Lossless cryo-grid preparation by controlled sample evaporation

Patent metadata

Field	Value
Publication number	US-11860071-B2
Application number	US-201716342529-A
Country	US
Kind code	B2
Filing date	Oct 17, 2017
Priority date	Oct 17, 2016
Publication date	Jan 2, 2024
Grant date	Jan 2, 2024

Abstract

Method for preparing a sample, wherein the sample is provided as a thin film on a support structure; the temperature of the support structure is adjusted to a value above the dew point temperature of the environment to decrease the film thickness, light is directed at the support structure, an intensity value of the transmitted light is measured, and the support structure is automatically inserted into a liquid cryogen dependent on the measured intensity value. The application further relates to a system comprising a support structure, a temperature-controlled stage for keeping the support structure at a pre-defined temperature, a transfer mechanism for moving the support structure into a container containing a liquid cryogen, a light source, a photodetector for measuring an intensity value of the transmitted light, and a control device for triggering the transfer mechanism dependent on the measured intensity value.

First claim

The invention claimed is:

1. Method for preparing a sample on a support structure ( 20 ), wherein
the sample is provided as a thin film having a film thickness on a support structure ( 20 );
the temperature of the support structure ( 20 ) is adjusted to a value above the dew point temperature of the environment, such that the film thickness decreases;
light is directed at the support structure ( 20 );
at least one intensity value of the light transmitted by the support structure ( 20 ) is measured;
the support structure ( 20 ) is automatically inserted into a liquid cryogen ( 80 ) dependent on the at least one measured intensity value, such that the sample is cooled down to an amorphous solid.

2. The method according to claim 1 , wherein the support structure ( 20 ) is automatically inserted into the liquid cryogen ( 80 ) when the at least one measured intensity value exceeds a pre-determined threshold.

3. The method according to claim 1 , wherein the temperature of the support structure ( 20 ) is adjusted to a value 2° to 8° above the dew point temperature of the environment.

4. The method according to claim 1 , wherein the film thickness is decreased by blotting or re-aspiration prior to adjusting the temperature to a value above the dew point temperature of the environment.

5. The method according to claim 1 , wherein the wavelength of the light is in the near infrared range, wherein particularly the wavelength is 780 nm or more.

6. The method according to claim 1 , wherein the light is directed at the support structure ( 20 ) along a light path (L) which is perpendicular to the surface of the support structure ( 20 ).

7. The method according to claim 1 , wherein the sample is provided onto the support structure ( 20 ) by aspirating a pre-defined amount of the sample into a capillary and dispensing the sample onto the support structure ( 20 ) by means of the capillary.

8. The method according to claim 1 , wherein an additional calibration step is performed, wherein the light is directed at a reference support structure ( 20 ) that is free from the sample, and wherein at least one reference intensity value of the light transmitted by the reference support structure ( 20 ) is measured, and wherein the support structure ( 20 ) carrying the sample is automatically inserted into the liquid cryogen ( 80 ) dependent on the ratio of the at least one measured intensity value and the at least one reference intensity value.

9. The method according to claim 1 , wherein the light directed at the sample comprises at least light of a first wavelength, particularly in the range between 600 nm and 900 nm, more particularly in the range between 780 nm and 880 nm or in the range between 630 nm and 670 nm, and light of a second wavelength, particularly in the range between 350 nm and 500 nm, more particularly in the range between 405 nm and 450 nm.

10. System ( 100 ) for preparing a sample on a support structure ( 20 ) comprising
a support structure ( 20 ) that is configured to accommodate a sample,
a temperature-controlled stage ( 1 ) that is configured to keep the support structure ( 20 ) at a pre-defined temperature when the support structure ( 20 ) is arranged on the temperature-controlled stage ( 1 ),
a transfer mechanism ( 60 ) that is configured to move the support structure ( 20 ) from the temperature-controlled stage ( 1 ) into a container ( 12 ) containing a liquid cryogen ( 80 ) so that the sample on the support structure ( 80 ) contacts the cryogen ( 80 ),

characterized in that the system ( 100 ) comprises
a light source ( 13 ) that is configured to provide light directed at the support structure ( 20 ),
a photodetector ( 14 ) that is configured to measure at least one intensity value of the light transmitted by the support structure ( 20 ),
a control device ( 30 ) that is configured to receive said measured intensity value from the photodetector ( 14 ) and trigger the transfer mechanism ( 60 ) dependent on the at least one measured intensity value, such that the support structure ( 20 ) is inserted into the container ( 12 ) containing the liquid cryogen ( 80 ).

11. System ( 100 ) according to claim 10 , characterized in that the light source ( 13 ) is adapted to emit the light along a light path (L), wherein the support structure ( 20 ) and the photodetector ( 14 ) are arranged along the light path (L), such that at least a part of the light travels from the light source ( 13 ) along the light path (L) via the support structure ( 20 ) to the photodetector ( 14 ), wherein particularly the support structure ( 20 ) is arranged perpendicular to the light path (L).

12. System ( 100 ) according to claim 10 , characterized in that the system ( 100 ) comprises a first adapter ( 3 ) configured to hold the support structure ( 20 ), wherein the transfer mechanism ( 60 ) is configured to be connected to the first adapter ( 3 ) holding the support structure ( 20 ), and wherein the transfer mechanism ( 60 ) is configured to pivot the first adapter ( 3 ) together with the support structure ( 20 ) into a vertical position above the container ( 12 ) and to move the first adapter ( 3 ) and the support structure ( 20 ) downwards after said pivoting so that the sample on the support structure ( 20 ) contacts the cryogen ( 80 ) in the container ( 8 ).

13. System ( 100 ) according to claim 12 , characterized in that the system ( 100 ) comprises an adjustment means ( 5 ) configured to hold the first adapter ( 3 ) with respect to the temperature-controlled stage ( 1 ) in an adjustable manner, wherein the adjustment means ( 5 ) comprises a holding means ( 50 ), particularly in the form of an electromagnet ( 50 ), that is configured to releasably hold the first adapter ( 3 ) and to automatically release the first adapter ( 3 ) when the sample is positioned on the support structure ( 20 ).

14. System ( 100 ) according to claim 12 , characterized in that the transfer mechanism ( 60 ) comprises a second adapter ( 7 ), wherein the first adapter ( 3 ) and the second adapter ( 7 ) are designed to engage with each other when the holding means ( 50 ) releases the first adapter ( 3 ), and wherein the transfer mechanism ( 60 ) is designed to pivot the second adapter ( 7 ) above said container ( 12 ) when the first adapter ( 3 ) is engaged with the second adaptor ( 7 ) and released from the holding means ( 50 ).

15. System ( 100 ) according to claim 10 , characterized in that the system ( 100 ) comprises a liquid handling system comprising a dispensing head ( 11 ), wherein the liquid handling system is configured to aspirate and dispense a volume of a sample via the dispensing head ( 11 ), wherein the system ( 100 ) further comprises a first reservoir, into which the dispensing head ( 11 ) containing a nanoliter volume of a sample can be immersed so as to allow diffusion-controlled sample conditioning, e.g. for desalting or exchange of buffer prior to deposition on the support structure ( 20 ).

Assignees

• Univ Basel

Inventors

• Braun Thomas
• Arnold Stefan
• Stahlberg Henning

Classifications

• G01N1/2813 Primary

  Producing thin layers of samples on a substrate, e.g. smearing, spinning-on (G01N1/30 takes precedence) · CPC title

• G01N1/42

  Low-temperature sample treatment, e.g. cryofixation · CPC title

• G01N1/44

  Sample treatment involving radiation, e.g. heat · CPC title