ELECTRON MICROSCOPE AND METHOD FOR TRANSMISSION ELECTRON MICROSCOPY IMAGING OF SAMPLE ARRAYS

Abstract

A method of electron microscopy imaging of samples, using an electron microscope (100) having a microscope column (10) and a transfer device (11) with a grid carriage (12), comprises the steps of preparing multiple samples (1) on a single electron microscopy grid (2), including dispensing the samples (1) with a dispenser device (30) on distinct positions on the grid (2), introducing the grid (1) with the transfer device (11) into the microscope column (10), and electron microscopy imaging of the samples (1), wherein the preparing step includes holding the grid (2) on the grid carriage (12) of the transfer device (11) or on a grid holder device (20) provided at the electron microscope (100) and dispensing the samples (1) on the grid (2) while holding it on the grid carriage (12) or on the grid holder device (20). Furthermore, an electron microscope (100) for electron microscopy imaging of samples is described.

Claims

1. A method of electron microscopy imaging of samples, using an electron microscope having a microscope column and a transfer device with a grid carriage, comprising the steps of preparing multiple samples on a single electron microscopy grid, including dispensing the samples with a dispenser device on distinct positions on the grid, introducing the grid with the transfer device into the microscope column, and electron microscopy imaging of the samples, wherein the preparing step includes holding the grid on the grid carriage of the transfer device or on a grid holder device provided at the electron microscope and dispensing the samples on the grid while holding it on the grid carriage or on the grid holder device.

2. The method according to claim 1, wherein, for dispensing the samples onto the grid on the grid carriage, the grid is provided on the grid carriage being inserted in the transfer device adjacent to an input port of the microscope column, or the grid is provided on the grid carriage being separated from the transfer device.

3. The method according to claim 1, wherein the grid has at least one identification feature, which identifies at least one of the grid itself and positions of the samples on the grid.

4. The method according to claim 3, wherein the identification feature includes at least one of a dot code arranged adjacent to the samples, a characteristic sample pattern formed by the samples, and a characteristic grid pattern formed by a grid label.

5. The method according to claim 4, wherein at least one of the dot code and the characteristic sample pattern is deposited on the grid after arranging the grid on the grid carriage or on the grid holder device.

6. The method according to claim 1, wherein the step of dispensing the samples onto the grid includes varying at least one of buffer solutions of the samples, concentrations of the samples, surfactants added to the samples, and concentrations of the surfactants.

7. The method according to claim 1, wherein the step of preparing the samples on the grid includes at least one of staining the samples with a staining substance, wherein the dispenser device is used for supplying the staining substance to the samples, and removing excess liquid with the dispenser device from the samples.

8. The method according to claim 7, wherein the step of staining the samples on the grid includes varying at least one of the staining substance, surfactants added to the staining substance and concentrations of the staining substance.

9. The method according to claim 1, including a step of optical imaging the grid on the grid carriage or on the grid holder device for collecting at least one optical image of the grid or an identification feature thereof.

10. The method according to claim 9, wherein the preparing step is controlled using the optical image such that the samples are dispensed in at least one of a central portion of the grid and a predetermined orientation of the grid relative to the grid carriage.

11. The method according to claim 7, wherein the preparing step includes determining locations of the samples using the optical image, and removing the excess liquid at the locations of the samples.

12. The method according to claim 1, wherein the samples are dispensed on the grid while holding it on the grid holder device, and the grid is moved to the transfer device by picking the grid from the grid holder device.

13. An electron microscope for electron microscopy imaging of samples, comprising a microscope column having a transfer device with a grid carriage, wherein the transfer device is configured for introducing an electron microscopy grid into the microscope column, and a dispenser device being arranged adjacent to the microscope column, such that the dispenser device is capable of dispensing the samples onto the grid while the grid is held on the grid carriage of the transfer device or on a grid holder device adjacent to the microscope column.

14. The electron microscope according to claim 13, wherein the dispenser device is arranged for dispensing the samples onto the grid on the grid carriage being coupled with the transfer device adjacent to an input port of the microscope column.

15. The electron microscope according to claim 14, wherein the dispenser device is coupled with the microscope column.

16. The electron microscope according to claim 13, wherein the dispenser device is arranged for dispensing the samples onto the grid on the grid carriage being separated from the transfer device.

17. The electron microscope according to claim 13, wherein the dispenser device comprises at least one piezoelectric dispenser.

18. The electron microscope according to claim 13, further comprising a sample plate carrier coupled with the dispenser device.

19. The electron microscope according to claim 13, further comprising an optical imaging device being arranged for collecting at least one optical image of the grid arranged at the transfer device or the grid holder device.

20. The electron microscope according to claim 13, wherein the electron microscope is provided with the grid holder device, and the grid holder device comprises a planar grid holder card being adapted for carrying an array of grids.

21. The electron microscope according to claim 20, wherein the grid holder card has at least one of the features the grid holder card has a size of a microscope slide, the grid holder card has at least one holder card label, and the grid holder card has a self-adhesive surface holding the grids.

Description

[0025] Further details and advantages of the invention are described in the following with reference to the attached drawings, which show in:

[0026] FIG. 1: features of preferred embodiments of an electron microscope according to the invention;

[0027] FIG. 2: a schematic partial plan view of a transfer device used according to the invention;

[0028] FIG. 3: a schematic plan view of a grid holder card;

[0029] FIG. 4: illustrations of grids with grid labels;

[0030] FIG. 5: schematic illustrations of depositing samples from a sample plate onto a grid; and

[0031] FIG. 6: further schematic illustrations of depositing samples onto a grid (FIG. 6A) and removing excess liquid from the samples (FIG. 6B).

[0032] Features of preferred embodiments of the invention are described in the following with particular reference to the provision of a transmission electron microscope with a dispenser device and the preparation of a sample array on a grid just before introducing the grid into the electron microscope. Details of the electron microscope and the dispenser device as such and methods of operating thereof are not described as far as they are known from conventional electron microscopes or dispenser devices. Furthermore, exemplary reference is made to the use of a translation device having a sliding grid carriage for introducing a grid into the microscope column of the electron microscope, wherein the grid carriage is permanently coupled with the transfer device. The implementation of the invention is not restricted to the use of this type of transfer device, but rather possible with other types of transfer devices, e. g. having a removable grid carriage.

[0033] FIG. 1 represents a schematic illustration of the electron microscope 100 having a microscope column 10 comprising a transfer device 11 with a grid carriage 12, a grid holder device 20, a dispenser device 30, an optical imaging device 40 and a control device 50 with a display 51. The electron microscope 100 further includes a support table 14 and additional components, like a power supply and a vacuum equipment (not shown in FIG. 1). As an example, an electron microscope Tecnai G2 (manufacturer: FEI) is used for implementing the invention.

[0034] The transfer device 11 includes the sliding grid carriage 12 (exemplary plan view shown in FIG. 2) and a vacuum lock 13 providing an injection port of the microscope column 10. The transfer device 11 is adapted for accommodating and shifting the grid carriage 12 with the grid 2 through the vacuum lock 13 and further into the beam path of the microscope column 10. The grid carriage 12 is shifted manually or using a motion drive. The transfer device 11 may have a removable grid carriage, like e. g. a sample holder of the manufacturer Gatan, USA.

[0035] The dispenser device 30 includes a piezoelectric droplet dispenser 31, a translation stage 32 and a sample plate carrier 33. The translation stage 32 and the sample plate carrier 33 have a fixed position relative to the microscope column 10. These components are connected with a support structure 34 comprising multiple vertical support rods 35 and at least one horizontal support beam 36 for moving the translation stage 32. The support structure 34 is positioned e. g. on the support table 14 as shown with drawn lines in FIG. 1. Alternatively, the support structure 34 can be directly connected with the microscope column 10 as shown at 34A with dashed lines in FIG. 1. Further exemplary features of the support structure 34 are illustrated in FIG. 5. With a preferred example, the dispenser device 30 comprises the apparatus sciFLEXARRAYER (manufacturer: Scienion AG, Germany).

[0036] The sample plate carrier 33 provides a support e. g. for a microtiter plate 37, wherein samples e. g. with varying buffer solutions or surfactants and optionally washing and/or staining solutions are arranged in the wells of the microtiter plate 37. Preferably, the sample plate carrier 33 is coupled with the support structure 34, e. g. with one of the vertical support rods 35.

[0037] The optical imaging device 40 comprises a CCD camera, which is mechanically connected with the piezoelectric droplet dispenser 31, the translation stage 32 or the support structure 34. The optical imaging device 40 is arranged for collecting an optical image of the sliding carriage 12 including the grid 2.

[0038] The grid holder device 20 comprises a grid holder card 21 (exemplary plan view shown in FIG. 3), which carries a plurality of grids 2. With the illustrated preferred example, the grids 2 are provided with a matrix arrangement with two rows A and B and five columns 1 to 5. The number/letter of the columns and rows provides an identification of the grids 2, which can be assigned to an identification feature of the grids 2. The grid comprise e. g. pore grids made of nanoporous amorphous SiN films with a 100 m thick frame, a 500 m*500 m window, an average pore diameter of 30 nm and a porosity of about 25% (manufacturer SiMPore, USA).

[0039] FIGS. 4A to 4C illustrate examples of identification features of the grid 2, wherein the identification features comprise'grid labels 3, which are formed as through-holes in a circumferential portion of the grid 2 (grid frame 4) and/or as a dot code (see FIG. 4B). The identification features allow a traceability of grids and grid recognition inside or outside the microscope.

[0040] FIGS. 5A to 5F illustrate temporal phases of dispensing the samples onto the grid 2 with schematic front views (FIGS. 5A to 5C, see also FIG. 1) and schematic side views (FIGS. 5D to 5F) of the transfer device 11 and the support structure 34. According to FIG. 5A, the transfer device 11 (shown in part) includes the grid carriage 12 with the grid 2. The support structure 34 includes two vertical support rods 35 extending in a vertical direction (z-direction) and two horizontal support beams 36. The horizontal support beams 36 provide a frame extending in horizontal directions (x-y-directions) perpendicular to the vertical support rods 35 and being movable with translation stages (not shown) in the vertical (z) and one (y) of the horizontal directions. The droplet dispenser 31 is coupled to one of the horizontal support beams 36. It is movable with a further translation stage (not shown, see translation stage 32 in FIG. 1) in the other one (x) of the horizontal directions. A microtiter plate 37 is coupled via a plate carrier (not shown) with one of the vertical support rods 35.

[0041] With a preferred embodiment of the invention, the electron microscopy imaging of the samples on the grid 2 is conducted with the following steps. Firstly, grids 2 are provided in the grid holder device 20 (see FIG. 1). One particular grid 2 is selected in dependency on the number and size of samples to be arranged as an array on the grid surface. The selected grid 2 is positioned on the sliding carriage 12 of the transfer device 11. The grid 2 is moved to the sliding carriage 12 using tweezers or a picking device (not shown).

[0042] Subsequently, with the embodiment of FIG. 5, the samples are dispensed on distinct positions on the grid 2 when it is held at the transfer device 11. The samples are taken with the piezoelectric droplet dispenser 31 from the microtiter plate 37 and deposited on the grid 2. The dispenser device 30 and the translation stages of the support structure 34 are controlled with the control device 50 in dependency on an optical image collected with the optical imaging device 40 and presented for monitoring purposes on the display 51 (see FIG. 1).

[0043] According to FIGS. 5A and 5D, the samples are aspirated from the microtiter plate 37 on the plate carrier. To this end, the piezoelectric droplet dispenser 31 is moved into one of the wells of the microtiter plate 37 and operated for accommodating the sample liquid. Subsequently, the piezoelectric droplet dispenser 31 is lifted in the vertical direction and moved to the transfer device 11, as shown in FIGS. 5B and 5E. Finally, the sample is dispensed onto the grid 2. According to FIGS. 5C and 5F, the piezoelectric droplet dispenser 31 is lowered towards the transfer device 11.

[0044] FIG. 6A schematically illustrates the creation of flying droplets 5 and the deposition on the grid 2 using the piezoelectric droplet dispenser 31. With the translation stage 32 (see FIG. 1), the piezoelectric droplet dispenser 31 is adjusted relative to the grid 2 such that the liquid droplets 6 are deposited at the distinct positions on the grid surface.

[0045] Subsequently, the dispenser device 30, e. g. another dispenser 31A thereof is used for removing excess liquid from the deposited droplets 6, so that the samples 1 to be imaged remain on the grid surface (FIG. 6B). If the samples are to be stained, the staining substance is supplied and excess staining substance is removed with the dispenser device 30, e. g. just another piezoelectric droplet dispenser thereof.

[0046] Finally, the grid 2 with the sample array is moved through the injection port into the microscope column 10. Transmission electron microscopy images of the samples 1 are collected as it is known from conventional transmission electron microscopy. The electron microscopy images are assigned to specific samples using the identification features 3 of the sample grids 2.

[0047] The features of the invention disclosed in the above description, the drawings and the claims can be of importance individually or in combination for the realisation of the invention in its different embodiments.