the Delrin tube. In this configuration, a shielded cable passes through the bore of the Delrin tube, and the conductive inner member of the shielded cable is attached to the fixture 902. The cable provides a means of electrically connecting the fixture 902 to a picoammeter, such as a Keithley Model 6400 picoammeter, for the purpose of measuring the ion current as ions are induced to land on the fixture 9092 or on a cryo-EM attached to the fixture 902. Measuring the ion current as ions are induced to land on the cryo-EM grid provides information useful for calculating the total number of ions deposited during a given collection time interval.

[0083] In one embodiment, the fixture 902, rod 16 is replaced by a tube assembly consisting of an inner tube 40 and an outer tube 41, as shown in FIG. 9. A cooling fluid, such as chilled antifreeze or liquid nitrogen blow-off gas, is directed into the inner tube 40 to provide a cryogenic gas or liquid that contacts the surface below the cryo-EM grid 39 for the purpose of cooling the cryo-EM grid 39. The cooling fluid then passes through the annular space 42 between the tubes and is either discarded or returned to the cooling fluid source. A temperature sensor 43 located on the surface below the cryo-EM grid 39 provides a signal useful for controlling the temperature of the cryo-EM grid 39. More specifically, as shown in 903, the tubular portion 38 is provided with a slot 44 so that a device, such as tweezers, can be inserted into the slot 44 to pick up or drop off the cryo-EM grid.

[0084] 10A and 10B show a means for flowing cryogen through the pedestal 900. The inner tube 40 and the outer tube 41 are used to deliver the cryogen 1000 to provide cooling to the end of the pedestal 900 where the cryo-EM grid can be attached. A temperature sensor 1002 is attached to the inside side of the end of the pedestal 900. The temperature sensing element can be a thermistor, platinum resistance temperature detector, or thermocouple. The temperature sensing element is connected to a temperature readout device by a wire 1004 that passes through the annular space between the inner tube 40 and the outer tube 41. The temperature readout device can be a digital temperature display that includes a provision for amplifying the temperature signal for the purpose of providing an auxiliary signal that can be used to control the flow of the cryogen. Controlling the flow of the cryogen provides a means for establishing a constant temperature for cooling the cryo-EM grid. It can be appreciated that a higher flow rate of cryogen provides a lower temperature and a lower flow rate of cryogen provides a higher temperature. One example of a means for controlling the temperature of the end of the pedestal is a temperature controller available, for example, from Omega Engineering, Inc. of Norwalk, Connecticut. The temperature controller receives a temperature signal, such as a mV signal generated by a thermocouple, and opens and closes an electrical switch useful for controlling the flow of cryogen. The final temperature established at the end of the pedestal is a function of the flow of cryogen, the thermal mass of the end of the pedestal, and the flow of ion-containing gas past the end of the pedestal.

[0085] In one embodiment, the flow of cryogen 1000 is delivered to the pedestal 900 by the inner tube 40. After leaving the end of the inner tube 40 located inside the outer tube 41, the cryogen 1000 flows through the annular space between the inner tube 40 and the outer tube 41, and exits the open end of the outer tube 41, as shown by 1006. The cryogen is delivered to the pedestal 900 by an insulating tube 1008 connected to a dewar 1010. In one embodiment, the dewar holds liquid nitrogen 1012, and the inner tube is sealed to the dewar 1010 such that the end of the inner tube 40 extends into the dewar 1010 to allow liquid nitrogen blow-off gas to enter the inner tube 40. The liquid nitrogen does not enter the inner tube 40. It can be seen that the liquid nitrogen in the dewar 1010 slowly boils, producing cold nitrogen blow-off gas. A feature of the apparatus described in this disclosure is to control the rate at which the liquid nitrogen boils. By controlling the boiling rate, the flow of the blow-off gas is controlled. A means for controlling the boiling rate of liquid nitrogen is shown in Figures 10A-10C. A heating element 1014 is immersed in the liquid nitrogen 1012 and current supplied to the heating element 1014 causes the liquid nitrogen to boil. The amount of heat supplied to the liquid nitrogen is controlled by a temperature control module 1016. An example of an electrical circuit for supplying heat to the liquid nitrogen is shown in Figure 10C. It can be seen that the battery in the circuit diagram is a lithium ion battery (e.g., a 1650 type battery) and the resistors are metal film resistors having resistance values ​​of + / -1% of the values ​​shown. It can be seen that the DVM in the circuit diagram is useful for displaying temperature by converting the resistance shown to temperature using a temperature-resistance conversion formula. In one embodiment, the switch (SW1) in the electrical circuit can be replaced with a switch closure connection on a temperature controller (e.g., manufactured by Omega Engineering). It can also be seen that the heating element in Figure 10B is the circuit component labeled 1N4148, which is a reverse biased diode. It should be noted that control of the boiling rate of liquid nitrogen is not limited to the use of the heater and heater circuit shown in FIG. 10C.A simple combination of an electrical resistor, such as a 100K ohm resistor, and an adjustable low voltage power supply provides another means of adding heat to the liquid nitrogen by passing a current through the 100K ohm resistor immersed in the liquid nitrogen. Increasing the voltage provided by the power supply increases the boiling rate, while decreasing the voltage decreases the boiling rate. Using the circuit shown in FIG. 10C provides automatic control of the temperature of the fixture 902.

[0086] FIG. 11 shows another feature of the ion focusing chamber 9 and component 702 (FIG. 7). A means is needed to insert and remove the pedestal 900 from the ion focusing chamber 18 before and after ions are deposited on the cryo-EM grid attached to the end of the pedestal 900. It can be seen that the pedestal 900 slides through an opening 706 in 704, which provides a means for aligning the longitudinal centerline of the pedestal 900 with the longitudinal centerline of the focusing funnel 9. It is also necessary to position the end of the pedestal 900 a predetermined distance downstream of the smallest ring in the focusing funnel 14. A stop collar 1100 is attached to the pedestal 900 to control the length of the pedestal 900 that can be inserted into 702. The stop collar also acts as a safety mechanism, preventing the pedestal 900 from being over-inserted and creating a discharge if the pedestal 900 contacts the smallest ring of the focusing funnel 14.

[0087] Referring to FIG. 12, cylindrical chambers 17 and 18 provide a means to protect the ions from exposure to room air after they have been deposited on the cryo-EM grid. Chambers 17 and 18 have a sufficient diameter to give the operator sufficient volume to perform tasks related to loading, unloading, and transferring sample substrates to and from the transfer case, or to auxiliary chambers for storing samples. An appropriate size for each of these chambers is 6 inches in diameter and 10 inches in height. Those skilled in the art of cryo-EM analysis will understand the need to preserve samples, particularly protein molecules, at low temperatures. After the ions have been deposited on the cryo-EM grid, they need to be preserved by storing them in liquid nitrogen. To do so, the loaded cryo-EM grid is removed from the end 15 of rod 16 and pushed into a vial of liquid nitrogen held inside chamber 18. Chamber 17 is designed to mate with chamber 18 to allow the two chambers to be easily assembled or disassembled. Chamber 17 is equal in diameter to chamber 18, allowing the end of chamber 17 to be aligned with the end of chamber 18 to form a single cylinder. Fixing an O-ring 45 in place in an O-ring groove in the end wall of chamber 18 provides a means of sealing the two cylinders together. When the two chambers are attached together, a flow of dry air or liquid nitrogen blow-off gas provided by gas source 46 passes through chamber 18 and exits through port 19 in chamber 17. When chambers 17 and 18 are disassembled, the flow of gas from 46 exits the top of chamber 18, as indicated by 47. By retracting end 15 of rod 16 into the interior of chamber 18, the end 15 of rod 16 is immersed in the liquid nitrogen blow-off gas provided by 46, allowing chambers 17 and 18 to be disassembled without exposing rod 16 or cold finger 41 to moist room air.The cryo-EM grid attached to the end 15 of rod 16 is immersed in dry gas supplied by 46 to prevent water vapor from condensing on the cryo-EM grid when it is attached to the end of cold finger 41. The cryo-EM grid attached to the end of 41 can then be picked up with tweezers and transferred to a vial 48 located inside chamber 18. In one embodiment of the technique described in this disclosure, vial 48 holds an EM grid holder(s) 49 and contains liquid nitrogen.

[0088] Theoretical validation of the focusing funnel 14 design can be provided using SIMION simulations of the trajectories ions follow as they pass through the focusing funnel. SIMION software can be purchased from Scientific Instrument Services (SIS), Inc., Ringoes, NJ. To simulate ion trajectories in the apparatus described in this disclosure, the SDS user program, also available from SIS, was modified to support implementation of the range of gas velocities predicted to carry ions through the focusing funnel. Those familiar with the use of computational fluid dynamics models to simulate gases emerging from ports projecting into the surrounding chamber will recognize that the emerging streams diverge. The extent to which the streams diverge depends on gas conditions such as gas pressure, gas flow rate, diameter of the port, physical size of the chamber, and the rate at which gas is evacuated from the chamber. To make the fluid flow simulation more accurate, additional parameters can be introduced into the fluid flow simulation, such as the molecular weight of the gas, the pressure differential across the chamber, gas temperature, and whether the flow is laminar or turbulent.

[0089] To simulate the gas flow in the apparatus described in this disclosure, it was assumed that the gas flow in the focusing funnel was laminar and occurred at atmospheric pressure with the ports and chamber held at room temperature. It was further assumed that the gas flow emerging from port 8 located along the centerline of the focusing funnel 14 spreads as it flows through the focusing funnel 14, as shown in FIG. 13. FIG. 13 shows the gas flow streamlines emerging from port 8 and the radial spread of the flow as it passes through the focusing funnel 14. In addition to the ion-containing gas flow induced in the focusing funnel 14 by port 8, the sheath flow provided by the conductor 11 merges with the ion-containing flow. The following simulation used a simple mathematical formula to model the spread of the gas emerging from port 8. It can be seen that the ion-containing gas flow spreads around the centerline of the focusing funnel. The formula y-velocity=2e-6 * A mathematical condition was established to simulate the spreading of the flowing gas using (x position - 5.1) / ion sequence number^2. Those skilled in the art of CFD practice will appreciate that this is a simplified equation and that more accurate equations are available in the scientific literature. This simplified equation adequately simulates the spreading of the gas flowing through the focusing funnel and provides an initial estimate of the magnitude of the voltage required to achieve ion focusing within the ion focusing funnel.

[0090] 14-23 show SIMION simulations of ion trajectories as a flow of ion-containing gas is directed through the focusing funnel 9. The gas flow carries the ions through the funnel while the ions are directed toward the centerline of the funnel 9 by voltages applied to electrodes in the funnel 9. In FIGS. 14-23, the figure captions indicate the ion focusing conditions. It should be understood that the use of "voltage applied to the focusing funnel" or related phrases refers to the voltage applied to the smallest ring during focusing. Referring again to FIG. 5, the voltage divider 30 is supplied with a voltage at one end and is provided with a connection to electrical ground at the other end. "Voltage applied to the focusing funnel" refers to the voltage applied to the distributor which is similarly provided to the smallest ring in the funnel 14. The voltage divider 30 distributes the voltage to each ring in the funnel 14.

[0091] In Figs. 14-23, the left panels show ion trajectory simulations when the voltages shown in the headings are applied to the electrode rings constituting the focusing funnel. The right panels of these figures show for comparison purposes ion trajectory simulations when no voltage is applied to the electrode rings in the focusing funnel. The right panels are reproduced in Figs. 14-23. It can be seen that the small squares in these figures represent the positions of the focusing wires, and the squares show a cross-section in the xy plane cut along the centerline of the focusing funnel. For further clarity, the ring electrodes are shown in Fig. 8 by thick vertical lines. The left panels of Figs. 14-23 show simulated ion trajectories while a voltage is applied to the funnel 14. The ion trajectories are represented by solid black lines. The dots superimposed on the trajectories represent timing marks. Ions flow from left to right in the device described in Figs. 14-23, and the left to right sequence of dots in these figures shows the position of the ions at each subsequent 10 millisecond interval. The cluster of 5x5 small squares near the right side of each panel represents the pedestal where ions are deposited. A cryo-EM grid can be attached to the end of the pedestal. The cluster of 5x5 small squares near the left side of each panel represents the port where ions enter the funnel 14.

[0092] The performance of the ion focusing funnel 14 and the realization of ion focusing versus the magnitude of voltage applied to the funnel 14 are shown in Figures 14-23. Ion containing gas is directed into the ion focusing funnel 14 as shown in Figure 14 and spreads out as it travels through the funnel 14. The flow of ion containing gas exiting the exit end of tube 8 is shown by flow streamlines 34. Those familiar with gas flow streamlines will recognize that streamlines are the trajectories followed by particles suspended in the flowing gas. They will also recognize that the flow exiting tube 8 expands in diameter the farther it flows into the funnel. The ion trajectories 34 diverge as the ions are entrained by the gas flow and expand as the gas flow expands. Referring now to the left panel of Figure 14, the ion trajectories continue to expand and then begin to converge at the end 15 of rod 16 due to the focusing properties of the electric field inside funnel 14. As the ion-containing gas penetrates further into the funnel, the gas continues to spread, but the ion trajectories no longer spread out in region 35. The electric field inside the funnel moves the ions toward the longitudinal centerline of the funnel. As the ion-containing gas penetrates further into the funnel and reaches region 36, the electric field also increases, providing a stronger ion focusing effect, as shown by the ion trajectories being focused toward the end 15 of rod 16. The focusing effect on the ions in region 36 is due to a decrease in the diameter of the focusing rings, which gives a stronger focusing effect to the ions in region 36.

[0093] The effectiveness of the ion-focusing funnel 14 can be understood by comparing the flux of ions delivered by the conductor 8 to the flux of ions deposited at the end 15 of the rod 16 or the end of the cryo-EM fixture shown in Figures 14-23. Ion trajectories terminating at the end 15 of the rod 16 indicate the flux of ions striking the end 14, and it can be seen that the flux is greater when a relatively large number of trajectories terminate at the end 15 of the rod 16. With 0 volts applied to the exit ring of the funnel 14, no ion focusing occurs. Nevertheless, some of the ions delivered by the conductor 8 pass straight through the funnel 14 and strike the cryo-EM grid attached to the end 15 of the rod 16. Application of a voltage to the exit ring of the funnel 14 increases the number of trajectories terminating at the end 15 of the rod 16. Those familiar with the operation of ion-focusing funnels will know that applying a higher voltage to the exit ring of the funnel 14 induces more ions to strike the end 15 of the rod 16.

[0094] Starting with the description of the right panel of Figure 14, ion trajectories begin at port 8 and progress toward the pedestal. Some of the ions close to the centerline of the focusing funnel pass directly through the focusing funnel and strike the pedestal, as shown by their trajectories, but not all ion trajectories terminate at the pedestal. The percentage of trajectories that terminate at the pedestal is a measure of the focusing efficiency of the focusing funnel. Compared to the voltage condition when no voltage is applied to funnel 14, the left panels of Figures 14-23 show the results of applying a voltage to funnel 14.

[0095] Figures 14-23 show a series of SIMION simulations using different focusing voltages, ions with different molecular weights, and ions with different charge masses. Tocilizumab is an antibody with a molecular weight of nearly 150 kDa and can be considered as an exemplary substance for testing the soft landing device.

[0096] Exemplary headings for Figs. 14-23.

[0097] (a) 300 kDa M+: ion mass and charge. 800, 1600, 2400, 3200, 4000: voltages applied to the ring electrodes, listed starting with the voltage applied to the largest ring and then the voltage applied to the smaller ring.

[0098] (b) Vx = 0.5 m / s: average gas velocity in the funnel.

[0099] FIG. 14 shows a simulation of ion focusing using

[0100] (a) aM+: ion mass and charge.

[0101] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0102] (c) Vx = 0.5 m / s: average x-directional gas velocity within the funnel. Note that the gas flows from the region enclosed by the largest ring to the region enclosed by the smallest ring.

[0103] FIG. 15 shows a simulation of ion focusing using

[0104] (a) aM+: ion mass and charge.

[0105] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0106] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity in the funnel.

[0107] FIG. 16 shows the conditions for preventing ions from entering the funnel 14 using:

[0108] (a) 300 kDa M+: ion mass and charge.

[0109] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0110] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity within funnel 14.

[0111] FIG. 17 shows the conditions for preventing ions from entering the funnel 14 using:

[0112] (a) 300 kDa M+: ion mass and charge.

[0113] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0114] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity within funnel 14.

[0115] FIG. 18 shows a simulation of ion focusing using

[0116] (a) 300 kDa M+: ion mass and charge.

[0117] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0118] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity within funnel 14.

[0119] FIG. 19 shows a simulation of ion focusing using

[0120] (a) 300 kDa M+: ion mass and charge.

[0121] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0122] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity in the funnel.

[0123] FIG. 20 shows a simulation of ion focusing using

[0124] (a) 300 kDa M+: ion mass and charge.

[0125] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0126] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity within funnel 14.

[0127] FIG. 21 shows a simulation of ion focusing using

[0128] (a) 300 kDa M+: ion mass and charge.

[0129] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0130] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity within funnel 14.

[0131] FIG. 22 shows a simulation of ion focusing using

[0132] (a) 300 kDa M+: ion mass and charge.

[0133] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0134] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity within funnel 14.

[0135] FIG. 23 shows a simulation of ion focusing using

[0136] (a) 300 kDa M+: ion mass and charge.

[0137] (b) 800, 1600, 2400, 3200, 4000: Each voltage applied in series, starting from the largest ring to the smallest ring.

[0138] (c) Vx = 0.5 m / s: average x-direction (left to right) gas velocity within funnel 14.

[0139] The SIMION simulation overviews shown in Figures 14-23 show that embodiments of the apparatus described in this disclosure provide improved deposition of ions onto a collecting surface compared to the absence of ion focusing. It should be understood that the range of conditions explored using SIMION simulations, along with the diagrams shown, are not intended to constrain or limit the techniques described in this disclosure to implementations of only the simulated conditions. There are other conditions outside of the range of simulated conditions of gas velocity and applied voltage that provide enhanced ion focusing and deposition of ions onto a collecting surface such as a cryo-EM grid. The simulated conditions, including the ion trajectories shown in Figures 14-28, not only provide a demonstration of conditions that enhance the percentage of ions deposited on the collection surface compared to conditions where no voltage is applied to the focusing funnel 14, but also provide a metric to quantify the kinetic energy of the deposited ions. As stated multiple times in this disclosure, the purpose of the apparatus described in this disclosure is to provide a means for soft landing of ions and charged particles on the collection surface. Simulations using SIMION software reveal the energy of the ions as they impact the collection surface. Table 1 shows the ion energies as they impact the surface for the focusing conditions shown in Figures 14-28. More specifically, Table 1 summarizes the conditions simulated by the SIMION software. Ion molecular weight refers to the mass of the ion entering the simulation. z represents the number of charges the ion carries. Vx refers to the background gas velocity within the focusing funnel 14. Funnel voltage refers to the voltages assigned to the rings within the focusing funnel 14. The highest voltages listed are assigned to the smallest rings. KE (kinetic energy) refers to the ion energy as the ion strikes the collection surface. Those familiar with the practice of depositing ions and charged particles onto surfaces will recognize that deposition below 1 eV is considered to be a very low energy impact. The low impact energies listed in Table 1 provide a deposition condition known as a soft landing.

[0140] Experimental verification of the focusing effect of the voltage applied to the focusing funnel 14 is shown in Figures 24-28. Figures 24-28 can be seen to be ion mobility spectra where the y-axis represents the ion current deposited on the collecting surface attached to the end 15 of the rod 16. Figure 24 shows a signal of about 0.02 (dashed line), corresponding to 0.04 picoamps, generated by depositing a flux of ions on the end 15 of the rod 16 when zero voltage is applied to the exit ring of the funnel 14. The flux of ions impinging on the end 15 of the rod 16 was measured by attaching a picoammeter to the rod 16 and measuring the ion current. The flux of ions impinging on the end 15 of the rod 16 increases when 1000 volts is applied to the funnel 14. The solid line in Figure 24 represents the ion mobility spectrum obtained with 1000V applied to the focusing funnel with the voltage applied to the inner member of the nDMA 5 ramped from 0 to 1000 volts. The peaks in this mobility spectrum are assigned to the flux of different types of ions as nDMA5 permeant ions of different mobilities as a result of the voltage applied to the inner member of the nDMA5. +++ Relatively high mobility ions, such as the doubly charged monomer ion of tocilizumab (M), strike the pedestal when a low voltage, such as 175 volts, is applied to the inner member of the nDMA5, thus allowing the high mobility ions to pass through the nDMA5. Higher ramping voltages applied to the inner member of the nDMA5 allow increasingly lower mobility ions to pass through the nDMA5. ++ ) ions, followed in sequence by singly charged monomers (M + ), singly charged dimer (2M + ) and single-charge trimmer (3M + ) can be seen to pass through the nDMA 5, be focused by funnel 14, and then strike end 15 of rod 16 as the voltage applied to the inner member of the nDMA 5 increases from a low to a high value. The peaks in Figure 24 are labeled according to the type of ion they represent, and can be taken as a whole to represent an ion mobility spectrum.

[0141] FIG. 25 shows the effect of applying 2 kV to the focusing funnel while increasing the voltage applied to the inner member of the nDMA 5. Comparing the mobility spectrum of FIG. 24 with that of FIG. 25, it can be seen that when 2 kV is applied to the funnel 14, the M +++ and M. ++ The smaller peaks assigned to the ions are shown, but + , 2M + and 3M + The increase in the height of the peaks is shown. As the voltage applied to the focusing funnel 14 is increased from 1 kV to 2 kV, the M +++ and M. ++ The ions were deflected away from the centerline of funnel 14, but M + , 2M + and 3M + It can be seen that the ions are more efficiently focused. FIG. 26 shows that when the voltage applied to the funnel 14 is further increased to 3 kV, the ions are more efficiently focused. + and 3M + The height of the peak representing the ion further increases, and the M +++ and M. ++ M including ion diffusion + This indicates that diffusion of ions begins. Figure 27 shows that a similar trend occurs when 4 kV is applied to the funnel 14. This trend continues when 5 kV is applied to the funnel 14, as shown in Figure 28.

[0142] The results shown in Figures 24 to 28 are shown together in Figure 29. The three curves in Figure 29 are + , M ++ and 2M + represents the height of the ion peak in the ion mobility spectrum corresponding to the ion current associated with the ion, M + For ions, the optimal focusing voltage is 2 kV, and values ​​of 3 to 4 kV are 2M + Optimal focusing of ions and M ++ Ions are shown to be optimally focused at 1 kV.

[0143] It should be understood that the apparatus described in this disclosure is not limited by the depiction of the preferred embodiment of the technology described in this disclosure or the ion signal shown in FIG. 29. For example, an alternative design of the ion source can be a variable charge reduction source available from IonDX, Inc., Monterey, California. The humidifiers 12a and 12b can be replaced with a thermostatic water bubbler or air sparger, such as the porous metal sparger manufactured by Teesing (www.teesing.com). Additionally, the humidifiers 12a and 12b can operate as coolers to generate liquid droplets or small frozen ice balls.

[0144] From the description herein, it will be understood that the present disclosure encompasses multiple implementations of the present technology, including, but not limited to, the following.

[0145] An apparatus for preparing a sample for examination by cryo-EM, comprising an electrospray ion source and one or more of: (a) a means for reducing charge on the electrospray droplets and ions; (b) a mobility filter configured to select ions having a particular conformation from a mixture of ions; (c) a humidifier configured to control the dew point and temperature of the ion-containing air; (d) an ion focusing funnel; (e) a holder for a cryo-EM grid; (f) a cooling pedestal configured to preserve the conformation of the deposited ions; (g) a chamber for protecting the deposited ions; and (i) a means for transferring the deposited ions or particles into liquid nitrogen.

[0146] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, further comprising means for reducing charge on the electrospray droplets and ions.

[0147] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, further comprising a mobility filter configured to select ions having a particular conformation from a mixture of ions.

[0148] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, further comprising a humidifier configured to control the dew point and temperature of the ion-containing air.

[0149] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, further comprising an ion focusing funnel.

[0150] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, further comprising a holder for a cryo-EM grid.

[0151] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, further comprising a cooling pedestal configured to preserve the conformation of deposited ions.

[0152] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, further comprising a chamber for protecting accumulated ions.

[0153] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, further comprising means for transferring deposited ions or particles into liquid nitrogen.

[0154] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, wherein the means for reducing the charge on the electrospray droplets and ions comprises a 210 polonium source, and alpha particles are emitted from the 210 polonium to the electrospray droplets and ions.

[0155] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, wherein the means for reducing charge on the electrospray droplets and ions includes a source of electrons having an energy of less than about 20 eV, the electrons being emitted into the electrospray droplets and ions.

[0156] An apparatus for preparing a sample for examination by cryo-EM as described in any of the preceding implementations, wherein the means for transferring deposited ions or particles into liquid nitrogen comprises tweezers.

[0157] 1. An apparatus for depositing a sample onto a surface for subsequent analysis, the apparatus comprising an electrospray ion source and one or more of the following: (a) means for reducing charge on the electrospray droplets, ions and particles; (b) a mobility filter configured to select ions and charged particles having a particular conformation or size from a mixture of ions and particles; (c) means for controlling the dew point and temperature of an ion- or particle-containing gas; (d) means for controlling hydration of the ions and charged particles; (e) an ion and charged particle focusing device; (f) means for controlling the kinetic energy of the ions and charged particles; (g) a holder for a substrate onto which the ions or charged particles are deposited; (h) means for depositing the ions and charged particles onto the substrate; (i) means for controlling the temperature of a collection surface; (j) a chamber configured to protect the deposited ions or charged particles; (k) means for transferring the deposited ions or charged particles to an auxiliary analytical device; and (l) means for storing the deposited ions or charged particles in a cryoprotectant.

[0158] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a means for reducing charge on electrospray droplets, ions and particles.

[0159] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising a mobility filter configured to select ions and charged particles having a particular conformation or size from the mixture of ions and particles.

[0160] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising means for controlling the dew point and temperature of the ion-containing or particle-containing gas.

[0161] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a means for controlling hydration of ions and charged particles.

[0162] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising an ion and charged particle focusing device.

[0163] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a means for controlling the kinetic energy of the ions and charged particles.

[0164] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a holder for a substrate onto which the ions or charged particles are deposited.

[0165] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising means for depositing ions and charged particles onto the substrate.

[0166] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a means for controlling the temperature of the collection surface.

[0167] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising a chamber configured to protect the deposited ions or charged particles.

[0168] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising means for transferring the deposited ions or charged particles to an auxiliary analytical device.

[0169] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising means for preserving the deposited ions or charged particles with a cryoprotectant.

[0170] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for reducing the charge on the electrospray droplets, ions and particles comprises a 210 polonium source, and alpha particles are emitted from the 210 polonium to the electrospray droplets, ions and particles.

[0171] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for reducing charge on the electrospray droplets, ions and particles includes an electron source having an energy of less than about 20 eV, and the electrons are emitted to the electrospray droplets, ions and particles.

[0172] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for controlling the dew point of the ion-containing or particle-containing gas comprises a dew point controller configured to add or remove water vapor based on heating or cooling a source of water vapor.

[0173] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for controlling the temperature of the ion-containing or particle-containing gas comprises a temperature controller configured to sense the temperature of the gas flow.

[0174] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for controlling hydration of the ions and charged particles comprises a humidifier configured to hydrate the ions and charged particles based on condensing water vapor on the ions and charged particles.

[0175] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for controlling the kinetic energy of ions and charged particles comprises a mass flow controller or a pressure controller or an electric field generator, or any combination thereof.

[0176] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for controlling kinetic energy uses physical distance, gas density, and gas velocity to produce ions and charged particles with low kinetic energy, separation between electrically driven electrodes affects ion energy, higher gas density reduces ion velocity and therefore ion energy, and the velocity of the ion-containing and particle-containing gas directly affects ion kinetic energy.

[0177] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for depositing ions and charged particles onto a substrate comprises an ion funnel.

[0178] An apparatus for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for controlling the temperature of the collection surface comprises a temperature controller.

[0179] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for transferring deposited ions or charged particles to an auxiliary analytical device comprises tweezers.

[0180] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, wherein the means for preserving the deposited ions or charged particles in a cryoprotectant comprises a refrigeration source.

[0181] An apparatus for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, wherein the mobility filter comprises a nano-differential mobility analyzer.

[0182] 1. A system for depositing a sample on a surface for subsequent analysis, comprising: an electrospray source; (a) a polonium source, where alpha particles are emitted from the polonium into electrospray droplets, ions, and particles; or an electron source having an energy of less than about 20 eV, where the electrons are emitted into the electrospray droplets, ions, and particles; (b) a mobility filter with a nano differential mobility analyzer; (c) a temperature controller and a dew point controller configured to control a temperature and dew point of an ion and particle flow stream, where the temperature control is based on sensing a gas flow temperature and the dew point control is provided by adding or removing water vapor based on heating or cooling a source of water vapor; (d) a humidifier configured to hydrate the ions or particles based on condensing the water vapor into the ions and charged particles; (e) an ion funnel configured to increase the flux of the ions and charged particles to minimize the time required to deposit a predetermined number of ions or particles; (f) a low (g) a holder configured to support the collection surface; (h) an ion deposition device configured to use and control the electric field, gas density and gas velocity to induce the ions and charged particles to strike the collection surface; (i) an optional temperature controller configured to control the temperature of the collection surface and the collected ions and particles while they are being collected; (j) an optional temperature controller configured to control the temperature of the collected ions and particles after they are collected;(k) a transfer stage or apparatus for moving the collected particles from the holder into an auxiliary analytical apparatus in a storage vessel, (l) an optional temperature controller configured to control the temperature of the collected ions and particles as the collected particles are transferred to an alternative analytical device, and (m) a transfer stage or apparatus for transferring the collected particles to a long-term storage vessel, such as a liquid nitrogen dewar, while they are being collected.

[0183] A system for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising: a polonium source, where alpha particles are emitted from the polonium into electrospray droplets, ions and particles; or an electron source having an energy of less than about 20 eV, where the electrons are emitted into electrospray droplets, ions and particles.

[0184] A system for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising a mobility filter comprising a nano-differential mobility analyzer.

[0185] A system for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a temperature controller and dew point controller configured to control the temperature and dew point of the ion and particle flow stream, wherein the temperature control is based on sensing the gas flow temperature and the dew point control is provided by adding or removing water vapor based on heating or cooling a source of water vapor.

[0186] A system for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising a humidifier configured to hydrate the ions or particles based on condensing water vapor into ions and charged particles.

[0187] A system for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising an ion funnel configured to increase the flux of ions and charged particles to minimize the time required to deposit a predetermined number of ions or particles.

[0188] The system for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a mass flow controller or pressure controller or electric field generator, or any combination thereof, configured to control kinetic energy using physical distance, gas density, and gas velocity to generate ions and charged particles with low kinetic energy, where separation between the moving electrodes affects ion energy, higher gas density reduces ion velocity and therefore reduces ion energy, and the velocity of the ion and particle containing gas directly affects ion kinetic energy.

[0189] A system for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a holder configured to support a collection surface.

[0190] A system for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising an ion deposition device configured to use and control an electric field, gas density and gas velocity to induce ions and charged particles to strike the collection surface.

[0191] A system for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a collection surface and an optional temperature controller configured to control the temperature of the collected ions and particles while they are being collected.

[0192] A system for depositing a sample on a surface for subsequent analysis as described in any of the preceding implementations, further comprising an optional temperature controller configured to control the temperature of the collected ions and particles after they are collected.

[0193] A system for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a transfer stage or device for moving the collected particles from the holder into an auxiliary analytical device in the storage container.

[0194] A system for depositing a sample onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising an optional temperature controller configured to control the temperature of the collected ions and particles as the collected particles are transferred to an alternative analytical device.

[0195] A system for depositing samples onto a surface for subsequent analysis as described in any of the preceding implementations, further comprising a transfer stage or apparatus for transferring the collected particles to a long-term storage container, such as a liquid nitrogen dewar, while they are being collected.

[0196] A method comprising preparing a sample for examination by cryo-EM using an apparatus or system described in any of the preceding implementations.

[0197] As used herein, the term "implementation" is intended to include, but not be limited to, embodiments, examples, or other forms of practicing the techniques described herein.

[0198] As used herein, the singular terms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular does not mean "one and only one" unless explicitly stated, but rather "one or more."

[0199] A phrase construct such as "A, B and / or C" within this disclosure explains that either A, B, or C may be present, or any combination of items A, B, and C may be present. "At least one of" following a phrase construct listing a group of elements indicates that at least one of those group elements is present, including, where applicable, any possible combination of the listed elements.

[0200] Reference in this disclosure to "an embodiment," "at least one embodiment," or similar embodiment language indicates that a particular feature, structure, or characteristic described in connection with the described embodiment is included in at least one embodiment of the disclosure. Thus, these various embodiment phrases do not necessarily all refer to the same embodiment, or to a specific embodiment that is different from all other embodiments described. Language of embodiments should be interpreted to mean that the particular features, structures, or characteristics of a given embodiment can be combined in any suitable manner in one or more embodiments of the disclosed devices, systems, or methods.

[0201] As used herein, the term "set" refers to a collection of one or more objects. Thus, for example, a set of objects can include a single object or multiple objects.

[0202] Relational terms such as first and second, above and below, upper and lower, left and right, etc. do not necessarily require or imply an actual relationship or order between the entities or operations, but may be used only to distinguish one entity or operation from another.

[0203] The terms "comprises," "comprising," "has," "having," "includes," "including," "contains," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises, has, includes, or contains a list of elements does not include only those elements, but may include other elements that are not expressly listed or that are inherent to such process, method, article, or apparatus. "comprises...a," "has...a," "includes...a," or "contains...a" following an element "a" does not, without further constraints, preclude the presence of additional identical elements in the process, method, article, or apparatus that comprises, has, includes, or contains the element.

[0204] As used herein, the terms "approximately", "approximate", "substantially", "essentially" and "about", or any other version thereof, are used to express and describe small variations. When used in conjunction with an event or circumstance, these terms can refer to when the event or circumstance occurs exactly as well as when the event or circumstance occurs approximately. When used in conjunction with a numerical value, these terms can refer to a range of variation of ±10% or less of that numerical value, such as ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less. For example, "substantially" aligned can refer to a range of angular variation of ±10° or less, such as ±5° or less, ±4° or less, ±3° or less, ±2° or less, ±1° or less, ±0.5° or less, ±0.1° or less, or ±0.05° or less.

[0205] Furthermore, amounts, ratios, and other numerical values ​​may be presented in a range format herein. It is to be understood that such range formats are used for convenience and brevity, and include numerical values ​​explicitly specified as the limits of the range, but should be understood flexibly to include each numerical value and all individual numerical values ​​or subranges encompassed within the range, as if the subrange were explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also include individual ratios such as about 2, about 3, and about 4, and subranges such as about 10 to about 50, about 20 to about 100, etc.

[0206] As used herein, the term "coupled" is defined as connected, but not necessarily a direct connection, and not necessarily a mechanical connection. A device or structure that is "configured" in a particular way is at least so configured, but may be configured in other ways not recited.

[0207] Benefits, advantages, solutions to problems, and any element or elements that may cause or make more pronounced any benefit, advantage, or solution are not to be construed as a critical, required, or essential feature or element of the technology described herein or any or all of the claims.

[0208] Moreover, in the foregoing disclosure, various features may be grouped together in various embodiments for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Inventive subject matter may lie in less than all features of a single disclosed embodiment.

[0209] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

[0210] It will be understood that the practice in some jurisdictions may require the deletion of one or more parts of the present disclosure after the application has been filed. Therefore, the reader should refer to the application as filed for the original content of the present disclosure. Any deletion of the content of the present disclosure should not be interpreted as an abandonment, loss or contribution to the public of any subject matter of the application as originally filed.

[0211] The following claims are hereby incorporated into this disclosure, with each claim standing on its own as separately claimed subject matter.

[0212] Although the description herein contains many details, these should not be construed as limiting the scope of the disclosure, but merely as providing illustrations of some of the presently preferred embodiments, and therefore, the scope of the disclosure will be understood to fully encompass other embodiments that may become apparent to those skilled in the art.

[0213] All structural and functional equivalents to the elements of the disclosed embodiments known to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Furthermore, no element, component, or method step of the present disclosure is intended to be a contribution to the public, regardless of whether the element, component, or method step is expressly recited in the claims. No element of the claims herein should be construed as a "means-plus-function" element unless the element is expressly recited using the phrase "means for". No element of the claims herein should be construed as a "step-plus-function" element unless the element is expressly recited using the phrase "step for". [Table 1]

Claims

1. 1. An apparatus for preparing a sample for examination by cryo-EM, comprising: (a) an electrospray ion source; (b) a means for reducing the charge on the Electrospray droplets and ions; and (c) a mobility filter configured to select ions having a particular conformation from a mixture of ions; and (d) a humidifier configured to control the dew point and temperature of the ion-laden air; (e) an ion focusing funnel; and (f) a holder for a cryo-EM grid; (g) a cooling pedestal configured to preserve the conformation of the deposited ions; (h) a chamber for protecting the deposited ions; (i) a means for transferring the deposited ions or particles into liquid nitrogen; An apparatus comprising:

2. said means for reducing the charge on the electrospray droplets and ions comprises a 210 polonium source; 10. The apparatus of claim 1, wherein alpha particles are emitted from 210 polonium into electrospray droplets and ions.

3. said means for reducing charge on electrospray droplets and ions comprises a source of electrons having an energy of less than about 20 eV; The apparatus of claim 1 , wherein the electrons are emitted into electrospray droplets and ions.

4. The apparatus of claim 1 , wherein the means for transferring the deposited ions or particles into the liquid nitrogen comprises tweezers.

5. 1. An apparatus for depositing a sample on a surface for subsequent analysis, comprising: (a) an electrospray ion source; (b) a means for reducing the charge on the Electrospray droplets, ions and particles; (c) a mobility filter configured to select ions and charged particles having a particular conformation or size from the mixture of ions and particles; (d) a means for controlling the dew point and temperature of the ion- or particle-containing gas; (e) a means for controlling the hydration of ions and charged particles; (f) an ion and charged particle focusing device; (g) a means for controlling the kinetic energy of the ions and charged particles; (h) a holder for a substrate onto which the ions or charged particles are deposited; (i) a means for depositing ions and charged particles onto a substrate; (j) a means for controlling the temperature of the collection surface; and (k) a chamber configured to protect the deposited ions or charged particles; (l) a means for transferring the deposited ions or charged particles to an auxiliary analytical device; and (m) a means for preserving the deposited ions or charged particles in a cryoprotectant; and An apparatus comprising:

6. said means for reducing charge on electrospray droplets, ions and particles comprises a 210 polonium source; 6. The apparatus of claim 5, wherein alpha particles are emitted from 210 polonium into electrospray droplets, ions and particles.

7. said means for reducing charge on electrospray droplets, ions and particles comprises a source of electrons having an energy of less than about 20 eV; The apparatus of claim 5 , wherein said electrons are emitted to electrospray droplets, ions and particles.

8. 6. The apparatus of claim 5, wherein said means for controlling a dew point of the ion-containing or particle-containing gas comprises a dew point controller configured to add or remove water vapor based on heating or cooling of a source of water vapor.

9. 6. The apparatus of claim 5, wherein said means for controlling the temperature of the ion-containing or particle-containing gas comprises a temperature controller configured to sense a temperature of the gas stream.

10. 6. The apparatus of claim 5, wherein said means for controlling hydration of the ions and charged particles comprises a humidifier configured to hydrate the ions and charged particles based on condensing water vapor on the ions and charged particles.

11. 6. The apparatus of claim 5, wherein said means for controlling the kinetic energy of ions and charged particles comprises a mass flow controller or a pressure controller or an electric field generator, or any combination thereof.

12. 12. The apparatus of claim 11, wherein said means for controlling kinetic energy uses physical distance, gas density, and gas velocity to produce ions and charged particles with low kinetic energy, separation between electrically driven electrodes affects ion energy, higher gas density reduces ion velocity and therefore ion energy, and the velocity of the ion- and particle-containing gas directly affects ion kinetic energy.

13. The apparatus of claim 5 , wherein said means for depositing ions and charged particles onto a substrate comprises an ion funnel.

14. The apparatus of claim 5 , wherein said means for controlling the temperature of the collection surface comprises a temperature controller.

15. 6. The apparatus of claim 5, wherein said means for transferring deposited ions or charged particles to an auxiliary analytical device comprises tweezers.

16. 6. The apparatus of claim 5, wherein said means for preserving deposited ions or charged particles in a cryoprotectant comprises a refrigeration source.

17. The apparatus of claim 5 , wherein the mobility filter comprises a nanodifferential mobility analyzer.

18. 1. A system for depositing a sample on a surface for subsequent analysis, comprising: (a) an electrospray source; (b) a polonium source, where alpha particles are emitted from the polonium to the electrospray droplets, ions and particles, or an electron source having an energy of less than about 20 eV, where the electrons are emitted to the electrospray droplets, ions and particles; (c) a mobility filter comprising a nano-differential mobility analyzer; (d) a temperature controller and a dew point controller configured to control a temperature and a dew point of the ion and particle flow stream, the temperature control being based on sensing a gas stream temperature and the dew point control being provided by adding or removing water vapor based on heating or cooling a source of water vapor; and (e) a humidifier configured to hydrate the ions or particles based on condensing water vapor into ions and charged particles; (f) an ion funnel configured to increase the flux of ions and charged particles to minimize the time required to deposit a given number of ions or particles; (g) a mass flow controller or pressure controller or electric field generator, or any combination thereof, configured to control kinetic energy using physical distance, gas density, and gas velocity to produce ions and charged particles with low kinetic energy, where separation between the moving electrodes affects ion energy, higher gas density reduces ion velocity and therefore ion energy, and the velocity of the ion and particle containing gas directly affects ion kinetic energy; and (h) a holder configured to support a collection surface; (i) an ion deposition device configured to use and control electric fields, gas density and gas velocity to direct ions and charged particles to impinge on a collection surface; (j) an optional temperature controller configured to control the temperature of the collection surface and the collected ions and particles while they are being collected; (k) an optional temperature controller configured to control the temperature of the collected ions and particles after they are collected; (l) a transfer stage or device for moving the collected particles from the holder into the storage container and into an auxiliary analytical device; (m) an optional temperature controller configured to control the temperature of the collected ions and particles as the collected particles are transferred to an alternative analytical device; and (o) a transfer stage or device for transferring the collected particles to a long-term storage container, such as a liquid nitrogen Dewar; An apparatus comprising: while they are collected, while they are collected.

19. A method comprising preparing a sample for examination by cryo-EM using an apparatus according to any one of claims 1 to 18.

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