(19)
(11)EP 3 502 650 A1

(12)EUROPEAN PATENT APPLICATION

(43)Date of publication:
26.06.2019 Bulletin 2019/26

(21)Application number: 17210499.4

(22)Date of filing:  22.12.2017
(51)International Patent Classification (IPC): 
G01N 1/28(2006.01)
G01N 35/10(2006.01)
B01L 3/02(2006.01)
G01N 1/42(2006.01)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
MA MD TN

(71)Applicant: Universiteit Maastricht
6211 LK Maastricht (NL)

(72)Inventors:
  • NIJPELS, Franciscus Johannes Theresia
    3620 Veldwezelt (BE)
  • RAVELLI, Raimond
    6331 BW Maastricht (NL)
  • BEULEN, Bart Wilhelmus Anna Maria Mathis
    6101 JA Echt (NL)
  • THEWESSEM, Sanne
    6171 VA Stein (NL)
  • HENDERIKX, Rene Jacobus Maria
    6021 RE Haler (NL)
  • HUYSMANS, Pascal Paul Marie
    6245 RK Eijsden (NL)
  • LÓPEZ-IGLESIAS, Carmen
    6221 KN Maastricht (NL)
  • PETER, Jacobus Peter Johannes
    6013 RP Hunsel (NL)

(74)Representative: De Vries & Metman 
Overschiestraat 180
1062 XK Amsterdam
1062 XK Amsterdam (NL)

  


(54)METHOD OF AND APPARATUS FOR APPLYING LIQUID TO A SUBSTRATE


(57) The invention relates to a method of applying liquid (1), in particular one or more samples, to a substrate (2), comprising the steps of adhering an amount of the liquid (1) to an applicator surface (5), moving the applicator surface (5) towards the substrate (2) until the liquid (1) touches the substrate (2) and forms a capillary bridge (9) between the applicator surface (5) and the substrate (2), and moving the applicator surface (5) relative to the substrate (2) thus applying the liquid to the substrate (2).




Description


[0001] The invention relates to method of and apparatus for applying liquid, in particular one or more samples, e.g. for imaging or diffraction experiments under cryogenic conditions, to a substrate, in particular a sample carrier, such as a film on a grid. Other examples of substrates include nanoreactors and nanofluidic devices, such as nanocapillary array membranes. Samples include (single) particles, e.g. macromolecular compounds and/or complexes thereof, e.g. proteins, DNA, RNA, viruses, cells, bacteria, nanoparticles, and crystals, for imaging, e.g. in a cryo-electron microscope (EM), or diffraction experiments, e.g. inspection by ionizing radiation, under cryogenic conditions.

[0002] T. Jain et al. "Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM.", J Struct Biol. 2012 Jul;179(1):68-75, relates to method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process.

[0003] WO 2013/109405, WO 2013/109406 and US2014360286 disclose methods for preparing an electron microscopy sample on an EM sample grid, comprising: dispensing a plurality of discrete specimens onto an EM sample grid in an ordered array of specimen locations, each specimen of the plurality of discrete specimens being placed into an individual specimen location in the array of locations.

[0004] As explained in S. Arnold et al. "Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts", Journal of Structural Biology, Volume 197, Issue 3, March 2017, Pages 220-226, transmission electron microscopy (TEM) of vitrified specimens has become a powerful technique for the high-resolution structural analysis of biological matter, and is increasingly recognized as a mainstream tool in biology.

[0005] Cryogenic sample-grid (cryo-EM grid) preparation and imaging methods ensure that biological specimens withstand the vacuum inside electron microscopes, allow their investigation while trapped at physiological conditions that conserve the structural arrangement of the biomolecules and reduce the effect of radiation damage. Arnold continues: these preparation methods have not improved significantly over the last 20 years and have some major drawbacks: a 2-4 µL sample volume is required, and 99.9% of the sample volume is lost during grid preparation, due to an extensive blotting step made with filter paper. Furthermore, blotting with filter paper can lead to protein aggregation or denaturation. The current state of the art sample preparation methods are recognized as one of the most significant limitations in cryo-EM.

[0006] Arnold relates to a method for cryo-electron microscopy (cryo-EM) that requires only 3-20 nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. This method is also described in WO 2017/005297.

[0007] It is an object of the present invention to provide an improved method of and apparatus for a applying liquid to a substrate, in particular by further reducing the amount of liquid per application, e.g. the amount of liquid required for preparing an EM specimen, and/or improve reproducibility.

[0008] To this end, the method according to the present invention comprises the steps of adhering an amount of the liquid to an applicator surface, moving the applicator surface towards the substrate until the liquid touches the substrate and forms a capillary bridge between the applicator surface and the substrate, and moving the applicator surface relative to, in particular along, the substrate thus applying the liquid to the substrate.

[0009] In an embodiment, after the liquid touches the substrate, the entire volume of the liquid is captured between the applicator surface and the substrate (including liquid that penetrates into or spreads over the substrate).

[0010] With the method of the present invention, it is possible to apply amounts of liquid expressed in picoliters (pL) to a substrate. Thus, loss of sample is further reduced or, ideally, avoided altogether and/or reproducibility is improved.

[0011] In a further embodiment, after the liquid touches the substrate, the applicator surface is moved further towards the substrate, thus increasing the width of the capillary bridge (relative to the direction of the movement of the applicator surface) and/or providing a supply of liquid for application within the capillary bridge.

[0012] In another embodiment, the applicator surface is moved relative to the substrate in a (systematic) pattern, such as parallel straight lines or line segments (connected e.g. by turns), undulating lines or line segments, or a spiral, e.g. until the capillary bridge breaks. In an embodiment, the lines or segments of the same line partly overlap to form a wider layer. Alternatively, several droplets can be applied on different locations on the substrate.

[0013] In another embodiment, during application of the liquid to the substrate, the distance between the applicator surface and the substrate is reduced, e.g. the applicator surface is moved gradually or stepwise towards the substrate to compensate for the reduced volume of the liquid in the capillary bridge resulting from application of the liquid to the substrate.

[0014] During application, the distance is preferably maintained in a range from 1 to 20 pm. Further, it is preferred that the liquid is applied in a pattern or layer having a thickness in a range from 10 nm to 10pm, preferably in range from 10 to 200 nm, preferably in range from 10 to 150 nm. Layer thickness can be influenced inter alia by the (adjustable) speed of the relative movement between the applicator surface and the substrate, the width of the applicator surface, the distance between the applicator surface and the substrate, which, at least with flat surfaces, defines the height of the capillary bridge, the viscosity of the liquid, and the (relative) hydrophilicity of the applicator surface and the substrate.

[0015] In another embodiment, enabling accurate definition of and/or control over the amount and shape of the liquid on the applicator surface, the applicator surface is defined at an end of a pin, in particular a solid pin (comprising no lumen or capillary), or as at least one hydrophilic area, preferably a continuous hydrophilic area or an array of hydrophilic areas, in a hydrophobic area or surroundings and/or the applicator surface is continuous and flat or (slightly) concave or convex. In an embodiment, the applicator surface has an area in a range from 1,0.1E3 to 50.1E3 µm2, preferably in a range from 2,0.1E3 to 30.1E3 µm2.

[0016] In an embodiment, the amount of liquid adhered to the applicator surface is smaller than 1000 pL (picoliter), preferably smaller than 500 pL, preferably smaller than 400 pL, preferably in a range from 10 to 300 pL, and/or the liquid on the applicator surface has a contact angle in a range from 20 to 90 degrees, preferably in a range from 25 to 55 degrees and/or the height of the liquid on the applicator surface is in a range from 1 to 30 pm. These parameters can be influenced e.g. by selecting area (µm2), shape, and hydrophilicity of the applicator surface in dependence of the properties of the liquid.

[0017] Yet another embodiment comprises the step of determining the distance between applicator surface and the substrate, before the liquid touches the substrate, preferably before adhering an amount of the liquid to the applicator surface. The distance can be determined e.g. by optics or by an inductive or an capacitive sensor.

[0018] In a refinement, the position of the applicator surface and/or of one or more locations on the substrate is measured, e.g. by positioning the tip of a pin defining the surface and one or more locations on the substrate in the focal plane of an on-axis camera, the measured position or positions are stored, preferably in a memory of a computer or controller memory, and the measured position or positions are used for positioning of the applicator surface relative to the substrate.

[0019] In an embodiment, two or more different samples, e.g. different substances and/or different concentrations of the same substance, are applied to the substrate.

[0020] In another embodiment, at least one reservoir, e.g. at least one pipette, is provided to supply the applicator surface with sample or samples. E.g., a droplet or meniscus is temporarily expelled from the reservoir, sample is taken from the droplet or meniscus by contacting the droplet or meniscus with the applicator surface or applicator surfaces, once or repeatedly until sufficient sample or samples has/have been applied to the substrate, and the droplet or meniscus is withdrawn into the reservoir. The reservoir containing the sample can be stored for future use.

[0021] In another embodiment, the substrate is exposed to a plasma, before applying the sample to the substrate. The plasma will (glow) discharge the substrate rendering it hydrophilic, which in turn facilitates a substantially even application of the sample to the substrate. Instead of or in addition to (glow) discharge, the plasma cleans the substrate. It is preferred that the substrate is subjected to a plasma just prior to applying the sample to the substrate, e.g. less than 15 minutes, preferably less than 10 minutes before applying the sample to the substrate.

[0022] In another embodiment, at least prior to and preferably also during and after application of the liquid to the substrate, the temperature of the substrate and/or of the applicator surface is adjusted to a temperature around, at or above the dew point of the gas surrounding the substrate and/or the applicator surface, preferably to one or more tenths of a degree, e.g. in a range from 0,1 to 1,0 degrees, below or above the dew point temperature of the gas surrounding the substrate and/or the applicator surface. In an example, the dew point temperature is calculated from temperature and humidity measurements of the gas surrounding the substrate and/or the applicator surface. The temperature of the substrate and/or the applicator surface is measured as well. These measurements are used in a closed feedback loop to control e.g. a Peltier element that thermally contacts the substrate and/or the applicator surface. Thus, exchange of water, in particular evaporation, between the liquid and the surrounding gas is suppressed or, ideally, prevented and reproducibility is further enhanced.

[0023] In a further embodiment, one or more of the above steps, preferably all steps are carried out in a closed chamber, preferably providing controlled temperature and humidity.

[0024] The invention also relates to an apparatus for applying liquid, in particular one or more samples, to a substrate comprising

a substrate holder,

an applicator surface for adhering an amount of the liquid to, which surface is preferably defined at the end of an elongated applicator, the apparatus further comprising

one or more actuators for moving the applicator surface and the substrate holder towards and relative to each other, and

a controller configured to drive the actuator(s) to move the applicator surface and the substrate holder towards and relative to each other to form a capillary bridge between the applicator surface and a substrate in the substrate holder and to apply the liquid to the surface.



[0025] In an embodiment, the controller is configured to effect one or more of the steps of the method(s) described above.

[0026] In another embodiment, the applicator surface is defined at an end of a pin, in particular a solid pin (comprising no lumen or capillary), or as at least one hydrophilic area, preferably a continuous hydrophilic area or an array of hydrophilic areas, in a hydrophobic area or surroundings and/or the applicator surface is continuous and flat or (slightly) concave or convex. In an embodiment, the applicator surface has an area in a range from 1,0.1E3 to 50.103 pm2, preferably in a range from 2,0.1E3 to 30.1E3 µm2.

[0027] In another embodiment, the apparatus comprises an on-axis measuring system, e.g. comprising a camera and an objective, configured to determine the distance between applicator surface and a substrate in the substrate holder, in particular to measure the position of the applicator surface and/or of one or more locations on the substrate is measured, to store the measured position or positions, and to use the measured position or positions for positioning of the applicator surface relative to the substrate.

[0028] In another embodiment, the substrate holder and the applicator surface are located inside a climate chamber. In a refinement, the apparatus comprises a cooling and/or heating element for the substrate and/or the applicator surface, a temperature sensor and a humidity sensor, and a controller configured to calculate the dew point of the gas surrounding the substrate and/or the applicator surface and to adjust, by means of the element(s), the temperature of the substrate and/or the applicator surface to a temperature around, at or above the dew point of the gas surrounding the substrate and/or the applicator surface, preferably to within one or more tenths of a degree from the dew point.

[0029] Typically, the controller comprises a processor and a memory and is configured, e.g. programmed, to receive data on the positions of the substrate and the applicator surface and the temperature and humidity of the surrounding gas, to process such data, and to operate one or more drivers, e.g. stepper motors, that move the substrate and/or the applicator surface, and one or more cooling and/or heating elements. In general, the controller may be implemented in the form of any system including a processor and a memory that is capable of performing the functions described in this specification. Further, the controller may be coupled to one or more input/output (I/O) devices. Examples of input devices may include, but are not limited to, one or more camera's and/or sensors, one or more buttons, a (small) keyboard, touchscreen, or the like. Examples of output devices may include, but are not limited to, a monitor or a display, speakers, or the like. Input and/or output devices may be coupled to the data processing system either directly or through intervening I/O controllers.

[0030] Within the framework of the present invention, the term "liquid" includes substances comprising a liquid phase, such as liquids containing (large) proteins and, non-newtonian fluids (such as pastes and blood), which substances are capable of forming a capillary bridge. Typical samples are aqueous solutions of large biomolecules such as proteins. However, also samples such as fibres, vesicles, viruses, nanocrystals and polymers and any other solution or suspension of particulate sample could be applied to the grid by capillary coating.

[0031] The invention will now be explained in more detail with reference to the Figures, which show an embodiment of the present invention.

Figures 1A and 1B illustrate steps to determine the position of an applicator surface.

Figures 2A and 2B illustrate steps to determine the position of a substrate.

Figure 3 is a perspective view of a pin according to the present invention taking liquid from a reservoir.

Figure 4 illustrate the inspection of the liquid adhering to the applicator surface and positioning of the applicator surface relative to the substrate.

Figures 5 to 7 are side views and a perspective view showing the application of a liquid to a substrate.

Figure 8 is a side view of a mechanism for resiliently mounting an applicator.

Figures 1 to 7 show an example of method of applying liquid, such as one or more samples 1, to a substrate, such as a so-called EM-grid 2, by means of an applicator in the form of a metal pin 3, e.g. a tungsten pin having a diameter of 1.6 mm.



[0032] The pin 3 has a frusto-conical tip 4 with an (imaginary) top angle of approximately 20 degrees and defining a flat end surface with sharp edges, serving as an applicator surface 5. The applicator surface was polished to obtain a platform having a diameter in a range from 50 to 200 pm, e.g. 100 pm. In this example, the tip is ultrasonically cleaned in ethanol.

[0033] As shown in Figures 1A and 1B, the applicator surface 5 at the end of the pin 3 is gradually positioned in the focal plane 6 of an on-axis camera 7 depicted as a lense. The position at which the applicator surface is in focus is stored and used as a reference for later positioning of the pin 3 relative to the EM-grid 2.

[0034] Subsequently, and shown in Figures 2A and 2B, the grid 2 is positioned (on a holder and brought) into the focal plane 6 of the on axis camera 7. Together with the position of the applicator surface, this provides the information needed to position the applicator surface at a defined distance away from the surface of the grid.

[0035] For non-planar grids, a focal series of the entire surface of the grid can be made in advance, to obtain a height map of the non-planar grid surface. This enables varying the absolute position of the pin (and thus the applicator surface) while maintaining a constant relative position of the pin to the grid.

[0036] Subsequently, and shown in Figure 3, the clean pin 3 is loaded with sample 1. The sample is stored in a reservoir, in this example a pipette 8, from which a small (sub-microliter) mother droplet is temporarily aspirated, preferably inside a climate chamber. The pin is brought into contact with the droplet, thus adhering an amount of the sample to the applicator surface of the pin.

[0037] The liquid on the applicator surface is visually inspected, shown in Figure 4, and the contact angle between the liquid and the surface and the height of the liquid are determined. Typical contact angles and heights observed on surfaces having a diameter in a range from 50 to 200 µm are in a range from 25 to 55 degrees and in range from 10 to 30 pm, respectively. Evaporation of the liquid can be mitigated using a dewpoint control feedback loop on the pin and/or the grid.

[0038] The pin 3, now loaded with a sub-nanoliter droplet, is moved towards (glow discharged) grid and until a capillary bridge 9 is formed, as shown in Figure 5.

[0039] Relative movement of the grid 2 and the pin 3 will deform the capillary bridge due to capillary shear interaction and apply a line 10 or layer of the liquid 1 to the grid 2, as shown in Figures 6 and 7.

[0040] With the method of the present invention, it is possible to apply amounts of liquid expressed in picoliters (pL) to a substrate. Thus, loss of sample and/or changes in the concentrations in the sample are further reduced or, ideally, avoided altogether.

[0041] Suitable components for an apparatus for applying liquid to a substrate, such as a holder for the substrate, a climate chamber, electrodes for generating a plasma, a visualization system, sensors, and a Peltier element, are known in the art and described in, e.g., PCT patent application PCT/EP2017/065466.

[0042] The invention is not restricted to the above-described embodiments, which can be varied in a number of ways within the scope of the claims. In an example, shown in Figure 8, the pin 3 is mounted in a frame 11 by means of resilient elements 12, allowing the pin to move resiliently in the axial direction. By establishing a capillary bridge and retracting the frame away from the substrate 2 with a force smaller than the capillary force between the substrate and the applicator surface, the liquid holds the substrate and the pin together. During application, the pin will follow, within boundaries, the surface of the grid even if this surface is not flat.


Claims

1. Method of applying liquid, in particular one or more samples, to a substrate, comprising the steps of
adhering an amount of the liquid to an applicator surface,
moving the applicator surface towards the substrate until the liquid touches the substrate and forms a capillary bridge between the applicator surface and the substrate, and
moving the applicator surface relative to the substrate thus applying the liquid to the substrate.
 
2. Method according to claim 1, wherein, after the liquid touches the substrate, the entire volume of the liquid is captured between the applicator surface and the substrate.
 
3. Method according to claim 1 or 2, wherein, after the liquid touches the substrate, the applicator surface is moved further towards the substrate.
 
4. Method according to any one of the preceding claims, wherein the applicator surface is moved relative to the substrate in a pattern.
 
5. Method according to any one of the preceding claims, wherein, during application of the liquid to the substrate, the distance between the applicator surface and the substrate is reduced and/or wherein said distance is maintained in a range from 1 to 20 µm and/or wherein the liquid is applied in a pattern or layer having a thickness in a range from 10 nm to 10pm, preferably in range from 10 to 200 nm.
 
6. Method according to any one of the preceding claims, wherein the applicator surface is defined at an end of a pin, in particular solid pin, or as at least one hydrophilic area, preferably a continuous hydrophilic area or an array of hydrophilic areas, in a hydrophobic area or surroundings and/or wherein the applicator surface is continuous and flat or concave or convex.
 
7. Method according to any one of the preceding claims, wherein the amount of liquid adhered to the applicator surface is smaller than 1000 pL (picoliter), preferably smaller than 500 pL, preferably smaller than 400 pL, preferably in a range from 10 to 300 pL, and/or wherein the liquid on the applicator surface has a contact angle in a range from 20 to 60 degrees, preferably in a range from 25 to 55 degrees and/or the height of the liquid is in a range from 1 to 30 pm.
 
8. Method according to any one of the preceding claims, comprising the step of determining the distance between applicator surface and the substrate, before the liquid touches the substrate, preferably before adhering an amount of the liquid to the applicator surface.
 
9. Method according to claim 8, wherein the position of the applicator surface and/or of one or more locations on the substrate is measured, the measured position or positions are stored, and the measured position or positions are used for positioning of the applicator surface relative to the substrate.
 
10. Method according to any one of the preceding claims, wherein two or more different samples are applied to the substrate.
 
11. Method according to any one of the preceding claims, wherein at least one reservoir is provided to supply the applicator surface with sample or samples.
 
12. Method according to any one of the preceding claims, wherein, at least prior to and preferably also during and after application of the liquid to the substrate, the temperature of the substrate and/or of the applicator surface is adjusted to a temperature around, at or above the dew point of the gas surrounding the substrate and/or the applicator surface, preferably to within one or more tenths of a degree from the dew point.
 
13. Apparatus for applying liquid, in particular one or more samples, to a substrate comprising
a substrate holder,
an applicator surface for adhering an amount of the liquid to, which surface is preferably defined at the end of an elongated applicator, such as a pin,
one or more actuators for moving the applicator surface and the substrate holder towards and relative to each other, and
a controller configured to drive the actuator(s) to move the applicator surface and the substrate holder towards and relative to each other to form a capillary bridge between the applicator surface and a substrate in the substrate holder and to apply the liquid to the surface.
 
14. Apparatus according to any one of the claim 13, comprising an on-axis measuring system (e.g. comprising a camera and an objective) configured to determine the distance between applicator surface and a substrate in the substrate holder. In particular to measure the position of the applicator surface and/or of one or more locations on the substrate is measured, to store the measured position or positions, and to use the measured position or positions for positioning of the applicator surface relative to the substrate.
 
15. Apparatus according to claim 13 or 14, wherein the substrate holder and the applicator surface are located inside a climate chamber.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description