WO2010040918A1 - Device for three-dimensional display in microscopy and method using such a device - Google Patents

Abstract

The invention relates to a device for three-dimensional display in microscopy, said device comprising a substrate (2) having a substantially planar surface (4) in which furrows (6) are formed, the surface of the furrows (6) being reflective so as to form a mirror, and wherein an object to be displayed is placed in a furrow (6).

Description

 Device for three-dimensional visualization in microscopy and method using such a device

The present invention relates to a device for three-dimensional visualization in microscopy and a visualization method using such a device.

The field of the present invention is broad field fluorescence microscopy for obtaining three-dimensional information about the object, or cell, observed.

In cell biology, to achieve a three-dimensional image, it is known to use confocal fluorescence microscopy. This technique makes it possible to visualize a sample according to slices of controlled thicknesses (of the order of a micrometer) with a resolution generally of the order of 0.5 μm. It is used for three-dimensional imaging based on cutting the sample into regularly spaced slices. It is then necessary to realize the necessary number of shots. Each time, the observed object is exposed with a laser beam.

A first disadvantage of this technique is that it takes several shots to obtain a three-dimensional view, which limits the acquisition rate. When it is desired to increase the resolution, it is necessary to increase the number of shots and thus to further limit the acquisition rate.

In the case of observation of cells, they are each time subjected to laser radiation which can damage them. In addition, in the field of fluorescence microscopy, taking into account multiple shots, a large dose of illumination is imposed and is associated with wear of the fluorophores, which is known as the photobleaching phenomenon.

Finally, the material used to make such three-dimensional images is an expensive material. Calibration is also required when shooting, which makes the process equally expensive.

Other techniques are also known to obtain a three-dimensional visualization in microscopy. It is known for example to move the object observed so as to perform a scan. The image is then reconstituted. Here again we find problems of acquisition rate. A third technique, stereovision, uses two or more cameras to view the same object from different angles. The images are then analyzed using software to provide the third dimension. US-2008/0158666 discloses a device for performing a three-dimensional visualization of an object. This device has a plate having a truncated pyramid-shaped well, with at least three faces and a bottom. The faces and bottom of the truncated pyramid are reflective. When one observes a cell with such a device, associated with a microscope, difficulties arise on the one hand because of the parasitic light reflected from the bottom of the well and on the other hand by the large number of images provided. In addition, this device does not track the trajectory of an object (cell) observed.

The object of the present invention is therefore to provide a device and an associated method making it easy to perform three-dimensional microscopic visualization. Preferably, the equipment used will be of a limited cost price. Advantageously, this device and the associated method will allow a sufficient acquisition rate to allow a trajectory of an observed object to be followed. Also preferably, the implementation of the present invention for the observation of a cell will not damage, or as little as possible, the observed cell.

For this purpose, the present invention proposes a device for three-dimensional microscopy visualization which, according to the present invention, comprises a support having a substantially flat face in which a groove is made, and the surface of the groove is reflective so as to to form a mirror, an object to visualize being intended to take place in the furrow.

In this way, an object disposed in the groove faces two reflecting faces. By observing the object and its reflections, for example on a shooting in two dimensions such as a classic photo, we observe the object seen from different angles. In addition, the position of the reflections with respect to the object itself on the shooting makes it possible to determine the position of the object in a plane perpendicular to the plane of the shot. A three-dimensional reconstruction can thus be envisaged. It suffices to know the geometry of the support, which is fixed, to determine the third dimension.

Providing grooves on the support makes it possible to circulate in said furrows a solution. In this way, the device according to the invention can be integrated with a fluidic system. The change of the observed object can be achieved here in a perfectly controlled and easy manner.

In a preferred embodiment, said face of the support comprises a set of parallel grooves spaced at a predetermined pitch. This gives a preferred orientation given by the longitudinal axis of the grooves.

The grooves are preferably V-shaped grooves. Thus, the mirrors made are flat mirrors and the calculations to be made to make a reconstruction in three dimensions are easier and more reliable. Advantageously, the angle formed between a wall of a groove and the flat face is approximately 54 °. For example, an angle of between 52 and 56 ° can be provided. It has been noticed in laboratory tests that this angle gives more accurate results. To achieve the reflective surfaces, an advantageous embodiment provides that each groove is covered with a layer of a metal selected from the group consisting of gold, silver, platinum and aluminum. Such metals are on the one hand easy to deposit in a thin layer and on the other hand make it possible to obtain a mirror of good quality. The invention is more particularly intended for the observation of objects whose size is of the order of one micrometer (1 μm ≈ 10 μm). The grooves used then have, for example, a width of between 2 μm (2 10 ^-6 m) and 100 μm and have a length of between 20 μm and 0.01 μm. length is at least 10 times (or at least 100 times) greater than their width.

The support is advantageously made of silicon. This material allows the grooves to be made by wet etching. This method and this material make it possible to obtain supports with a well-defined geometry at an interesting cost price. The present invention also relates to a device for microscopic and three-dimensional visualization comprising a microscope, a microscope slide and microscope-related imaging means, characterized in that a device as described above is fixed on the microscope slide, and in that the imaging means are connected to an image processing device for rendering three-dimensional images from a received image and comprising on the one hand the image of the object to be observed and, on the other hand, its reflections in the corresponding groove.

In such a device for viewing in microscopy and in three dimensions, the image processing means are for example integrated in a microcomputer having a display screen, on which it is then possible to restore a three-dimensional image.

Finally, the present invention also relates to a method of microscopic visualization of an object in three dimensions. According to the invention, this method comprises the following steps:

placing the object to be observed in a groove made on a support and having a reflective concave surface,

- shooting through a microscope of the object observed, and its reflections on the reflective surface of the furrow, - analysis of the image of the object and its reflections in order to build a three-dimensional image of the object to observe.

Details and advantages of the present invention will emerge more clearly from the description which follows, given with reference to the appended diagrammatic drawings in which: FIG. 1 is a perspective view of a device for three-dimensional microscopic visualization according to the present invention,

FIGS. 2a to 2d schematically illustrate a method for obtaining the device of FIG.

Fig. 3 is a block diagram for an application of the present invention, and

FIG. 4 is a diagram from which calculations can be made for the construction of a three-dimensional image from a 2D image of an object to be observed and its reflections.

Figure 1 shows a preferred embodiment of a device used for three-dimensional microscopy visualization according to the present invention. In this figure, there is a plate 2. This is intended to serve as a support for objects to be observed. This plate is for example of rectangular shape and has a very small thickness compared to its length and width. It thus presents two main faces. We will assume, in the Following the description, this plate is arranged horizontally. It thus has an upper face 4 and a lower face parallel to the upper face. FIG. 1 represents on an enlarged scale a portion of the upper face 4 of the plate 2. The upper face 4 of the plate 2 is substantially planar and grooves 6 are noticeable in this upper face 4. In the preferred embodiment shown in this figure, each groove 6 has a V shape. The grooves 6 are arranged parallel to each other with a regular pitch. Other forms of furrows and other arrangements of the furrows could be considered.

To give an indication of the dimensions of the grooves 6, there is shown in Figure 1 a scale bar 8 which corresponds to 400 microns. This scale bar 8 is given for illustrative and not limiting purposes. Thus, for example, the width of a groove is for example between 2 and 100 microns while the length of a groove is for example between 20 microns and 1 cm. The pitch separating two grooves 6 can be adjusted according to the objects to be observed.

Each groove 6 has two facets 10, each facet 10 corresponding to a branch of V. Thus, a facet 10 joins the upper face 4 of the plate 2 at the bottom 12 of the groove 6 corresponding. Here we find a dihedral and the bottom 12 is reduced to a straight line. There is no flat surface at the bottom 12.

Each facet 10 of a groove 6 is covered with a reflective material. This is preferably a metal that can be deposited in a very thin layer. The metals used can be, for example, gold, platinum, aluminum or silver. The deposit is made on the facets 10 so as to obtain reflective facets. These facets 10 thus form "micromirrors" whose geometry is perfectly controlled.

FIGS. 2a to 2d illustrate the production of grooves 6 on one face of a plate 2. In the preferred embodiment described here and illustrated in FIG. 2, the plate 2 is made of silicon.

A resin 14 is coated and treated in patterns of determined dimensions. These patterns are transferred to a layer 16 of nitride on the silicon. Wet etching treatment (by potash action per example) is made on the silicon and then furrows are obtained as shown in Figure 2c. The layer 16 of nitride is then removed and the silicon is covered with a thin metal deposit 18. It is chosen in this embodiment to cover both the facets 10 of the grooves 6 and the rest of the upper face 4 of the plate 2 by the metal deposit 18. The latter is made so as to obtain a reflective surface.

The plate 2 provided with its micro-mirrors then forms a support for receiving the elements to be observed. The plate 2, as an observation support, is then fixed using a macroscopic mechanical device on a microscope slide (not shown). This mechanical device makes it possible to clamp between the plate 2 and the microscope slide. Such a clamping device is known to those skilled in the art and already used in the field of microscopic observation and is not described here. The assembly formed by the microscope slide, the plate 2 with its micromirrors and the sample to be observed is then placed on a microscope support (not shown). This is a microscope quite classic as those commonly used in laboratories. It can be a right or inverted microscope.

FIG. 3 illustrates the principle underlying the invention and makes it possible to obtain a three-dimensional restitution by using the plate 2 and its micromirrors to receive the sample to be observed.

In FIG. 3, two facets 10 of a groove 6 of the plate 2 are shown on an enlarged scale. This is a sectional view in a plane marked by a reference (Y, Z). Two fluorescent beads are disposed between the facets 10 of the groove 6 shown. In the case shown, the two balls 20 are aligned along the axis Z. Each of the balls 20 is reflected in each of the facets 10. This gives the ball located closest to the bottom 12 two reflections 20 ", 20 "and for the ball farthest from the bottom 12 two reflections 20 ', 20".

The balls 20 and their reflections 20 "and 20" are observed through an objective 22 associated with unrepresented means of shooting. The observation of the balls 20 is in the direction given by the Z axis. Since the facets 10 forming micro-mirrors are inclined with respect to the angle of observation, the reflected images have a side view of the balls 20 In addition, the position of the reflections 20 'and 20 "along the Y axis depends on the position along the Z axis of the reflected ball, in the image obtained (right part of FIG. the image a ball 20 which is arranged between the image of its reflections 20 'and 20 ".The right part of Figure 3 corresponds to a shooting of a mounting similar to that shown on the left part of Figure 3 with only the ball 20 being closest to the bottom 12 of the groove 6. As apparent from the previous paragraph, the position of the ball in the reference (X, Y) is given directly by the image from the shooting and the position along the Z axis can be calculated through the reflections 20 'and 20 "of the ball 20. This opens the way to a three-dimensional reconstruction from a single view of the scene captured by an optical microscope. This three-dimensional reconstruction can be performed using a relatively similar approach to the techniques used in stereovision. It is a question here of observing singular points in two views of the same scene taken with an offset point of view.

FIG. 3 represents a simple situation in which a luminous point, corresponding to the ball 20, and its two primary reflections. According to the image obtained, the X and Y coordinates of the ball 20 are known and it is desired to determine its coordinates in Z. Due to the invariance of the micromirrors along the X axis, the X coordinates are not not useful for three-dimensional reconstruction. In the following, relations between Y and Z are given. To establish these relations, two problems concern us: how do the reflections of an object undergoing a translation along the Y axis only behave? how do the reflections of an object undergoing translation along the Z axis behave? An important parameter here is the angle made by the facets 10 with respect to the upper face 4 of the plate 2. This angle is called α and is represented in FIG. 4. In this last figure, the orientation of the axis Z is inverted with respect to the orientation selected in FIG. 3. An angle β is defined as follows: β = 1 ~ 2 * ₍₁₎

In order to simplify the calculations, only the case of the left reflection (20 ') is developed here. The calculations concerning the right reflection (20 ") are similar calculations, to the near sign. First of all suppose a translation A2 which is the translation of the point A represented in FIG. 4 according to a vector ^e y of length Δy. The relative positions of the points A and A2 are compared as well as the relative positions of their respective reflections A ¹ and A2 '. In FIG. 4, arrows 24 illustrate the direction of observation of the scene by the user. In FIG. 4, point A2 is to the right of A. Similarly, point A2 'is to the right of point A'. We call δy the variation of the coordinate in y between A ¹ and A2 '. We then have the following equation:

Δy = - siniβ) ₍₂ )

It is now assumed that point A moves along the Z axis to take the place of point A3. The reflection A ¹ of the point A then moves to come to the position A3 ¹ . A3 is translated with respect to A according to Δz. Likewise, along the Z axis, a displacement δz between A 'and A3' is defined. In this case, we have the following equation: δz

Δz = cosψ) ₍₃₎ We can then generalize and suppose that the point A translates firstly along the Y axis and secondly along the Z axis. The reflection A 'of point A will then move, in the direction of the Y axis, of a difference δ _tota i which is given by the following equation:

"W _α i = δy + δz = Δy - SiTIiJ) + Δz - cos (β) (2) + (3) We deduce the expression of Δz

cosψ) ₍ 4 ₎

For the image A "of the point A, the only difference in the calculations is on the sign of Δz, thus obtaining the following equation:

L'image que l'on obtient par les moyens de prise de vue à travers l'objectif 22 et le microscope nous donne les quantités δ_totai et Δy. L'angle β est fixe et est déterminé par la gravure du silicium (cf. figure 2 et réalisation des sillons 6 plus haut). _{Δz ≈} Δy - si - δ _total

The image that is obtained by the means of shooting through the lens 22 and the microscope gives us the quantities δ _tota i and Δy. The angle β is fixed and is determined by the etching of the silicon (see Figure 2 and realization of the grooves 6 above).

We note here that this method of reconstruction does not require no calibration.

The stereovision principle for locating Z coordinates also applies using both reflections (left and right). For this it is necessary to combine equations (4) and (5):

* g ^{* ~} toy ^• sin (β) - left r right

Δz = Δy = "total ⁺ " total cos (β) 2 • sin (β) Δy - sin (β) - δ? ™ fg left _ f. right

Δz = Δz = total total

COS (β) 2 • COS (β) (β)

Thanks to the use of the plate 2 provided on its upper face 4 furrows 6 and micro-mirrors, it is possible to achieve a three-dimensional reconstruction with two views of the same scene. Thus, with a single image, three three-dimensional measurements are made. This redundant information can be used to evaluate the coherence of the three-dimensional measurements made, this characteristic being relevant for quantifying the quality of the reconstructions. To validate this reconstruction method, beads with a diameter of 1 μm were glued on a microscope slide and placed under the micromirrors formed by the facets 10. Thus, the balls used are located on the same surface with the same altitude z (different case of Figure 3). We have therefore in equations (4) and (5) a value Δz = 0. By moving the balls in the plane (X, Y), we measure δ _tota i and Δy and we calculate the Δz values. Experimental results indicate that the average gap between the beads and -45 nm ± 306 nm (with 80 measurements made, 1 nm = 10 ^{~ 9} m). Such a result thus makes it possible to validate the proposed method.

As indicated above, a preferred embodiment provides that the support for the samples to be observed is made from a silicon substrate. Of course, it is conceivable to use a support for the samples to be observed in another material. In the case of a silicon plate 2, micromachining strategies make it possible to design micromirrors with different geometries. This amounts to modifying the angle β introduced in particular in equation (1) above. Tests were performed with facets 10 inclined relative to the upper face 4 by an angle close to 35 ° and then with an angle close to 54 ° (corresponds to the value of the angle α). It was found that the measurements made with micromirrors inclined by 54 ° are much more accurate than those made with inclined micromirrors. about 35 °. A preferred embodiment of the invention therefore provides a silicon plate 2 with facets 10 of the grooves 6 inclined about 54 ° with respect to the upper face 4 of the plate 2.

For three-dimensional reconstruction from shots taken through the microscope, software is used to perform the above calculations. The camera is preferably a digital camera. An image processing software is then used to determine the position of the image of the ball and its reflections. Such software is already known and is not described here. Once the position of the ball, or more generally of the element to be observed, and the positions of its reflections determined, the specific software performs the three-dimensional reconstitution of the element observed. Display means are advantageously provided to show the reconstruction performed. In practice, a microcomputer is for example associated with the microscope and its camera (digital camera for example). The image processing, three-dimensional reconstruction and visualization software are then implanted in the microcomputer which advantageously also comprises a display screen.

The present invention can be used for example for observation in cell biology. As an illustrative and non-limiting example, it is possible to observe yeasts or mammalian cells. For such applications, a gene of the cells is fluorescently labeled by genetic engineering techniques known to those who typically perform cell observations under a microscope. The nucleus of the cell appears as a round structure around the focus of fluorescence thus achieved. It is then possible, thanks to the three-dimensional reconstruction described above, to observe in 3D the trajectory of a gene inside the corresponding nucleus.

Such observations can be made using standard optical microscopes, which are the tools widely disseminated in the scientific community. Such microscopes, unlike confocal microscopes, are inexpensive.

The device according to the invention thus allows the use of conventional microscopes to make observations such as those made with confocal microscopes. As a result, the invention makes it possible to produce three-dimensional observations at a low cost. In addition, it is noted that with a device according to the invention, no calibration is necessary. In addition, all wavelengths can be used.

As a result, the present invention is suitable for rapid microscopy. It is relatively inexpensive in light for three-dimensional reconstruction.

It is also noted that the plates described in the foregoing description can be made at reduced cost. The etching technique is a known and commonly used technique thus obtaining cheap media.

The present invention is also particularly suitable for a device with integrated fluidic system. Here it is possible to circulate a solution in the grooves described. The change of the observed object is easily achievable and can be done in a controlled manner. The present invention is not limited to the preferred embodiments of the device and method described above by way of non-limiting examples and the variants mentioned. It also relates to all the variants within the scope of those skilled in the art within the scope of the claims below.

Claims

1. Device for visualization in three-dimensional microscopy, characterized in that it comprises a support (2) having a substantially planar face (4) in which a furrow (6) is formed, and in that the surface a furrow (6) is reflecting so as to form a mirror, an object (20) to be viewed being intended to take place in the groove (6). 2. Device according to claim 1, characterized in that said face (4) of the support (2) comprises a set of parallel grooves (6) spaced at a predetermined pitch. 3. Device according to one of claims 1 or 2, characterized in that each groove (6) is a V-shaped groove. 4. Device according to claim 3, characterized in that the angle (α) formed between a wall (10) of a groove (6) and the face (4) plane is about 54 °. 5. Device according to one of claims 1 to 4, characterized in that the groove (6) is covered with a layer of a metal selected from the group consisting of gold, silver, platinum and silver. 'aluminum. 6. Device according to one of claims 1 to 5, characterized in that the groove (6) has a width of between 2 microns (2 ^10-6 m) and 100 microns and has a length between 2 microns and 0 , 01 m. 7. Device according to one of claims 1 to 5, caratérisé in that the groove has a width at least 10 times greater than its width. 8. Device according to one of claims 1 to 6, characterized in that the groove has a length at least 100 times greater than its width. 9. Device according to one of claims 1 to 8, characterized in that the support (2) is silicon. 10. Device for visualization in microscopy and in three dimensions comprising a microscope, a microscope slide and microscope-related means of shooting, characterized in that a device according to one of claims 1 to 9 is fixed on the microscope slide, and in that the imaging means are connected to an image processing device making it possible to restore three-dimensional images from a received image and comprising, on the one hand, the image of the object to be observed and, on the other hand, its reflections in the corresponding furrow. 11. Apparatus for viewing in microscopy and three dimensions according to claim 10, characterized in that the image processing means are integrated in a microcomputer having a display screen. 12. Method for microscopic visualization of an object in three dimensions, characterized in that it comprises the following steps: - placing the object (20) to be observed in a groove (6) made on a support (2) and having a reflective concave surface, - Shooting through a microscope of the object (20) observed, and its reflections (20 ', 20 ") on the reflective surface (10) of the groove (6), - Analysis of the image of the object (20) and its reflections (20 ', 20 ") to construct a three-dimensional image of the object to be observed.