Qualification process for cryo-electron microscopy samples as well as related sample holder

Abstract

A qualification process for a sample to be examined by means of cryo-electron microscopy. The, sample (12) is applied to a sample carrier (10) provided for cryo-electron microscopy and subsequently the sample (12) arranged on the sample carrier is examined by means of dynamic light scattering. The particle size distribution within the sample (12) is determined by means of the dynamic light scattering. Further, a sample holder designed to carry out the qualification process.

Claims

1. A qualification process for determining a presence of clumping or dissociation of particles selected from macromolecules and macromolecular complexes in a sample (12) to be examined by cryo-electron microscopy, the qualification process comprising: applying the sample (12) on a sample carrier (10) provided for cryo-electron microscopy, scanning the sample (12) on the sample carrier by means of dynamic light scattering, determining a particle size distribution within the sample (12) by means of dynamic light scattering, and carrying out a determination of clumping or dissociation of macromolecules or macromolecular complexes within the sample (12) from the determining of the particle size distribution.

2. The qualification process according to claim 1, wherein the sample contains macromolecular complexes.

3. The qualification process according to claim 1, further comprising examining the sample (12), on the sample carrier used for the qualification, by means of cryo-electron microscopy.

4. The qualification process according to claim 1, wherein the dynamic light scattering uses a laser-beam, wherein the laser-beam follows a laser-beam path (8), and wherein the laser-beam path (8) is guided through the sample (12) without encountering the sample carrier (10) provided for cryo-electron microscopy.

5. The qualification process according to claim 1, further comprising adapting a cryo-sample carrier parameter as part of a sample preparation for cryo-electron microscopy by at least one of an adjustment of a cryo-sample carrier-material, an adjustment of other properties of the sample carrier, and buffering the sample.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) Embodiments of the invention will be described in detail qirg reference to the accompanying illustrations, which are intended to illustrate the invention and are not to be regarded as limiting:

(2) There is shown in:

(3) FIG. 1 a schematic illustration of an exemplary embodiment of the DLS measuring apparatus;

(4) FIG. 2a, b a schematic representation of an exemplary embodiment of the grid holder 11 with the sample 12 which in turn is located on the grid;

(5) FIG. 3a, b a schematic representation of an embodiment of the grid holder 11 with inserted grid;

(6) FIG. 4a, b, c a schematic representation of the processes during grid-induced aggregation of the sample 12 and

(7) FIG. 5a, b a chemical representation of an embodiment of the diffusion times as a function of the diffusion constants and distances.

DETAILED DESCRIPTION OF THE INVENTION

(8) At this point, it should be pointed out that the exemplary embodiment or the embodiments serve for the explanation and are not necessarily to be considered as restrictive.

(9) In FIG. 1 a schematic representation of an embodiment of the DLS measurement apparatus is shown.

(10) FIG. 1 shows the basic DLS measurement setup consisting of the most important optics and electronics components. The confocal adjustment of laser beam 8 and detector 5, which must be ensured for the measuring principle, is implemented in the optical head 6. This measurement volume 9 is positioned within the drop that adheres to the grid.

(11) FIGS. 2a and 2b show a schematic illustration of an exemplary embodiment of the grid holder 11 with the sample 12 which adheres to the grid surface.

(12) FIGS. 3a and 3b are schematic diagrams in which an embodiment of the grid holder 11 with the inserted grid is shown.

(13) In the embodiment presented above (FIGS. 2a, 2b and 3a, 3b), a holder is inserted into a multiwell plate, which holder can accommodate a grid so that the laser beam 8 bypasses the grid, in this case parallel to the grid surface, passing into sample 12 (not shown in FIGS. 3a, b for clarity). The detector 5 looks at the focal point of the laser beam 8 (confocal arrangement, see FIG. 1). The sample 12 is sealed by the oil 14 located in the well and therefore cannot evaporate, so the concentration ratios remain constant. Due to the positioning possibility within the well, in the concrete embodiment implemented by motorized X, Y and Z positioning units, the focal point or the measuring volume 9 can be individually positioned within the sample 12. With a correspondingly precise holder, this is not necessary.

(14) A prepared cryo-EM grid (coating, charge are applied beforehand) is loaded with a few microliters of the sample 12, this is usually done by manual pipetting. The same work steps are carried out as in the further cryo-EM examination. The grid is then inserted into the grid holder 11. And the grid holder 11, in turn, is transferred to the container filled with paraffin oil. Sealed in this way, measurements can be carried out on the sample 12 over longer periods of time (possibly several hours). The subsequent measurement is suitable for determining whether and if so when the contact with the grid surface has an effect on the sample 12. The DLS measurements are usually carried out as a series of individual measurements, which are then summarized as a photon count rate/time diagram or as a radius/time diagram. A control sample is usually prepared in compliance with the identical conditions (paraffin oil sealing, temperature, measurement period, etc.), which, in contrast to sample 12, rests on a polystyrene surface (not shown).

(15) FIGS. 4a, 4b and 4c show a schematic representation of the processes in the case of grid-induced aggregation of the sample 12 and a surface attachment of the macromolecules as a result of electrostatic attractive forces.

(16) In FIGS. 5a and 5b schematic diagrams of an embodiment of the diffusion times as a function of diffusion constants and distances are shown. In FIGS. 5a and 5b dots represent time of first detection of agglomerates. For this, they must travel a distance of 0.5 mm between grid surface and measurement volume 9.

(17) The diffusion constant of aggregates is calculated using the following formula:

(18) $D=\frac{m^s}{s}=\frac{0.00000025m^s}{2500s}\text{∼}10-10\frac{m^s}{s}$

(19) FIG. 5b shows the dependency of diffusion time from distance from grid surface and measurement volume 9. The downward pointing arrow at 100,000 nm shows distance from grid surface to measurement volume 9 are transcended by aggregates by diffusion in 400 ms. The upward pointing arrow shows 10,000 nm distance from grid surface to measurement volume 9 are transcended by aggregates by diffusion in 1 ms.

(20) The measurements on the control sample are usually used as a comparison (control) before loading the grid and after the series of measurements on the grid has been completed. If the contact of the sample 12 with the grid surface has an unfavorable effect on the solubility of the sample 12, this becomes visible after a certain time as a change in the particle size or scattered light intensity (FIGS. 5a, 5b, photon count rate as a function of time). Ideally, a sample 12, both on the grid and in the control, should have unchanged values of the count rate and radius distribution even over a comparatively long period of time. In this case it can be assumed that the sample 12 does not aggregate due to interactions with the grid or that the complexes disintegrate if an identical grid for shock freezing is loaded with this sample 12. Several explanatory models are conceivable, as to why the contact of the sample 12 with the grid surface has effects on the aggregation behavior of the sample. FIGS. 4a and b show an obvious mechanism. The buffer has corrosive properties, which attack the grid material. The metal ions detaching from the metal grid of the grid accumulate in the sample volume. The copper ion concentration reaches a critical value within a certain time; if this is exceeded, the macromolecule rapidly aggregates. The time it takes to reach this critical ion concentration is dependent on both the buffer and the macromolecule. There is also another aggregating effect and this is the plasma charging, which is a central step in the preparation of the grid. Here, the parameters of the charging density can be varied. It has been shown that a certain charge density value on the one hand binds only a few macromolecules to surfaces, but at the same time still offers sufficient hydrophilicity of the grid surface, so that the sample covers the grid sufficiently.

(21) DLS measurements of the droplet volume of sample 12 on the grid allow conclusions to be drawn about the aggregation behavior of the individual molecules directly on the grid surface. At to, aggregation is not yet detected using DLS. The causative agent diffuses in the sample volume, until the critical ion concentration is exceeded. The DLS measurement volume 9 serves here as a representative partial volume in order to draw conclusions about the aggregation behavior of the sample 12 in the entire sample volume. The information can be used as a reliable timer for the subsequent rapid freezing process.

REFERENCE LIST

(22) 1 display device 2 autocorrelator 3 photomultipliers 4 light guides 5 detector 6 optical head 7 scatter light path 8 DLS laser beam 9 detector focus/measurement volume 10 cryo-electron microscopy—carrier (grid) 11 grid-holder [DLS] (for the cryo-EM-Grid with sample) 12 sample 13 container 14 paraffin oil