METHOD, DEVICE AND SYSTEM FOR THE TREATMENT OF BIOLOGICAL CRYOGENIC SAMPLES BY PLASMA FOCUSED ION BEAMS

Abstract

The invention relates to a method, a device and a system for the treatment of biological frozen samples using plasma focused ion beams (FIB). The samples can then be used for mass spectrometry (MS), genomics, such as gene sequencing analysis or next generation sequencing (NGS) analysis, and proteomics. The present invention particularly relates to a method of treatment of at least one biological sample. This method is particularly used for high performance microscopy, proteomics analytics, sequencing, such as NGS etc. According to the present invention the method comprises the steps of providing at least one biological sample in frozen form. The milling treats at least one part of the sample by a plasma ion beam comprising at least one of an O.sup.+ and/or a Xe.sup.+ plasma.

Claims

1. A method of treatment of at least one biological sample comprising the steps of: providing at least one biological sample in frozen form; and milling at least one part of the sample by a plasma ion beam comprising at least one of an O.sup.+ and a Xe.sup.+ plasma.

2. Method according to the preceding claim wherein with an Xe.sup.+ plasma based ion beam O.sub.2 gas has been added.

3. Method according to any of the preceding claims wherein the milling is performed by a plasma ion beam comprising at least 10% O.sup.+ plasma ions, more preferably at least 66%, and more preferably almost or exactly 100% O.sup.+ plasma ions.

4. Method according to any of the preceding claims with the further step of milling at least one part of the sample by at least one plasma ion beam at: an effective beam current of at least 6 nA, preferably of at least 500 nA, even more preferably of at least 1000 nA; and/or a mass removal rate of at least 100 ?m3/second, more preferably at least 500 ?m3/second, and more preferably at least 1,000 ?m3/second.

5. Method according to any of the preceding claims with the step of milling the sample in order to perform at least one of a removal of unwanted parts of the sample and an extraction of a target.

6. Method according to the preceding claim with the further step of accumulating a plurality of the targets in an accumulated sample.

7. Method according to any of the preceding claims wherein the treatment is a submicron treatment and the method further comprising the step of generating at least one microscopic image at least for one of visualizing and controlling the milling, in particular wherein the microscopic image is generated by at least one of SEM and TEM microscopy.

8. Method according to any of the preceding claims with the further step of analyzing the sample or the accumulated sample, in particular proteins in the sample, by mass spectrometry, preferably by an orbitrap fusion mass spectrometer.

9. Method according to any of claims 5 to 8 with the further step of sequencing the target(s) by next-generation-sequencing (NGS).

10. Method according to any of claims 5 to 9, wherein spatial information of the target is also tracked and further used for the analysis of it, preferably when analyzing proteins contained in the sample.

11. A device for the treatment of at least one biological sample, particularly for carrying out a method according to any of the preceding claims, comprising: at least one support for at least one biological sample in frozen form; and at least one plasma ion beam generator configured to generate at least one plasma ion beam comprising at least one of an O.sup.+ or a Xe.sup.+ plasma.

12. Device according to claim 11, comprising a dual plasma ion beam generator, in particular wherein the plasma ion beam generator is configured to generate a plasma ion beam comprising at least 10% O+ plasma ions, preferably at least 66%, and more preferably almost or exactly 100% O+ plasma ions.

13. Device according to the two preceding claims with a microscope, preferably a SEM or a TEM microscope, for generating a microscopic image at least for one of visualization and controlling the milling.

14. A system comprising a device according to the preceding claim and at least one of a microscope, preferably a SEM or a TEM microscope, a mass spectrometer, preferably an orbitrap fusion mass spectrometer, and a sequencing station, preferably a NGS station.

15. Use of a method according to any of the claims 1 to 10, at least for one of proteomic analyses, genomics, such as gene sequencing, and high performance microscopy, such as SEM or TEM.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0076] FIG. 1 depicts an embodiment of a workflow according to the prior art.

[0077] FIG. 2 shows a principle arrangement in an FIB milling station.

[0078] FIG. 3 depicts a result of milling in accordance with the present invention.

[0079] FIG. 4 shows the preparation of grids for embryo anterior and posterior, respectively.

DESCRIPTION OF EMBODIMENTS

[0080] FIG. 1 schematically depicts an embodiment of a typical workflow as it is practiced in the art. A biological sample 1 shown is intended to be further analyzed, the biological sample consisting of one or more organisms, cells, structures, proteins, DNA, RNA etc. In order to analyze cells, parts of cells, proteins, RNA and/or DNA contained in the biological sample the sample 1 is exposed to one or more stages for their selected breaking apart and disintegrating etc. by one or more digestions or enzymes, symbolized by a respective container 2. The parts, molecules, proteins etc. can be further separated in a high pressure liquid chromatograph 3 (HPLC) so that the parts of particular interest can then be better isolated. These can then be transferred into a mass spectrometer 4 in order to further isolate and specify the parts, molecules, proteins etc. of interest. One disadvantage is the rather unspecified chemical and physical separation without localizing these proteins etc.

[0081] Furthermore, laser dissection is known to isolate parts of interest. However, the samples should be kept alive while discarding the time aspect or should be chemically fixated that would affect the proteomics results and can degrade signals.

[0082] FIG. 2 shows an exemplifying example of an FIB sample preparation. As FIG. 2 shows, the gallium (Ga.sup.+) primary ion beam generated by an Ga.sup.+ ion generator 5 hits the sample surface and sputters a small amount of material, which leaves the surface as either secondary ions (i.sup.+ or i.sup.?) or neutral atoms (n.sup.0). The primary beam also produces secondary electrons (e.sup.?). As the primary beam rasters on the sample surface, the signal from the sputtered ions or secondary electrons is collected to form an image.

[0083] According to the present invention the primary ion beam now comprises O.sup.+ and/or Xe.sup.+ as described before and claimed below. Ar.sup.+, N.sup.+, Kr.sup.+, Ne.sup.+, He.sup.+ and/or H.sup.+ can be also comprised.

[0084] At low primary beam currents, very little material is sputtered and modern FIB systems can easily achieve 5 nm imaging resolution (imaging resolution with Ga ions is limited to ?5 nm by sputtering and detector efficiency). At higher primary currents, a great deal of material can be removed by sputtering, allowing precision milling of the specimen down to a sub micrometer or even a nano scale.

[0085] A gas assisted etching and/or an optional gas gun 6 can realize selected deposition. This can be applied according to the present invention, particularly when Xe.sup.+ is being used as the primary ion beam O.sub.2 is advantageous to be added by the gas gun 6.

[0086] If the sample is non-conductive, a low energy electron flood gun 7 can be used to provide charge neutralization. In this manner, by imaging with positive secondary ions using the positive primary ion beam, even highly insulating samples may be imaged and milled without a conducting surface coating, as would be required in an SEM.

[0087] According to FIG. 3 the biological sample 1 can be treated according to the present invention by selecting a target 11 and/or removing unwanted parts 12. As is shown this can be done by milling the target 11 out of the unwanted part 12 or by milling and sputtering away the unwanted part 12 from the target 11. The latter is a mass-removal of the unwanted part(s) or volumes.

[0088] In FIG. 4 two charts are shown. The left one exemplifies the preparation of grids for two different regions of an embryo, anterior (y-axis) and posterior (x-axis) and demonstrates one of the preferred advantages of the present invention, namely the additional use of spatial information. More particularly, FIG. 4 shows the spatial selection or distribution of the targets in a biological sample in accordance with the present invention. In the present case grids for two different regions of the embryo (Drosophila embryo) have been developed, anterior and posterior. More regions can be tracked as well.

[0089] The samples were run on orbitrap fusion mass spectrometer of the present assignee to identify the various proteins. As mentioned before, those can then also be spatially assigned.

[0090] To the right of FIG. 4 the distribution of the protein expression targets vis-?-vis the RNA expression targets are shown.

[0091] Whenever a relative term, such as about, substantially or approximately is used in this specification, such a term should also be construed to also include the exact term. That is, e.g., substantially straight should be construed to also include (exactly) straight.

[0092] Whenever steps were recited in the above or also in the appended claims, it should be noted that the order in which the steps are recited in this text may be the preferred order, but it may not be mandatory to carry out the steps in the recited order. That is, unless otherwise specified or unless clear to the skilled person, the order in which steps are recited may not be mandatory. That is, when the present document states, e.g., that a method comprises steps (A) and (B), this does not necessarily mean that step (A) precedes step (B), but it is also possible that step (A) is performed (at least partly) simultaneously with step (B) or that step (B) precedes step (A). Furthermore, when a step (X) is said to precede another step (Z), this does not imply that there is no step between steps (X) and (Z). That is, step (X) preceding step (Z) encompasses the situation that step (X) is performed directly before step (Z), but also the situation that (X) is performed before one or more steps (Y1), . . . , followed by step (Z). Corresponding considerations apply when terms like after or before are used.

Comparative Examples

[0093] Tests with Ga.sup.+, Xe.sup.+, and O.sup.+ have been conducted. Sample preparation requires selectively removing large portions of cryopreserved Drosophila (fruit fly embryo's). The following has been observed.

[0094] With a Ga.sup.+ plasma ion beam a current available was at around 50 nA. The milling rate appears to decrease as dose builds up on sample. The time required to mill appears not advantageous for the amount of samples required.

[0095] With Xe.sup.+ an increase in current was allowed for a 10 times improvement in the milling rate. However, the milling rate could even be improved with O.sub.2 gas.

[0096] The use of O.sup.+ as a primary ion beam showed the best results for this application, achieving milling rates up to 4500 ?m.sup.3/second (45? enhancement compared to Ga.sup.+). Further advantageously the samples appeared to be getting not damaged by the ion beam (probably by local heating) as expected with such high milling rates. This is even more noticeable as sample gets smaller or on samples that are not supported/submerged in ice.

[0097] The O.sup.+ plasma ion beam had a voltage of 30 keV in a 1 ?A setting with an effective current of 680 nA being measured. This is more than 13 times the current compared to the Ga.sup.+ ion beam set up.

[0098] In the before-mentioned example with the O.sup.+ plasma ion beam the beam has undergone a CCS Pattern with 15 ?m z-depth, 1 ?s dwell time, 65% x-overlap and 85% y-overlap. Half of a Drosophila embryo could thus be removed in ?7 min.

[0099] The O.sup.+ plasma ion beam according to the present invention can thus create a workflow allowing many samples to be prepared in a single session. In the end ?100 cells may be needed for one sample so that a very considerable time is saved compared to standard methods.