WO2006048562A1

WO2006048562A1 - Use of fluorescence markers for x-ray microscopy

Info

Publication number: WO2006048562A1
Application number: PCT/FR2005/050767
Authority: WO
Inventors: Jean-Pierre Moy; Jean-François ADAM
Original assignee: Commissariat A L'energie Atomique
Priority date: 2004-11-02
Filing date: 2005-09-20
Publication date: 2006-05-11
Also published as: FR2877445A1; FR2877445B1

Abstract

The invention relates to the use of a mineral nanocrystal containing a heavy element as a marker for X-ray microscopy.

Description

USE OF FLUORESCENCE MARKERS FOR X-RAY MICROSCOPY

TECHNICAL AREA

The present invention relates to novel markers usable in X-ray microscopy.

Such markers are moreover fluorescence markers, that is to say visualizable by fluorescence microscopy.

Therefore, these can be used to mark a sample that can be observed, simultaneously or delayed, by these two complementary methods of microscopy.

ETATDELATECHNIQUEANTÉRIEURE

X-ray microscopy is currently undergoing remarkable development. Indeed, the use in this technology of wavelengths much shorter than those of visible or ultraviolet light (of the order of 100 times), provides access to much improved spatial resolutions. Indeed, the ultimate resolution, limited by diffraction, is of the order of one nanometer. Moreover, tomography offers the possibility of restoring three-dimensional images.

X-ray microscopy is mainly studied in synchrotron radiation centers, because this technology requires sources of very high brightness. However, laboratory equipment is currently under study and should be available in the near future.

A particularly interesting, but not limiting, field for the exploitation of X-ray microscopy is the "window of water". This window corresponds to the soft X-ray energy range between the carbon threshold (284 eV, 4.4 nm) and the oxygen K threshold (543 eV, 2.3 nm). In this band, organic materials, whose carbon (present in proteins, lipids, ...) is the most important element, are 10 to 20 times more absorbent than water. In addition, the penetration of radiation into the water is of the order of 10 μm to 500 eV. Thus, this observation technique is situated between optical microscopy, which allows the observation of living organisms but with a resolution that is fundamentally limited to 0.3 μm, and transmission electron microscopy, of nanometric resolution, but which can only be performed on slices of thickness close to 0.1 microns, prepared according to a very heavy protocol, which alters the structure of the observed organism.

By using X-rays in the "water window", it is now possible to practice high-resolution, full-field transmission microscopy on whole cells in their natural environment. Under these conditions, good intrinsic contrast is obtained between the carbonaceous structures of the organelles, the cytoplasm more loaded with water, and the surrounding environment containing little carbon. Moreover, the preparation of the samples for this microscopy respects the native structures, since the cells remain whole and in an aqueous medium.

Today, micro-scopies are no longer aimed solely at observing morphological characters, but mainly at highlighting biochemical processes using specific markers. Indeed, it has become possible to locate, with the intrinsic resolution of the microscope, phenomena occurring at the molecular level.

This last application has undergone a spectacular development in the field of confocal or classical microscopy, thanks to the fluorescence markers.

Currently, the majority of fluorescent markers are organic molecules, not detectable by X-rays.

More recently, two families of nanocrystals that can be used as markers have appeared in the field of fluorescence microscopy:

• nanocrystals ("Quantum dots" or Qdots in English) used and marketed for some years for specific labeling fluorescence microscopy, or even for macroscopic observation in vivo optical fluorescence imaging. These semiconductor crystals, most often of formula CdS or CdSe, are characterized by: a very broad absorption: different nanocrystals, emitting in the visible / near Infra-Red, can be excited by the same wavelength

; it is therefore easy to separate the excitation from the emission spectrally; a thin emission band, which depends on the size of the nanocrystal: for example, for CdSe particles, the emission ranges from 480 nm (for particles of 2.1 nm in diameter) to 730 nm (4.6 nm of diameter). In general, the larger the nanoparticle, the more the emission shifts to red.

In addition, it is possible to functionalize these nanocrystals so that they can specifically bind to selected targets.

• Moreover, there are many fluorescent compounds, excitable in ultraviolet or visible, oxides type, oxysulfides and sulfides of heavy metals, doped, mostly by rare earths. These have an emission according to a characteristic spectrum of the dopant, often consisting of a small number of lines. In particular, compounds doped with Europium (Eu) trivalent (Gd ₂ O ₂ S: Eu, Y ₂ O ₃ : Eu, Lu ₂ O ₃ : Eu) are well known for their good fluorescence yield in the red, around 620 nm. These compounds are now capable of being obtained in the form of nanometer-sized crystals, which makes it possible to envisage their functionalization to render them selective for a biological target.

The potential of X-ray microscopy can only be fully exploited if identifiable X-ray markers are available. Nowadays, the only reliable marker that can be used in this microscopy consists of gold nanobeads. Apart from the cost of this marker, it is not observable in visible light and can therefore be located only under X-rays. Moreover, the functionalization of these nanobeads remains delicate because of the chemical inertia of the gold. .

However, besides the need for specific markers identifiable for X-ray microscopy, other than markers based on gold beads, there is also the need for markers that can be jointly visualized by lighter microscopies.

Indeed, obtaining X-ray images is relatively long and tedious, especially for three-dimensional images, and only covers a very limited field. It is therefore desirable to be able to identify the regions of interest at low magnification by a rapid technique such as optical microscopy, before starting the acquisition of a three-dimensional image. There are certainly architectures that allow to graft simultaneously on the same frame, an active molecule capable of binding to a target and several detectable molecules (radioisotopes, fluorochromes, or nano-gold beads). While this principle theoretically allows the construction of probe molecules specific to a given target and recognizable in fluorescence as well as in X-rays, the complex synthesis of such molecules, of unique design for each case, remains dissuasive.

SUMMARY OF THE INVENTION

For the first time, the Applicant has demonstrated that fluorescence markers were able to absorb X-rays and therefore used as markers for X-ray microscopy.

In this way, the present invention proposes the first markers of X-ray microscopy, alternative to gold nanobeads, which are furthermore observable by fluorescence microscopy. These markers thus make it possible to combine the efficiency and the simplicity of the localization by fluorescence microscopy with the spatial resolution of the X-ray microscopy.

Fluorescence markers particularly adapted to the invention are mineral nanocrystals containing a heavy element.

Advantageously, the heavy element corresponds to an element of high atomic number but less than or equal to 49. It is thus preferred nanocrystals containing Cadmium, for example of formula CdS or CdSe, or of Yttrium, Gadolinium or Lutecium , doped or not with Europium or Terbium.

The functionalization of such a marker allows its addressing to a specific target on a sample. The functionalization of the nanocrystals is known to those skilled in the art.

Advantageously, the marker according to the invention has a dimension less than 20 nm, compatible with the thickness of the nanocrystals and the absorption of X-rays.

According to a first aspect of the invention, a sample labeled with a marker according to the invention can be observed only by means of X-ray microscopy. Thus, the present invention relates to an observation method. of a sample, comprising the following steps: labeling with a fluorescence marker, capable of absorbing X-rays, of the sample to be observed; observation of the sample using the X-ray microscope

A sample is any object whose observation at a high magnification is of interest. More particularly, in the context of the invention, it is a biological sample, for example cells.

According to a second aspect of the invention, the same labeled sample is observed, simultaneously or delayed, using a fluorescence microscope.

Preferably, the method according to the invention is implemented as follows: in a first step, the fluorescence micro-scopie makes it possible to locate the area of interest in the sample; - Then, the X-ray microscopy allows to visualize with a high resolution this area of interest.

By fluorescence microscopy is meant both transmission and reflection microscopy. Reflective fluorescence microscopy includes both confocal and epifluorescence microscopy. These various microscopic techniques are well known to those skilled in the art.

With the help of the data thus acquired, it is possible to restore the image of the sample in 2 or 3 dimensions (2D or 3D).

The combination of the two microscopies also makes it possible to superimpose or combine the images obtained using these two complementary technologies.

It appears that according to this last embodiment, a new device is necessary. Thus, the invention also relates to a sample observation device, equipped with a fluorescence microscope, an X-ray microscope, and a location for the sample located in the field of observation of the samples. two microscopes. This device makes it possible to observe the same sample labeled with a single marker, simultaneously or successively, without manipulation, using two distinct and complementary microscopic techniques. EXAMPLE OF REALIZATION

The invention and the advantages which result therefrom will emerge more clearly from the following example of embodiment, in support of the appended figure. This is however in no way limiting.

FIG. 1 schematically illustrates a device allowing the observation of a sample in transmission and in epifluorescence, simultaneous with the observation in X-rays.

FIG. 2 schematically illustrates a device allowing the observation of a sample by confocal fluorescence microscopy, simultaneous with the observation in X-rays.

The table below reports the ability of various 10 nm thick heavy metal nanocrystals to absorb X-rays at different energy levels (300, 400 or 500 eV):

This table reveals that nanocrystals with a thickness of 10 nm have a lower absorption than gold, but nevertheless significant and sufficient to provide a detectable contrast in X-ray images. Thus, some nanocrystals in a 30x30x30 nm voxel ³ have an absorption of the order of 5%, unambiguously visible in images with 1,000 to 2,000 photons per pixel.

In the soft X-ray domain, we find that the absorption is not directly proportional to the atomic number, because it varies by jumps in the vicinity of the transitions L, M and N of the elements. This explains why Y (atomic number 39) is more absorbent than Gd (atomic number 64). Moreover, nanocrystals containing cadmium are particularly well suited to microscopy in the "window of water" in the vicinity of the threshold K of oxygen, for example at 500 eV, because cadmium has its thresholds M in this region . More generally, elements with the highest atomic number, but less than or equal to 49, are particularly favorable. This is also the case of Yttrium.

According to the invention, the observation of a sample of interest can therefore be carried out as follows:

A) Marking of the sample with the aid of a marker as defined in the invention

It is possible to use existing specific markers, for example functionalized nanocrystals of CdSe or Lu ₂ O ₃ : Eu, by choosing those with the highest X-ray absorption.

B) Installation of a device according to the invention

The diagrams shown in FIGS. 1 and 2 illustrate examples of possible arrangements, based on the dimensions of an existing objective (MITUTOYO LWD 5Ox 0.55).

The lighting system for optical microscopy is identical to that of a conventional microscope:

1) in transmission:

The sample (7), placed on its support (2), is observed in transmission, thanks to the light (3) introduced by the rear face of the sample (Figure 1). 2) in epifluorescence:

With a semi-transparent or dichroic plate (4), light at the excitation wavelength of the marker (visible or near UV; 3bis) is introduced through the objective (5) (FIG. 1).

3) confocal: According to the confocal configuration, we illuminate (3) by the traditional "dichroic cube" and a semitransparent plate (4), and we detect by a detector (9) the light received through a small conjugated hole from the source (10) (Figure 2). Transmission lighting is not necessary.

The simultaneity of the observations is made possible by the great difference of openness of the objectives:

Fresnel ("Flat Zone") lenses (6) used in X-rays have an aperture that does not exceed F / 10 or at best F / 8, ie a numerical aperture of 0.06; The 2Ox or 5Ox (5) long distance and magnification objectives have numerical apertures of 0.4 to 0.55.

It is therefore possible to illuminate and observe by 45 ° mirrors (7) drilled to let the X-ray radiation (8) by losing very little light, and keeping the resolution of the microscope objectives. Figures 1 and 2 show that a provision compatible with the actual objective dimensions can be found.

C) Observation of the sample by fluorescence and X-ray microscopy

The optimal observation using the device shown in the figures therefore comprises three steps:

- locating, with an average magnification 200 to 50Ox, areas of interest, first in normal lighting by transmission (Figure 1);

- The finer localization, by an epifluorescence observation (Figure 1), or better in confocal fluorescence (Figure 2), regions where the marking was made under the best conditions;

an X-ray observation, either in simple transmission or in tomography with rotation of the sample, showing the markers by their absorption.

The images obtained by the optical means and the 2D or 3D images provided by the X-ray microscope can advantageously be combined so as to make X-ray resolved parts better match their environment, which may be more contrasted in visible light. .

With this device, it is also possible to record, through the microscope objective, the fluorescence image of the markers excited by X-rays, if care has been taken to remove any visible light from the X-ray source, for example, thanks to a thin sheet of beryllium placed in front of the X-ray condenser. This image would obviously have a resolution limited by optics, ie of the order of 1 μm, but could provide additional information compared to the image obtained by X-rays.

Claims

1. Use of a mineral nanocrystal containing a heavy element as a marker for micro-scopie X-rays.

2. Use of a mineral nanocrystal containing a heavy element according to the claim

1, characterized in that the nanocrystal contains an element of high atomic number, but less than or equal to 49.

3. Use of a mineral nanocrystal containing a heavy element according to the claim

2, characterized in that the nanocrystal contains cadmium.

4. Use of a mineral nanocrystal containing a heavy element according to claim

3, characterized in that the chemical formula of the nanocrystal is CdS or CdSe.

5. Use of a mineral nanocrystal containing a heavy element according to claim 2, characterized in that the nanocrystal contains yttrium, or Gadolinium, or Lutecium.

6. Use of a mineral nanocrystal containing a heavy element according to claim 5, characterized in that the chemical formula of the nanocrystal is chosen from the group comprising: Y ₂ O ₃ , Y ₂ O ₃ : Eu, Y ₂ O ₃ : Tb, Gd ₂ O ₃ , Gd ₂ O ₃ : Eu, Gd ₂ O ₃ : Tb, Lu ₂ O ₃ , Lu ₂ O ₃ : Eu, Lu ₂ O ₃ : Tb.

7. Use of a mineral nanocrystal containing a heavy element according to one of claims 1 to 6, characterized in that the nanocrystal is functionalized.

8. Use of a mineral nanocrystal containing a heavy element according to one of claims 1 to 7, characterized in that the nanocrystal has a size less than 20 nm.

9. A method of observing a sample, comprising the following steps: labeling with a mineral nanocrystal containing a heavy element of the sample to be observed; - observation of the sample using an X-ray microscope

10. A method of observing a sample according to claim 9, characterized in that the labeled sample is observed simultaneously or deferred using a fluorescence microscope.

11. A method of observing a sample according to claim 10, characterized in that: first, it implements fluorescence microscopy, so as to allow the location of the area or areas of interest to within the sample; then, to implement X-ray microscopy, so as to allow viewing with high resolution of this or these areas of interest.

12. A method of observing a sample according to one of claims 10 and 11, characterized in that in a subsequent step, the image of the sample is restored in 2D or 3D.

13. A method of observing a sample according to one of claims 10 to 12, characterized in that in a subsequent step, the images obtained by fluorescence microscopy and X-ray microscopy, respectively, are superimposed and combined.