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 + and/or a Xe + plasma.

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 +  and a Xe +  plasma.   
     
     
         2 . Method according to the preceding claim wherein with an Xe +  plasma based ion beam O 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 +  plasma ions, more preferably at least 66%, and more preferably almost or exactly 100% O +  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 μm3/second, more preferably at least 500 μm3/second, and more preferably at least 1,000 μm3/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 +  or a Xe +  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.