WO2007100293A1

WO2007100293A1 - Method of applying alignment markers for cryo-microscopy

Info

Publication number: WO2007100293A1
Application number: PCT/SE2007/000191
Authority: WO
Inventors: Sergej Masich; Bertil Daneholm
Original assignee: Sidec Technologies Ab
Priority date: 2006-03-01
Filing date: 2007-03-01
Publication date: 2007-09-07

Abstract

The present invention relates to a method for applying markers to a cryo sample used in an electron microscope, comprising the steps of using nano markers dissolved in toluene, diluting these in isopentane to a total concentration of 0.1 to 0.2 mg/ml.

Description

Method of applying alignment markers for cryo-microscopy

TECHNICAL FIELD

The present invention relates to a method for image alignment and in particular a method of applying nano sized markers to a cryo sample.

BACKGROUND OF THE INVENTION

Cryo-sections of vitreous biological samples are successfully used in several laboratories to study the ultrastructure of cellular organelles and their molecular components in a native hydrated state. However, three-dimensional imaging of these structures using electron tomography (ET) still remains problematic.

Alignment of the images is one of the most crucial steps in ET. Two methods are generally used for the alignment of tilt series: cross-correlation of the collected images and alignment of images using fiducial markers applied to the specimen. When the methods have been compared in studies with stained plastic sections, it has been shown that the cross-correlation gives higher error than alignment with aid of fiducial markers. The low image contrast due to the low electron dose, required for vitreous specimens, makes the cross-correlation method even less reliable. Thus, the use of fiducial markers constitutes the first-hand alternative for high-resolution tomographic reconstructions of vitreous sections.

Colloidal gold particles of 5 to 20 nm in diameter are conventionally used as fiducial markers to align images of plastic sections for ET. Gold particles suspended in water cannot be applied directly as markers onto vitreous specimens, which have to be kept at temperatures below -135°C. To overcome this problem, gold particles have been applied on a support film attached to the grid prior to the shift to the low temperatures. However, only a minor fraction of the sections could be aligned and reconstructed by using this approach since usually the section and the support film were several micrometers apart from each other. SUMMARY OF THE INVENTION

The present invention resolves some of these problems realized in a number of aspects, wherein a first aspect, a method of applying alignment markers to a cryo sample for use in a microscope is provided, comprising the steps of: diluting nano sized markers with a liquid diluting hydrocarbon, liquid at cryo temperatures; applying the diluted nano sized markers to the cryo sample kept at cryo temperatures;

The method may further comprise the steps of filling a staining chamber with the diluted nano sized markers; dipping the cryo sample in the staining chamber; rinsing the cryo sample in a rinsing hydrocarbon, liquid at cryo temperatures and that has a sufficient evaporation at cryo temperatures; and blotting the cryo sample.

The diluted nano sized markers may have a concentration of 0.01 to 1.0 mg/ml in the liquid diluting hydrocarbon.

The diluted nano sized markers may even have a concentration range of 0.1 to 0.2 mg/ml in the liquid diluting hydrocarbon.

The liquid rinsing hydrocarbon may be one of ethane, methane, and propane.

The nano sized markers may be pre dissolved in an organic solvent liquid at room temperature, such as toluene, acetone or hexane before diluting the markers.

The diluting hydrocarbon may be one of isopentane, propane, or butane.

The nano sized electron dense marker may be a quantum dot from the list of PbS based, PbSe based, CdTe/CdS based, CdSe/ZnS based, and CdSe based.

With cryo temperature is meant a temperature below -100° C.

The alignment markers are electron dense. The method may further comprise the step of using the cryo sample with applied nano sized markers in an electron tomography measurement

Another aspect of the present invention concerns the use of quantum dots as section markers for making high resolution reconstruction, such as high definition images, wherein the quantum dots are attached directly to the section, and more specifically the use of section attached quantum dots wherein the quantum dots are fiducial markers used in a cryo-electron tomography system.

Another aspect of the present invention, a use of electron dense nano sized particles diluted in a diluting hydrocarbon liquid at cryo temperatures as alignment markers in an electron tomography measurement on a cryo sample in an electron microscope is provided.

Yet another aspect of the present invention, a method of applying nano sized alignment markers to a cryo sample for use in an electron microscope in an electron tomography measurement is provided, comprising the steps of: diluting nano sized electron dense markers, pre dissolved in an organic liquid, such as toluene, with a second organic liquid, such as isopentane, to a final concentration of 0.1 to 0.2 mg/ml; applying the diluted nano sized markers to the cryo sample kept at cryo temperatures; and removing surplus organic liquid by rinsing in a third organic liquid, such as liquid ethane.

The method may further comprise applying nano sized alignment markers to a cryo sample for use in an electron microscope in an electron tomography measurement wherein the second organic liquid is isopentane and the third organic liquid is different from the second organic liquid. The third organic liquid can be liquid ethane.

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in a non-limiting way and in more detail with reference to exemplary embodiments illustrated in the enclosed drawings, in which:

Fig. 1A shows a workstation for quantum dot application inside the cryo-chamber of an ultramicrotome;

Fig. 1B shows an electron micrograph of PbS Core EviDots Espresso quantum dots on a carbon support film;

Fig. 1 C shows an electron micrograph of a cryo-section of human skin with attached quantum dot markers;

Fig. 2 showing plots of error in the alignment of quantum dot markers versus tilt angle for a number of different configurations;

Fig. 3 shows cryo-transmission electron micrograph of vitreous section of native human skin;

Fig. 4 shows reconstruction of a mitochondrion from a vitreous skin section;

Fig. 5 shows reconstruction of a tonofilament bundle from a vitreous skin section; and

Fig. 6 shows reconstruction of a multilamellar organelle from a vitreous skin section.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Here described is an approach for accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, which form liquid suspensions in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. These semiconductor crystals are well defined and electron dense, which justify their use as tags for antibodies in immunoelectron microscopy. Quantum dots are routinely used in life sciences in their water-soluble modification, but they are initially manufactured in organic solvents. These solvents can be substituted with other organic solvents to make suspensions of quantum dots possible to apply onto sections at cryo-temperatures. Data collection can be performed from different regions of the vitreous sections, even if these are not flat. Our experiments showed that quantum dots attach to the section surface and can serve as reliable fiducial markers for ET. We present high-resolution tomograms of some organelles in cryo-sections from human skin cells using the method. The average error in the image alignment did not exceed 12 A and the resolution was estimated to be better than 5 nm. Thus, the use of section attached quantum dots as fiducial markers in cryo- electron tomography provides a novel technique for high-resolution 3D imaging of organelles and macromolecular complexes in their native hydrated state.

Electron microscopy of quantum dots and application of the dots onto vitreous cryo- sections

Earlier colloidal gold fiducial markers were used, which cannot be applied to cryo-sections at very low temperatures. The present invention relates to quantum dots (QDs). The QDs may differ in size and shape, but commercially available QDs are usually standardized as to these parameters. The size of approximately 10 nm should be chosen, since small particles are known to be difficult to detect in images acquired at high tilt angles while large particle sizes usually result in higher alignment errors. The quantum dots selected for the experiments are shown in Figure 1B on a carbon support film. The particles are spherical, although their diameter varies from 10 to 17 nm. The QDs were delivered dissolved in toluene at a concentration of 5 mg/ml. The commercial stock solution of the quantum dots was diluted with isopentane. The solvent solidifies at as low temperature as -160⁰C, thus providing the opportunity to work comfortably from room temperature to cryo-temperatures. On the other hand, isopentane does not evaporate effectively at the low temperatures. Therefore, after deposition of the QDs onto the section, the sections were rinsed in liquid ethane, which facilitated removal of the surplus liquid. The QDs were usually evenly distributed over the section, although occasionally some clusters of the quantum dots (Fig. 1C, 3) were observed. The grids were immersed in the isopentane solution and the grid with the cryo-sections had no support film. This resulted in deposition of QDs on both surfaces of the section, which is shown in inset in Figure 3C. The quantum dots contain heavy metal atoms (lead in the experiments), which provide good visibility in the electron microscope (Fig. 1C, 3). At high tilt angles, when the effective thickness of the specimen is increased and the contrast reduced, it may be difficult to identify the markers at low electron dose and therefore irradiation and underfocus was adopted accordingly.

Alignment of images for tomography using QDs as fiducial markers

One of the important features of the approach is the absence of support film. It has been confirmed in earlier observations that there is usually a gap between the section and the "support" film. In such cases, interference of the markers on the film and the section would make it difficult or even impossible to track images during data collection in the microscope.

The choice of grid is also very important. The grid must allow microscopy over a large area at high tilts, but still have closely spaced bars to ensure that the section is stable. 600 mesh thin bar grids were found to be convenient for the tomographic data collection.

Figure 2A shows alignment errors for a tilt series with 1 μm underfocus and a dose of approximately 30 electrons / nm² per image. The average error amounted to 2.1 pixels (12 A), but it was apparent that some images had been poorly aligned, especially at high tilts (two spikes in Fig. 2A). Visual analysis confirmed that markers were difficult to distinguish on the corresponding tilts. After increase in the dose to approximately 60 electrons / nm2 per image and underfocus to 4 μm, alignment was considerably improved. Two examples of these series are presented in Figures 2B and C. The average error in these series was reduced to less than 1.5 pixels (about 8 A) and the aberrant spikes were diminished. Still, an increase of the alignment error was observed at high tilt angles. This effect can be seen for gold markers on plastic sections as well (Fig. 2D) and can be explained by deterioration of the contrast.

Reconstruction of human skin cell organelles from vitreous cryo-section

Epidermis represents a suitable model system for cryo-transmission electron tomography of vitreous tissue sections as it can be directly vitrified in its native, fully hydrated state without the use of cryo-protectants or any other pre-treatment. To estimate the resolution that can be obtained by our approach, we reconstructed several organelles from cells of the human skin.

Figure 3 shows cryo-transmission electron micrograph of a 120 nm thick vitreous section of the human skin, representing the cytoplasm of a keratinocyte situated in the upper part of the viable epidermis (stratum granulosum). The image shown was acquired subsequent to the collection of the tomographic low dose data. Compression of the section in the cutting direction (open white arrow) is on the order of 50%, and is most easily recognized from the oval shape of mitochondria (large black box). Large amounts of tonofilament bundles (TF), composed of keratin intermediate filaments in a parallel arrangement, are present. Individual 8-10 nm thick keratin intermediate filaments are clearly visible (small black box). Five QDs deposited on the vitreous section are pointed out with black arrows. A tilt series from -60 to +56 degrees with two-degree increments was collected. All five markers were used in the alignment and the average alignment error was less than 1.3 pixels or about 7 A. Areas reconstructed in 3D are indicated by rectangles in the Figure 3.

Three different sections through the reconstructed mitochondrion are presented in Figure 4. Earlier reconstructions of plastic embedded neuronal mitochondria have revealed the outer and inner mitochondrial membranes to be approximately 7 nm thick and separated with approximately 8 nm, thus, providing 15 nm as the centre-to-centre distance between the membranes. We found this parameter to vary in our reconstruction from 70 A to 145 A. An obvious correlation between the thickness and membrane orientation was observed. The thinnest areas were oriented perpendicular to the sectioning direction thus reflecting well-documented compression of the cryo-sections. However, since the two layers are clearly resolved in both directions, we claim the resolution in XY-plane to be significantly better than 7 nm (centre-to-centre distance between the two layers in the thinnest area).

The second reconstruction from the same section includes a bundle of keratin tonofilaments (Fig. 5). The original image and the projection of the reconstructed volume are in good agreement (Fig. 5C). Figure 5D show six clearly resolved individual tonofilaments (green), which have been demarcated from the rest of the bundle. As the diameter of intermediate keratin filaments is known to be approximately 8 nm with a centre-to-centre distance in a bundle of about 15 nm, the resolution must be within a few nanometers needed to allow the filaments to be discerned. Figure 6 show a reconstruction of a cytoplasmic organelle with a multilamellar content, most probably representing a lamellar body or a melanosome, which is seen within the black box in Figure 1C. In this case, a tomographic tilt series was collected with one- degree increments from -50 to +60 degrees, and the average error in the alignment of 28 fiducial markers was 2.1 pixels (12 A). The orientation of the lamellae with respect to the cutting direction is approximately 30 degrees, reducing, but not eliminating, the effect of compression on the periodicity. The lamellae are well resolved through the whole depth of the reconstruction with a centre-to-centre distance of approximately 8 nm.

Electron microscopy of vitreous cryo-sections has revealed fine structure of cell organelles at a remarkably higher resolution than classical methods. Electron tomography of such sections is highly desirable and several successful attempts have been reported. The main obstacle for routine usage of the method is to achieve good image alignment. When fiducial markers are positioned on the support film, the gap between the section and the film causes a number of problems in data collection as well as in alignment. We suggest a simple method to place fiducial markers directly onto vitreous cryo-sections, which circumvent the limitations. It is worth mentioning that the method does not require any costly equipment and can be easily applied in any laboratory.

One of the known drawbacks with fiducial markers on the support film is displacement of the section relative to the film due to a charging effect. This effect becomes irrelevant when the markers are applied directly to the section. The charging may cause changes in the geometry of the section, but these changes are, probably, negligible at the resolution considered in tomographic reconstruction of cryo-sections.

The absence of support film is advantageous from another point of view, as the specimens on bare grids exhibit higher contrast and resolution than those on the grids with a support film. On the other hand, it could be argued that the lack of film could be unfavourable due to charging of the section during electron microscopy. However, in the experiments performed, no signs of image deterioration was observed except for the areas at the edges of the sections that were flickering or bending in the electron beam. During the course of method development, some "know-how" experience that simplifies the work has been acquired. One is a special design of a workstation for application of the quantum dots. The slot for the quantum dot suspension has a funnel-like widening. It prevents spreading of isopentane over the surface and simplifies insertion of the grid into the solution. This is important when working in limited space inside the ultramicrotome cryo-chamber.

In the experiments PbS quantum dots were used. However, any type of quantum dots or nano sized particles dissolved in an organic solvent should be feasible to use.

The alignment error for the tissue cryo-sections with QDs is considerably higher than for stained plastic sections with gold markers (usually below 0.5 pixels). This can be explained by the difference in the imaging conditions since plastic sections sustain much higher electron doses providing better contrast. Moreover, it should be kept in mind that the cryo-sections are usually compressed in the sectioning direction. This causes a remarkable increase of the section thickness (up to two-fold), reducing the image contrast especially at high tilts.

It has been found difficult to trace some markers at low dose conditions at high tilts. Detailed analysis led to the conclusion that these markers contributed considerably to the average alignment error (Fig. 2A). To further confirm that the error was indeed caused by uncertainty in the determination of the marker coordinates, the dose was increased from 30 to 60 electrons / nm² per image and the underfocus to 4 μm. If the error had been caused by instability of the quantum dots on the section in the electron beam, this would have resulted in a higher error. Since the error decreased (Fig. 2B, 2C), it was concluded that the quantum dots remain firmly attached to the vitreous sections.

The reconstructions allowed to conclude that the precision in image alignment by application of quantum dots is not a limiting factor to obtain high-resolution reconstruction of cell organelles in vitreous cryo-sections. Resolution of 5 to 7 nm can be easily achieved without any precautions. Limitations in alignment are not caused by the nature of quantum dots as markers per se, but rather by the specificity of the imaging condition, i.e. combination of the low dose and extraordinary thickness of the vitreous cryo-sections.

Preparation of high pressure frozen skin samples Skin biopsies (area, 1x1 mm2; thickness, 100-150 μm) were collected from the left forearm of a male Caucasian. To avoid dehydration, the biopsies were instantly placed in 1-hexadecane (MERCK). Subsequently, the samples were placed in the cavity between two aluminium cylindrical platelets and the cavity space was filled with 1-hexadecane to prevent air admittance. Finally, the samples were vitrified in a high-pressure freezer HPM 010 (Baltec, Balzers, Liechtenstein), at a pressure of 2300 bar. The high-pressure freezing procedure was essentially performed as can be understood by the person skilled in the art.

Cryo-sectioning

The vitrified sample was mounted with cryo-glue (ethanol/isopropanol, 3:1) directly in the holder of an Ultracut UCT ultramicrotome (Leica, Germany). The sample was subsequently trimmed with a trimming knife (Diatome, cryotrim 90°, Switzerland) and sectioned with a diamond knife (Diatome, cryo immuno 3.0 mm 35°, Switzerland). Mounting of the sample and sectioning was performed at -145⁰C, and the nominal thickness set at 50 nm. Cryo-electron microscopy of vitreous specimens is more thoroughly described in 3,12. The cryo-sections were transferred to 600 mesh thin bar grids (Agar scientific #G2655C, England) with an eyelash glued to a wooden stick. Pressing of the sections was performed mechanically with a stamping tool (Leica, Germany). The grids were stored in liquid nitrogen until quantum dots were applied.

Application of quantum dots onto cryo-sections

PbS Core EviDots Espresso quantum dots (QDs) (Evident Technologies, USA) dissolved in toluene at a concentration of 5 mg/ml were used. Before application, the stock solution was diluted with isopentane (2-Methylbutane, Fluka, cat. no 59075) to a final concentration of 2 - 4 % (i.e. 0.1 to 0.2 mg/ml markers in the isopentane diluted solution); however, other concentrations are possible within a range of 0.01 to 1.0 mg/ml depending on size and type of markers used. To apply the QDs to a grid, a specially designed aluminium workstation (Fig. 1A) was used (as described earlier). All the subsequent procedures were performed inside the ultramicrotome cryo-chamber at a temperature of - 155°C. One of the chambers of the station was filled with the quantum dot suspension and the other one with the liquid ethane. Grids with sections were first transferred with pre-cooled anti-capillary tweezers into the suspension of QDs for a few seconds and then rinsed briefly in liquid ethane. The grids were blotted with a filter paper and moved back into the storage container. 5

Data collection and electron tomography

A single axis GATAN cryo-holder (model 626) at -180⁰C was used. Tilt series were collected at 200 kV in a FEG CM200 FEI microscope equipped with a cooled slow scan

10 2048x2048 TemCam-F214 CCD camera (pixel size 14 μm) and a software for automated data collection (TVIPS₁ Gauting, Germany). The images were recorded with the CCD camera in either 1 or 4 μm underfocus at the dose of approximately 30 or 60 electrons per nm2 per image and the total magnification 1.66x15000. Tilt series were collected with two- degree increments in the range -60 to +60 degrees. In cases when sections were not flat,

15 two to four images had to be excluded from the reconstructions at high tilts since the region of interest was not visible. The images were displayed in the XPIX program, and fiducial marker positions determined in a semi-automated way. A least square procedure was used to align the images. The alignment procedure employed "natural" parameters that may give higher errors than "formal" matrices since fewer parameters are used. In

20 cases of large errors, the marker positions were additionally checked in the XPIX program and tuned by changing the mask and search area sizes. The reconstructions were computed using weighted back projection and analysed in either the BOB or XTV programs.

25 The present invention will be further explained with references to the accompanying figures. Figure 1. Application of quantum dots on cryo-sections, figure 1A - Workstation for quantum dot application inside the cryo-chamber of an ultramicrotome. Three hollows can be seen: A slot 1 for the quantum dot suspension which has a funnel-like widening so that the EM grid can be easily immersed into the suspension and extracted with fine tip

30 tweezers. A reservoir 2 for the liquid ethane. Liquid ethane was prepared in a separate device by cooling gas jet in a metal container submerged in liquid nitrogen. The liquid ethane was transferred into the reservoir 2 with a 1 ml Eppendorf pipette. A Storage container 3 for grids. A piece of filter paper for ethane blotting was placed in a grid holder beside the workstation. In figure 1B an electron micrograph of PbS Core EviDots

35 Espresso quantum dots on a carbon support film at high concentration is shown. Figure 1 C shows an electron micrograph of a cryo-section of human skin with attached quantum dot markers. The markers are evenly distributed over the section; some are pointed out with black arrows. QDc denotes a cluster of quantum dots. Open white arrow indicates the direction of the sectioning. Inset: side view of the tomographic reconstruction of the area included in the square in C; quantum dots are attached to both surfaces of the section. Scale bars: 10 mm (A); 50 nm (B and inset in C); and 200 nm (C).

In figure 2 plots showing error in the alignment of quantum dot markers versus tilt angle are shown. Figure 2A represents a tilt series collected at 32 electrons / nm2 per image in 1 μm underfocus. Six fiducial markers give the average error of 1.7 pixels (10 A). In figure 2B-2C the dose was increased to 60 electrons / nm2 per image and underfocus to 4 μm. Number of quantum dots in the alignment was 12 (in figure 2B) and 5 (in figure 2C). The average errors were 1.3 pixels (7 A) in both cases. Figure 2D shows an example of the alignment in a plastic section using gold markers. 22 gold markers attached to a plastic section were used. Note that an increase in the error at high tilts is also observed.

Figure 3. Cryo-transmission electron micrograph of vitreous section of native human skin, representing the cytoplasm of a keratinocyte located in the upper part of the viable epidermis (stratum granulosum). Large amounts of tonofilament bundles (TF), composed of keratin intermediate filaments in a parallel arrangement, are present. Individual 8-10 nm thick keratin intermediate filaments are clearly visible (small black box). Compression of the section in the cutting direction (open white arrow) is on the order of 50%, and is recognized from the oval shape of mitochondrion compressed space between the two plasma membranes of the mitochondrion seen in the direction of sectioning (large black box). Arrows indicate five quantum dots, deposited on the vitreous section and used for alignment of the tilt series. The section thickness: 120 nm. Scale bar: 200 nm.

Figure 4. Reconstruction of a mitochondrion from a vitreous skin section (cf. Figure 3, large black box). Three non-overlapping sections through the reconstructed volume are shown. The outer and inner membranes are well resolved. Scale bar: 50 nm.

Figure 5. Reconstruction of a tonofilament bundle from a vitreous tissue section (cf. Figure 3, small black box). Figure 5A - An extract from the image corresponding to the reconstructed area. Individual 8-10 nm thick keratin intermediate filaments are visible. Figure 5B shows surface rendered presentation of the reconstructed volume. Figure 5C shows comparison of the reconstruction with original data. A set of six filaments (marked in green) is singled out. Figure 5D'- 5D" shows a stereo-view of a cross-section through the reconstructed volume. The six resolved filaments are coloured in green. Scale bars: 20 nm.

Figure 6. Reconstruction of a multilamellar organelle from a vitreous skin section (cf. Figure 1C, black box). Two non-overlapping sections through the reconstructed volume are shown. Scale bar: 30 nm.

Alternative embodiments

The nano sized markers are pre dissolved in a toluene solution by the manufacturer, however it should be understood that they may be dissolved in other solutions (e.g. hexane and acetone) which may operate together with the dilution substance or independently at cryo temperatures. The same is true for the dilution substance, in the above examples isopentane, that it may be substituted for another substance that is liquid at the cryo temperatures of interest in this application for instance propane or butane. The rinsing in liquefied ethane may also be conducted in any other suitable hydrocarbon substance that is liquid at cryo temperatures, for instance propane.

The term "dissolved", used with respect to the nano sized markers and a solution e.g. "pre dissolved in a toluene solution", is considered to be analogous with the nano sized markers being suspended in a solution due to the inherent insolubility of the nano sized markers in these solutions.

PbS based quantum dots has been used as an example; however it should be understood that other materials may be used; for instance, but not limited to: PbSe based, CdTe/CdS based, CdSe/ZnS based, and CdSe based. For an application in an electron microscope they should be electron dense, i.e. give a high contrast image using the electron microscope. With nano sized alignment markers are meant particles with a mean diameter in the nano meter scale that are electron dense, i.e. absorb or scatter electrons.

It should be noted that the word "comprising" does not exclude the presence of other elements or steps than those listed and the words "a" or "an" preceding an element do not exclude the presence of a plurality of such elements. It should further be noted that any reference signs do not limit the scope of the claims, and that several "means", "devices", and "units" may be represented by the same item of hardware.

The above mentioned and described embodiments are only given as examples and should not be limiting to the present invention. Other solutions, uses, objectives, and functions within the scope of the invention as claimed in the below described patent claims should be apparent for the person skilled in the art.

Claims

1. A method of applying alignment markers to a cryo sample for use in a microscope characterized in that the method comprise the steps of:

- diluting nano sized markers with a liquid diluting hydrocarbon, liquid at cryo temperatures;

- applying said diluted nano sized markers to said cryo sample kept at cryo temperatures;

2. The method according to claim 1 characterized in that the method further comprise the steps of

- filling a staining chamber with said diluted nano sized markers;

- dipping said cryo sample in said staining chamber;

- rinsing said cryo sample in a rinsing hydrocarbon, liquid at cryo temperatures and that has a sufficient evaporation at cryo temperatures; and

- blotting said cryo sample.

3. The method according to claim 1 characterized in that said diluted nano sized markers have a concentration of 0.01 to 1.0 mg/ml in said liquid diluting hydrocarbon.

4. The method according to claim 1 characterized in that said diluted nano sized markers have a concentration of 0.1 to 0.2 mg/ml in said liquid diluting hydrocarbon.

5. The method according to claim 2 characterized in that said liquid rinsing hydrocarbon is one of ethane, methane, butane, and propane.

6. The method according to claim 1 characterized in that said nano sized markers are pre dissolved in an organic solvent liquid at room temperature, such as toluene, acetone or hexane before diluting said markers.

7. The method according to claim 1 characterized in that said diluting hydrocarbon is one of isopentane, propane or butane.

8. The method according to claim 1 characterized in that said nano sized electron dense marker is a quantum dot from the list of PbS based, PbSe based, CdTe/CdS based, CdSe/ZnS based, and CdSe based.

9. The method according to claim 1 characterized in that cryo temperature is defined as below -100° C.

10. The method according to claim 1 characterized in that said alignment markers are electron dense.

11. The method according to claim 10 characterized in that the method further comprising the step of using said cryo sample with applied nano sized markers in an electron tomography measurement

12. Use of quantum dots as section markers for making high resolution reconstruction such as high definition images characterized in that the quantum dots are attached directly to the section.

13. Use of section attached quantum dots according to claim 12 characterized in that the quantum dots are fiducial markers used in a cryo-electron tomography system.

14. Use of electron dense nano sized particles diluted in a diluting alkane liquid at cryo temperatures as alignment markers in an electron tomography measurement on a cryo sample in an electron microscope.

15. A method of applying nano sized alignment markers to a cryo sample for use in an electron microscope in an electron tomography measurement, comprising the steps of:

- diluting nano sized electron dense markers, pre dissolved in an organic liquid, such as toluene, with a second organic liquid, such as isopentane, to a final concentration of 0.1 to 0.2 mg/ml;

- applying said diluted nano sized markers to said cryo sample kept at cryo temperatures; and removing surplus organic liquid by rinsing in a third organic liquid, such as liquid ethane.

16. A method of applying nano sized alignment markers to a cryo sample for use in an electron microscope in an electron tomography measurement according to claim 15 characterized in that the second organic liquid is isopentane and the third organic liquid is different from the second organic liquid.