Fei Comapny

Philips Electron Optics

Michael Frederick Hayles

Rudolf Johannes Peter Gerardus Schampers

Uwe Luecken

Mathijs Petrus Wilhelmus Van Den Boogaard

Andreas Theodorus Engelen

Remco Theodorus Johannes Petrus Geurts

Johannes A H W G Persoon

Dirk Arie Mattheus De Winter

Christianus Thomas Wilhelmus Maria Schneijdenberg

Gerardus N A Van Veen

Ruud Schampers

Ron Van Den Boogaard

Johannes Persoon

Chris Schneijdenberg

Matthijs De Winter

Described is a system and method for in situ sample preparation and imaging. The system includes a multi-axis stagehaving a bulk stageand a grid stagewith various degrees of freedom to allow for sample preparation. In some embodiments, a focused ion beam system is used to prepare a lamella on the bulk stage. The lamella can then be transferred to the grid stagefrom the bulk stagewithout needing to move the multi-axis stagefrom the focused ion beam system.

Sample preparation stage

A method of forming a sample from a capillary with high-pressure frozen sample material comprises providing a high-pressure capillary with vitrified sample material at a temperature Tbelow the glass transition temperature T, cutting the capillary, warming the capillary to a temperature Tbetween temperature Tand temperature T, cooling the capillary to a temperature Tbelow temperature T, as a result of which material is extruded from the capillary, and freeing a sample from the extruded sample material at a temperature below temperature T. Repeating this temperature cycle results in further extrusion of the sample material. The extruded material can then be sliced by, for example, ion beam milling.

Forming an electron microscope sample from high-pressure frozen material

The invention relates to the extraction of a frozen hydrated sample for TEM (Transmission Electron Microscope) inspection, such as a vitrified biological sample, from a substrate and the attaching of said sample to a manipulator. Such a hydrated sample should be held at a cryogenic temperature to avoid ice formation. By melting or sublimating a part of the sample material outside the area to be studied in the TEM and freezing the material to the manipulator (), a bond is formed between sample () and manipulator. This makes it possible to transport the sample from the substrate to e.g. a TEM grid. In a preferred embodiment a part () of the manipulator () is held at a cryogenic temperature, and the melting or sublimation is caused by heating the tip () of the manipulator by electric heating of the tip and then cooling the tip of the manipulator to a cryogenic temperature, thereby freezing the sample () to the manipulator.

Method for attaching a sample to a manipulator by melting and then freezing part of said sample

In relatively thick samples for electron microscopy imaging, details of interest are often located in the bulk of the sample, so that they cannot be directly imaged in the form of a SEM image. According to the invention, so as to expose the cross-section containing the details of interest, the frozen sample is subjected to ion milling, in such a manner that the desired cross-section is exposed. Thereafter, the exposed cross-section is further eroded in a controlled manner via sublimation, whereby the detail of interest is approached in a very accurate manner, and its fine details become visible. Hereafter, the finally desired SEM image can be made. By repetition of this process, a large number of successive cross-sections can be imaged, so that a spatial representation of the sample is obtained.

Method of obtaining a particle-optical image of a sample in a particle-optical device

The SEM according to the invention is arranged to detect scattered electrons transmitted by the specimen (22). The detector is constructed so as to have a flat detector surface (26) for a large angular range. In order to select a given angular range, a mask (24) with an eccentric opening (40) is arranged between the specimen and the detector surface and it is also arranged so as to be rotatable. An optimum angle relative to a given direction in the specimen can thus be selected for a given type of contrast. The mask is preferably displaceable in the specimen direction, the detector surface being subdivided into a plurality of parts (26  ) and the assembly formed by the specimen and the detector surface is tiltable through an angle relative to the optical axis (4). A high degree of flexibility is thus achieved for the selection of the electrons to be detected which are scattered by the specimen, so that adjustments can be made so as to achieve an optimum contrast situation.

SEM for transmission operation with a location-sensitive detector

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Eindhoven, Netherlands

Michael Frederick Hayles