Method for preparing biological samples for study in a charged particle device

Abstract

Method for preparing a biological sample for investigation in a charged particle device. The present invention relates to a method for preparing a biological sample for investigation in an analytical device, the method comprising the following steps: providing a biological material to be investigated; providing a sample holder configured to be placed in the analytical device; and transferring the biological material to the sample holder in order to prepare the biological sample. According to the present invention, the method comprises the following steps: obtaining a sample of the biological material provided on the sample holder; transferring the sample to a screening device for screening the sample; and evaluating the biological sample based on the results obtained by the screening device. Using the method, time and resources can be used more efficiently for investigating biological samples, for example, using a charged particle microscope in the form of a cryo-EM.

Description

[0001] The present invention relates to a method of preparing a biological sample for study in a charged particle device.

[0002] Biology is the natural science that studies life and organisms, including their physical structure, chemical processes, molecular interactions, physiological mechanisms, development and evolution.

[0003] Cell biology is a branch of biology that studies the structure and function of the cell, the basic unit of life. Cell biology is concerned with the physiological properties of cells, metabolic processes, signal transduction pathways, life cycle, chemical composition, and the interaction of cells with their environment. In cell biology, molecular recognition between macromolecules controls all of the most complex processes in cells. The most common macromolecules include biopolymers (nucleic acids, proteins, carbohydrates, and lipids) and large non-polymeric molecules (such as lipids and macrocyclic compounds).

[0004] Many researchers are interested in studying macromolecular complexes in their natural environment at high resolution in order to reveal their structural dynamics and interactions. For this purpose, charged particle microscopy can be used. Charged particle microscopy is a well-known and increasingly important technique for imaging microscopic objects, particularly in the form of electron microscopy (EM). Historically, the basic class of electron microscopes has evolved into many well-known types of equipment, such as transmission electron microscopes (TEM), scanning electron microscopes (SEM) and scanning transmission electron microscopes (STEM), as well as various subclasses, such as so-called "dual beam" tools (e.g., FIB-SEM), which additionally employ a "machining" focused ion beam (FIB), for example allowing supporting activities such as ion beam milling or ion beam induced deposition (IBID) to be performed.

[0005] EM offers a variety of ways to study biological samples: conventional TEM is used to study the overall morphology of biological samples; electron crystallography and single particle analysis are dedicated to the study of proteins and macromolecular complexes; while (cryogenic) electron tomography and Cryo-EM of vitreous sections (CEMOVIS) target organelles and molecular architecture. In Cryo-EM and CEMOVIS, samples are preserved by rapid freezing using vitrification techniques and observed by cryo-TEM. CEMOVIS also includes cryosectioning of samples, which can be done using cryo-FIB technology.

[0006] The preparation of biological samples for investigation in an analytical device is often time-consuming and laborious. One of the drawbacks associated with these investigations is that only after the biological sample has been prepared and investigated in the analytical device does the user know whether the preparation of the biological sample was successful.

[0007] Preparation of cryo-EM specimens, for example, involves the following steps: taking an aqueous sample of biological material (usually a purified protein complex), applying it to a support structure (grid), reducing its size to be as thin as possible (approximately 100 μm, depending on the size of the biomolecule), and ) and then freezing this layer quickly enough to prevent the water from crystallizing. Many aspects of this method of preparing biological samples are problematic.

[0008] One of the challenges is obtaining a thin layer of sample on a support structure.

[0009] In US 2010 / 181495 A1, a method and a device for preparing a specimen for cryo-electron microscopy are described. Here, a carrier is fixed to a holder, a sample liquid is applied to the carrier, and a blotting device is applied for removing excess sample liquid from the carrier by means of an absorption medium. The absorption medium is irradiated with light, and changes in the optical properties of the absorption medium are detected by an optical sensor device. A control device moves the blotting away from the carrier as a function of the detected changes in the optical properties. This improves the reproducibility of the steps for obtaining the thin layer.

[0010] Since it is difficult to set up a blotting step in a reproducible manner, US 2017 / 350798 A1 proposes a method and apparatus in which the need for blotting excess liquid can be minimized or eliminated by reducing the volume of the sample from the microliter scale to the picoliter scale.

[0011] Even though the blotting step presents challenges, many other aspects of the above methods for preparing biological samples are challenging and unpredictable. Purified complexes may encounter microscopic surfaces, materials, and dynamics that change the purified complexes. Often, these changes are only visible after the sample on the grid is examined in Cryo-EM. Therefore, valuable resources, including scientist preparation time and expensive EM time, may be wasted.

[0012] In view of the above, it is an object of the present invention to provide an improved method relating to the preparation of biological samples for investigation in a charged particle device and in particular a method as described above which prevents the waste of valuable resources.

[0013] To this end, the present invention provides a method for preparing a biological sample for investigation in an analytical device, as defined in claim 1. The analytical device as defined herein is a charged particle device, such as a charged particle microscope in the form of an electron microscope. However, it should be noted that the analytical device may generally be any analytical device that requires significant resources, in particular time and money, to analyze a biological sample.

[0014] Specifically, the analytical device can be an EM device, such as a cryo-TEM. These devices are relatively expensive and require a lot of time to analyze the sample. Other microscopes, such as optical microscopes, fluorescence microscopes, and scanning probe microscopes are also relatively expensive. For example, the same is true for x-ray diffractometers.

[0015] The method according to the invention comprises the step of providing a biological material to be studied in the analytical device. The biological material may be a cell, a cell component, a single cell organism, a macromolecule, such as a protein complex, etc. It is conceivable that the biological material needs to be stored and studied in an aqueous liquid. The liquid may be water, an electrolyte, a cell fluid, blood plasma, etc. The aqueous liquid may comprise one or more buffer solutions.

[0016] The method further comprises the step of providing a sample holder configured to be placed in the analytical device. In some embodiments, the sample holder can be any suitable container that can be used in the analytical device. In other embodiments, the sample holder is specifically configured to be placed in the analytical device, for example due to the fact that the sample holder is configured to cooperate with a specific recess in the analytical device. In a specific embodiment, the sample holder is an EM grid.

[0017] Once the biological material and the sample holder are provided, the biological material is transferred to the sample holder in order to prepare the biological sample. It is conceivable that after the transfer, the sample holder is ready for investigation in an analytical device. Alternatively, one or more additional steps, such as a vitrification step in the case of cryo-EM, may be required to compete for the preparation of the biological sample. Therefore, in one embodiment, the method may further include the step of rapidly cooling the biological material on the sample holder using a cryogen in order to prepare the biological sample.

[0018] In order to use resources in an efficient manner, the method as defined herein provides the additional step of obtaining a sample of the biological material and screening the sample in a screening device. The results of the screening can then be used to evaluate the biological sample. The evaluation can include the step of defining the quality grade of the biological sample. For example, based on the results obtained by the screening device, it can be determined that the biological sample is not adequately prepared for study in an analytical device. Therefore, it can be decided not to continue the study in the analytical device, thereby saving valuable resources.

[0019] Thus, a portion of the sample already provided on the sample holder is used as a sample, which is then screened in turn. In doing so, many possible interactions between the sample and the environment during sample preparation are taken into account, thereby achieving reliable and representative screening results. In particular, the sample can be screened before the actual sample is studied in the analytical device, and therefore, the evaluation of the results can include a decision whether to study this particular biological sample in the analytical device. In that case, the method as described above ensures an efficient use of resources. Therefore, the purpose of the present invention is achieved.

[0020] In one embodiment, the method may comprise the steps of transferring the sample holder with the biological sample to the analysis device and analyzing the biological sample. This step may be conditionally performed, for example based on an evaluation of the results of a screening device. In one embodiment, the steps of transferring and analyzing the biological sample are performed only if a certain minimum quality level is defined for the biological sample.

[0021] Alternatively or additionally, the evaluation step is used to enhance the information obtained in analyzing the biological sample. Thus, screening can be used to interpret the results of a study of a biological sample in an analytical device. Thus, the evaluation of a biological sample can be used to improve the clarity of a study of a biological sample. For example, screening may reveal the oligomeric composition of an analyte, or provide evidence of post-translational modifications that would otherwise not have been noticed in the sample analysis.

[0022] According to the present invention, there is provided a method for preparing a biological sample for study in a charged particle device, the method comprising the following steps:

[0023] - Provide biological materials to be studied;

[0024] - providing a sample holder configured to be placed in said charged particle device;

[0025] - transferring the biological material to the sample holder in order to prepare the biological sample; and

[0026] - obtaining a sample of said biological material provided on said sample holder and performing ambient ionization mass spectrometry on said sample.

[0027] The method may comprise the steps of preparing a biological sample, in particular a protein complex, for investigation in a charged particle device, in particular an electron microscope, such as Cryo-EM. The biological sample may be provided on a suitable support structure, such as an EM grid. Then, once the biological sample is provided on the support structure, a portion of the sample is used as a sample and the sample is subjected to ambient ionization mass spectrometry for orthogonal analysis (i.e. screening). Thus, in practice, the screening device as defined herein comprises a mass spectrometer.

[0028] Sample screening by native mass spectrometry generates relevant information about one or more aspects of the biological sample, in particular protein complexes. These aspects include, for example, sample purity, integrity and homogeneity of the biological sample; protein complex stoichiometry; protein identity and sequence information; post-translational modifications; small molecule binding. Based on this information, it can be assessed whether the prepared sample can be successfully studied using charged particle microscopy. For example, structural information obtained by mass spectrometry can be reported directly to the user to eliminate useless samples. The information can be obtained by a protein-centric approach, in which the sample carrier is not further processed before analysis and the (structural) information is obtained from the intact biomolecule; or by a peptide-centric approach, in which the sample carrier is analyzed before processing, which analysis is a bottom-up proteomics approach well known to the skilled person. Therefore, screening biological specimens using mass spectrometry can be used to enhance or interpret the results obtained by charged particle microscopy.

[0029] Ambient ionization mass spectrometry may comprise one or more techniques selected from the group consisting of: paper spray ionization; liquid extraction surface analysis; desorption electrospray ionization. Other techniques are also contemplated.

[0030] From the above it can be concluded that combining biological sample preparation for study in charged particle microscopy with mass spectrometry can provide a complete workflow for rapid screening of samples on grids prior to cryo-electron microscopy.

[0031] In one embodiment, the method comprises the steps of providing a biological material to be studied in the form of a solution. For example, a biological material to be studied can be provided, and the method can include the step of preparing a solution with the biological material. The solution, such as a protein complex provided in an aqueous liquid comprising one or more buffers, can then be transferred to the sample holder.

[0032] In one embodiment, the step of obtaining a sample of the biological material transferred to the sample holder comprises the step of obtaining the sample using an absorption technique. Such a technique is suitable for easy, reliable and rapid acquisition of the biological material provided on the sample holder. It is to be noted in this regard that absorption techniques as defined herein include both absorption phenomena, i.e. accumulation occurring in the bulk of a carrier, and adsorption phenomena, i.e. accumulation occurring at the surface of a carrier.

[0033] For example, it is noted that, particularly when preparing Cryo-EM samples, it is important that the layer provided on the sample holder is as thin as possible. For this reason, once an aqueous sample of biological material is applied to the support structure (grid), blotting paper is often used to ensure that excess sample is removed and that the layer of biological sample is as thin as possible. This general absorption technique can be advantageously used to obtain biological samples for screening devices.

[0034] In one embodiment, the blotting as an absorption technique can be advantageously used in the method as described herein, particularly when using a blotting material, such as blotting paper, because the blotting material is at least partially used in the step of performing ambient ionization mass spectrometry. It is found that ambient ionization mass spectrometry is particularly useful for biological samples. In addition, the sample concentrations required for performing EM and MS are roughly the same. In this embodiment, the blotting material that has been used (and usually discarded) in EM sample preparation is advantageously used in a screening method to evaluate whether EM samples can be studied in EM. Here, the sample present in (or above) the blotting material has a high degree of similarity to the sample present on the EM grid, thereby producing reliable and predictable screening device results. In addition, it is conceivable that this embodiment of the method can also be easily automated so that sample preparation and screening can be controlled.

[0035] Blotting material, such as the manipulation of blotting paper is conceivable. For example, it is conceivable that before performing the ambient ionization mass spectrometry step, a portion of the blotting material is removed or cut. In one embodiment, the blotting material is used multiple times, each time for different samples, and the blotting material is cut into corresponding pieces to test each individual sample using ambient ionization mass spectrometry. This is very useful in automated sample preparation.

[0036] The invention will now be described in detail based on exemplary embodiments and the accompanying schematic diagrams, in which:

[0037] Figure 1 - shows a longitudinal cross-sectional view of a charged particle microscope according to a first embodiment of the present invention;

[0038] Figure 2 - a first embodiment of a method for screening a biological sample being prepared as a sample for investigation in a charged particle microscope is shown;

[0039] Figure 3 - a second embodiment of a method for screening a biological sample being prepared as a sample for investigation in a charged particle microscope is shown;

[0040] Figure 4 - a third embodiment of a method for screening a biological sample being prepared as a sample for investigation in a charged particle microscope is shown;

[0041] Figure 1 (not to scale) is a highly schematic depiction of an embodiment of a charged particle microscope M, which may be used as an analytical device in one embodiment of the method according to the invention. More specifically, it shows a transmission type microscope M, which in this case is a TEM / STEM (although, in the context of the present invention, it may effectively just be a SEM (see Figure 2 ), or for example ion-based microscopy. Figure 1 In the embodiment of the present invention, within a vacuum housing 2, an electron beam B generated by an electron source 4 propagates along an electro-optical axis B′ and passes through an electro-optical illuminator 6 for directing / focusing the electrons onto a selected portion (which may, for example, be (locally) thinned / planarized) of a biological sample S. A deflector 8 is also depicted, which may (among other things) be used to achieve a scanning motion of the electron beam B.

[0042] The biological sample S is held on a sample holder H, which in this case is a support structure in the form of a grid (not shown), and this sample holder H can be positioned with multiple degrees of freedom by a positioning device / stage A, which moves a bracket A' to which the holder H is (removably) attached; for example, the sample holder H can contain fingers that can (especially) move in the XY plane (see the Cartesian coordinate system depicted; in general, movements parallel to Z and tilts about X / Y are also possible). This movement allows different parts of the sample S to be illuminated / imaged / inspected by an electron beam B (in the Z direction) traveling along an axis B' (and / or allows scanning movements to be performed as an alternative to beam scanning). If necessary, a cooling device (not shown, but known to those skilled in the art) can be brought into close thermal contact with the sample holder H to keep it (and the sample S thereon) at, for example, low temperatures.

[0043] The electron beam B will interact with the sample S so that various types of "stimulated" radiation are emitted from the sample S, including (for example) secondary electrons, backscattered electrons, X-rays, and optical radiation (cathodoluminescence). If desired, one or more of these radiation types may be detected by means of an analysis device 22, which may be, for example, a combined scintillator / photomultiplier tube or an EDX (energy dispersive X-ray spectrometer) module; in this case, an image may be constructed using essentially the same principles as in a SEM. Alternatively or in addition, however, electrons that traverse (pass through) the sample S, exit / emit from the sample S, and continue to propagate (generally, but usually with some deflection / scattering) along the axis B' may be studied. This transmitted electron flux enters an imaging system (projection lens) 24, which will typically include various electrostatic / magnetic lenses, deflectors, correctors (such as an stigmator), etc. In normal (non-scanning) TEM mode, this imaging system 24 may focus the transmitted electron flux onto a phosphor screen 26, which may be retracted / withdrawn (as indicated by arrow 26') if desired, so that it is away from the axis B'. An image (or diffraction pattern) of (a portion of) the sample S will be formed by the imaging system 24 on the screen 26, and this can be viewed through a viewing port 28 located in a suitable portion of the wall of the housing 2. The retraction mechanism of the screen 26 is mechanical and / or electrical in nature and is not depicted here, for example.

[0044] As an alternative to viewing the image on the screen 26, use may be made of the fact that the depth of focus of the electron flux leaving the imaging system 24 is typically quite large (e.g. of the order of 1 meter). Therefore, various other types of analysis devices may be used downstream of the screen 26, such as:

[0045] - TEM camera 30. At camera 30, the electron flux may form a static image (or diffraction pattern) which may be processed by controller / processor 20 and displayed on a display device (not shown), such as a flat panel display. When not needed, camera 30 may be retracted / withdrawn (as schematically shown by arrow 30') to move it away from axis B'.

[0046] - STEM camera 32. The output from camera 32 can be recorded as a function of the (X, Y) scanning position of the electron beam B on the biological sample S, and an image can be constructed, which is a "map" of the output from camera 32 as a function of X, Y. Camera 32 may contain a single pixel of, for example, 20 mm in diameter, as opposed to the matrix of pixels characteristically present in camera 30. In addition, camera 32 will typically have a higher speed than camera 30 (e.g., 10 pixels per second). 2 images per second) at much higher acquisition rates (e.g., 10 6Again, when not needed, the camera 32 can be retracted / withdrawn (as schematically indicated by arrow 32') so as to be away from the axis B' (although in the case of, for example, an annular dark field camera 32, such retraction is not necessary; as in such a camera, the central hole would allow flux to pass when the camera is not in use).

[0047] As an alternative to imaging using cameras 30 or 32 , it is also possible to call upon a spectroscopic device 34 , which can be, for example, an EELS module.

[0048] It should be noted that the order / position of the items 30 , 32 and 34 is not strict and many possible variations can be envisaged. For example, the spectroscopic device 34 can also be integrated into the imaging system 24 .

[0049] In the illustrated embodiment, microscope M further includes a retractable X-ray computed tomography (CT) module, generally indicated by reference numeral 40. In computed tomography (also known as tomographic imaging), a sample is viewed along different lines of sight using a source and (diametrically opposed) detectors to obtain penetrating views of the sample from a variety of angles.

[0050] Note that a controller (computer processor) 20 is connected to the various depicted components via control lines (buses) 20'. This controller 20 may provide various functions, such as synchronizing actions, providing set points, processing signals, performing calculations, and displaying messages / information on a display device (not shown). Needless to say, the (schematically depicted) controller 20 may be (partially) inside or outside the housing 2 and may have a unitary or composite structure as desired.

[0051] The skilled person will appreciate that the interior of the housing 2 need not necessarily be kept in a strict vacuum; for example, in a so-called "environmental TEM / STEM", a background atmosphere of a given gas is intentionally introduced / maintained within the housing 2. The skilled person will also appreciate that, in practice, it may be advantageous to limit the volume of the housing 2 so that it substantially surrounds the axis B' where possible, in the form of a small tube (e.g., of the order of 1 cm in diameter) through which the electron beam used passes, but widened to accommodate structures such as the source 4, the sample holder H, the screen 26, the camera 30, the camera 32, the spectroscopic device 34.

[0052] Reference now Figures 2 to 4 , shows the preparation for use in an analytical device such as Figure 1 Different examples of biological samples studied in a charged particle microscope are depicted in FIG.

[0053] Usually, these Figures 2 to 4 The method shown in contains the following substeps:

[0054] (1) A biological material 52, such as an aqueous sample of a purified protein complex, is provided and applied to a sample holder 51 (here a support structure, such as an EM grid);

[0055] (2) In the illustrated embodiment, the biological sample 52' of the sample provided on the sample holder 51 is obtained by an absorption technique using an absorbent material 61, such as blotting paper 60, 61;

[0056] (3)-(5) Processing the biological sample 52' (in Figure 3 and 4 ), prepare the biological sample 52' for screening in a screening device. In the illustrated embodiment, different types of ambient ionization mass spectrometry are used to screen the biological sample. These types of ambient ionization mass spectrometry techniques will be described in more detail below.

[0057] Figure 2 The so-called paper spray ionization of blotting paper is shown. In step 1, a biological sample 52 is provided on a sample holder 51 by a supply nozzle 53. In step 2, blotting paper 61 is used to remove excess sample 52 and reduce the size of sample 52 to a layer as thin as possible so that the sample can be vitrified (not shown) in a subsequent step. The details of the method for vitrifying these samples can be obtained from, for example, US 9,865,428 B2; and these methods themselves are known to those skilled in the art. This blotting shown in step 2 can be performed on multiple identical or different samples. As shown in step 3, blotting paper 60 can contain multiple cake sheets 61, which can now contain multiple identical or different screening samples 52', which represent the corresponding samples 52 on the sample holder 51. The blotting material 60 is then transferred to a holder 70, which pierces triangles from the blotting paper to separate the screening samples 52' of various blottings. These triangles 61 are then transferred to the stage of a mass spectrometer. To extract and ionize a sample for mass spectrometry analysis, a compatible extraction buffer 71 is drawn onto blotting paper 61 and a high voltage (1-5 kV) is applied, thereby producing a jet of small droplets 74 into the source of a mass spectrometer for analysis.

[0058] exist Figure 3 In the present invention, liquid extraction electrospray ionization of the blotted material is used as a screening aid. The process is similar to Figure 2The process is similar to that of the present invention, but here a blotting device 80 is used to obtain a sample 52' on the blotting material 61. The blotting device 80 can then be transferred as a whole to a liquid extraction surface analysis (LESA) stage. Here, as shown in step 3, a pipette 91 filled with a buffer compatible with a mass spectrometer applies droplets to the surface of the blotting material 61 containing the biological sample 52'. The droplets stay here for a few seconds, allowing the analyte molecules to diffuse from the surface by forming liquid microjunctions. Now, the pipette 91 contains a portion of the sample 52' (step 4) and can be moved to the source of the mass spectrometer (step 5), where a voltage 72 is applied to generate a jet of small droplets 74 to enter the source of the mass spectrometer for analysis. The solution can also be re-injected and then introduced into the mass spectrometer using a transmitter through conventional (nano) ESI.

[0059] Figure 4 Desorption electrospray ionization of blotting material is shown. The process is similar to that described for Figure 2 and 3 Here, as Figure 3 As shown, a blotting device 80 is used, which is transferred to act as a desorption ESI stage. Here, a conductive emitter 91 is filled with a buffer 71 compatible with mass spectrometry, to which a voltage 72 is applied, thereby generating a stream of charged droplets 71 that wet the blotting material. Upon interaction with the blotting sample 52', some of the droplets are ejected again as charged droplets 74 containing the analyte. These droplets 74 can then be introduced into the vacuum of a mass spectrometer for analysis.

[0060] The present invention has been described above by using several examples, but the present invention is not limited to the above examples.

[0061] Typically, methods as described herein are particularly advantageous when the preparation of a biological sample for analysis in an analytical device requires a significant expenditure of resources, for example due to the fact that the actual preparation requires a great deal of time; that the process of successfully preparing the biological sample is uncertain; and / or that the cost per unit time of the analytical device is relatively high. This is particularly true for studying biological samples in charged particle devices. In these cases, collecting a sample of the biological sample and using an orthogonal screening device to assess and evaluate whether the biological sample can be successfully analyzed in the analytical device can at least increase confidence in the biological sample analysis process.

[0062] The methods described herein are particularly suitable for EM sample preparation. In EM sample preparation, current limitations include: sample loss; low throughput; and sample consumption. Using the methods described herein, at least one or more of these limitations are addressed. Currently, preparing grids for Cyro-EM results in a 99.9% loss of proteins, which are absorbed by blotting material (e.g., blotting paper). This blotting paper currently has no use and is discarded. According to one embodiment of the method described herein, the blotting material is advantageously used to screen biological samples on the grid, in particular using mass spectrometry. This technology is relatively cheap and fast (seconds to minutes per paper sample), and therefore provides valuable information early in the study of biological samples. In addition, it can also be used for high-throughput screening of even large sample sets. In this way, it is possible to select the correct buffer solution, for example, or other conditions required for the successful preparation of biological samples for study in an electron microscope.

[0063] Furthermore, by screening the biology before taking samples to the electron microscope, users will achieve higher throughput by eliminating unnecessary time-, resource-, and sample-consuming pre-screening steps in electron microscopy to, for example, negative stain EM that limit high-throughput analysis.

[0064] The desired protection is defined by the following claims.

Claims

1. A method for preparing a biological sample for study in a charged particle device, the method comprising the following steps: Provide biological materials to be studied; providing a sample holder configured to be placed in the charged particle device; and The biological material is transferred to the sample holder to prepare the biological sample; characterized in that Follow these steps: A sample of the biological material provided on the sample holder is obtained and ambient ionization mass spectrometry is performed on the sample.

2. The method according to claim 1, wherein the biological material to be investigated is supplied in the form of a solution and the solution is transferred to the sample holder.

3. The method according to claim 1 or 2, wherein the step of obtaining a sample of the biological material transferred to the sample holder comprises the step of obtaining the sample using an absorption technique. The method of claim 3 , wherein the absorption technique comprises blotting using a blotting material.

5. The method of claim 4, wherein the blotting material is blotting paper.

6. The method of claim 4, wherein the blotting material is used at least in part in the step of performing ambient ionization mass spectrometry.

7. The method of claim 1 or 2, comprising the step of evaluating the biological sample based on the results obtained by the ambient ionization mass spectrometry.

8. The method according to claim 7, wherein said evaluation comprises the step of defining a quality grade of said biological sample.

9. The method according to claim 1 or 2, comprising the steps of transferring the sample holder with the biological sample into the charged particle device and analyzing the biological sample.

10. The method of claim 8, wherein the step of transferring and analyzing the biological sample using the charged particle device is performed only if a certain minimum quality level is defined for the biological sample.

11. The method of claim 7, wherein the evaluating step is used to enhance information obtained in analyzing the biological sample.

12. The method of claim 1 or 2, wherein ambient ionization mass spectrometry comprises one or more techniques selected from the group consisting of: paper spray ionization; liquid extraction surface analysis; desorption electrospray ionization.

13. The method of claim 1 or 2, wherein the sample holder comprises a grid.

14. The method according to claim 1 or 2, further comprising the step of rapidly cooling the biological material on the sample holder using a cryogen to prepare the biological sample.

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