WO2008146226A1

WO2008146226A1 - Sample holder for an optical device

Info

Publication number: WO2008146226A1
Application number: PCT/IB2008/052050
Authority: WO
Inventors: Dirk L. J Vossen; Sjoerd Stallinga
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2007-05-25
Filing date: 2008-05-23
Publication date: 2008-12-04

Abstract

The invention relates to an optical device which comprises at least three objectives (24, 25, 26, 27), each objective (24, 25, 26, 27) comprising an optical axis ((24', 25', 26', 27'), the device further comprising a polyhedron-shaped sample holder (28, 36, 38), comprising a plurality of faces defining a volume within which the sample is to be placed, each objective (24, 25, 26, 27) facing one corresponding face of the polyhedron-shaped sample holder (28, 36, 38), said face being substantially perpendicular to the optical axis (24', 25', 26',27') of the corresponding objective (24, 25, 26, 27).

Description

Sample holder for an optical device

Field of the invention

The invention relates to optical devices which comprise at least three objectives through which a light beam is irradiated on or collected from a sample for analysis, imaging or manipulation of the sample.

Background of the invention

Conventional microscopes use a single objective lens to image an object; a light beam is focused on the object and the light reflected or transmitted is detected so as to obtain an image of the object. A good image resolution is a constant goal in microscopy. The resolution increases with the numerical aperture of the optical device. The numerical aperture may be defined as n.sin(α), where α is the angle between the chief ray and the marginal ray of the converging cone of light produced by the objective lens (that is to say, α is the half-angle of the maximum cone of light that can enter or exit the objective lens) and n is the refractive index of the incidence medium. However, the free working distance, that is to say, the maximal possible distance between the objective lens and the sample, decreases when the numerical aperture increases. A trade-off therefore has to be found between a high free working distance and a high numerical aperture.

To solve this problem, microscopes have been developed that comprise more than one objective lens; those microscopes are called 4π-microscopes (also referred to as theta-microscopes). Thanks to the use of a plurality of objective lenses, the 4π- microscopes permit to use a moderate numerical aperture and a high free working distance, without compromising the image resolution or to increase the resolution for a given numerical aperture and free working distance. As a matter of fact, for instance if two facing objective lenses are used, the resolution is increased in the direction of the common optical axis of the two lenses. The principle may be extended to three, four or even more objective lenses. Fig.l schematically represents a 4π-microscope of the prior art with two objective lenses, for illustration and comprehension purposes. Light is emitted by a light source 1 , for instance a laser. The emitted beam of light 2 is collimated by a lens 3 and split into two parts by a beam splitter 4. The first part is reflected by a mirror 5 towards a first objective lens 6 centered around a first optical axis 7 and the second part is reflected by mirrors 8 and 9 towards a second objective lens 10 centered around a second optical axis 11, collinear with the first optical axis 7. The two beams are incident on and focused by two objective lenses 6, 10 on a sample 12, thereby forming a tiny focused spot that can scan the sample 12. The reflected, scattered and/or fluorescent light is captured by the two objective lenses 6, 10, transported back to the beamsplitter 4 and the part of the light that is transmitted by this latter is converged by a lens 13 onto a photo-detector 14.

Fig.2 schematically represents a 4π-microscope of the prior art with four objective lenses 15, 16, 17, 18. The objective lenses 15, 16, 17, 18 are positioned around a sample 19, which is positioned in a holder device comprising two parallel glass or plastic plates 20, 21 sandwiching the sample 19, in a classical manner. The angle between the optical axes 15', 16', 17', 18' of the lenses 15, 16, 17, 18 and the direction 22 normal to the surfaces of the plates 20, 21 being quite important (it is at least equal to the arc sinus of the numerical aperture of each lens in the (symmetrical) arrangement of Fig.2), the spots focused by the objective lenses suffer from aberrations, namely astigmatism and coma. The focused spots will have a size larger than the diffraction limit. This aberration problem always occurs when the optical axis of an objective is not perpendicular to the sample surface, which is necessarily the case when more than two objective lenses are used.

The same aberration problem exists with optical tweezers. In optical tweezers, a laser beam is focused on a particle in order to apply a force to this particle, as will be explained in more details later. The laser beam is focused by an objective lens. Again, a plurality of laser beams may be used, that is to say, an optical tweezers device may comprise a plurality of objective lenses, all focusing light on the same particle in a configuration comparable to the one of the 4π-microscope of Fig.2. For the same reasons as explained above, the laser light beams suffer from aberrations. As a consequence, the focused spot is widened and the forces exerted on the particle to be manipulated decrease. Therefore, the quality of the optical tweezers is worse.

US 2004/0173760 describes a dual beam confocal microscope in which a prism or prisms can be used in front of a sample to reduce or eliminate coma and/or astigmatism. However, a problem of such a device is that the prism(s) has(ve) to be very well aligned in the system, which is difficult to obtain, and may introduce extra reflections.

Furthermore, the complexity of the microscope is increased because of the use of such additional element(s). It is to be noted that this problem is even more important when such a 4π-confϊguration device must comprise at least three objective lens. By way of example, a 4π-microscope having four objectives lenses should comprise four prims postioned around the sample.

Summary of the invention It is therefore an object of the present invention to provide an optical device which overcomes the above-mentioned problems.

In particular it is desired an optical device which avoids or corrects the above mentioned aberrations without adding to much complexity.

In accordance with the present invention there is provided an optical device which comprises at least three objectives, each objective comprising an optical axis, the device further comprising a polyhedron- shaped sample holder, comprising a plurality of faces defining a volume within which the sample is to be placed, each objective facing one corresponding face of the polyhedron- shaped sample holder, said face being substantially perpendicular to the optical axis of the corresponding objective.

The sample holder should be understood as an element holding, that is to say supporting, the sample. It may also be designated as a sample support.

Thanks to the invention, each objective having an optical axis substantially perpendicular to the corresponding face of the polyhedron- shaped sample holder, the aberrations are greatly reduced. Indeed, in the prior art, the origin of the aberration is the big angle between the normal to the surface of a plate sandwiching the sample and the optical axis of the objective facing this plate. By an "objective", it should be understood any optical device comprising lenses, adapted to optically guide light beams to or from a sample. An objective may comprise a single objective lens or a plurality of objective lenses, such as a doublet of lenses or more complicated combinations of lenses. An objective in any case presents a global optical axis for the light beams passing through.

According to an embodiment, at least two objectives are aligned in pairs with collinear optical axes.

According to an embodiment, the optical device comprises a plurality of singlet lenses as objectives, with a numerical aperture in the range of 0.4-0.55 or 0.80- 0.9 and preferably 0.55-0.70.

According to an embodiment, the optical device comprises a cubic sample holder and objective lenses adapted for CD, BD and preferably for DVD readouts.

According to an embodiment, the optical device comprises six objective lenses, the sample holder being cubic, and it further comprises means for generating six light beams arranged to irradiate respective faces of said holder, the generating means and this holder being arranged so that each light beam has a linear polarization parallel to a diagonal of the respective face

According to an embodiment, the optical device comprises eight objective lenses, the sample holder being an octahedron, and it further comprises means for generating eight light beams arranged to irradiate respective faces of said holder, the generating means and this holder being arranged such that each light beam has a linear polarization, and such that, on one hand, the polarization of light beams irradiated on four adjacent faces points towards a first apex common to those four faces, and on the other hand, the polarization of light beams irradiated on the four other adjacent faces points towards a second apex common to those four other faces, the first apex being opposite to the second apex.

According to an embodiment, the sample holder comprises as many faces as objectives.

According to an embodiment, the sample holder is hollow and may be filled with the sample.

According to an embodiment, the sample holder is made of a homogeneous solid material, with a recessed channel extending through the volume of the sample holder, in order to permit to image, analyze of manipulate a fluid flowing through the channel.

According to an embodiment, the optical device is a 4π-microscope and/or an optical tweezers. According to the invention there is also provided a sample holder for an optical device comprising at least three objectives, each objective comprising an optical axis, the sample holder being of a polyhedron shape and comprising a plurality of faces defining a volume within which the sample is to be placed, the faces being arranged so that each objective may face one corresponding face of the sample holder with its optical axis substantially perpendicular to said face

According to an embodiment, the sample holder is a octahedron.

According to an embodiment, the sample holder comprises two pyramids formed of a homogeneous solid material, adapted to be pressed the one against the other to hold the sample therebetween. According to an embodiment, the sample holder is a cube.

According to an embodiment, the sample holder is made of a homogeneous solid material with a recessed channel extending through the volume of the sample holder, in order to permit to image, analyze of manipulate a fluid flowing through the channel. These and other aspects of the invention will be more apparent from the following description with reference to the attached drawings.

Brief description of the drawings

- Fig.l is a schematic view of an exemplary 4π-microscope of the prior art with two objective lenses;

- Fig.2 is a schematic view of an exemplary 4π-microscope of the prior art with four objective lenses;

- Fig.3 is cross-section schematic view of a 4π-microscope according to the invention, wherein the polyhedron- shaped sample holder is an octahedron combined with four objective lenses;

- Fig.4 is a perspective view of the octahedral sample holder of the 4π- microscope of Fig.3; - Fig.5 is a perspective schematic view of 4π-microscope according to the invention, wherein the polyhedron- shaped sample holder is a cube combined with six objective lenses;

- Fig.6 is a perspective schematic view of a cubic sample holder according to a particular embodiment;

- Fig.7 comprises four diagrams showing the lateral (left diagrams) and axial (right diagrams) light distribution in an oil immersion irradiated through an objective lens with a numerical aperture NA=I.4 at a wavelength λ=0.650μm, the top diagrams being relative to a conventional microscope with a unique objective lens and the bottom diagrams being relative to a 4π-microscope, comprising two facing objective lenses;

- Fig.8 is a schematic view of a cubic sample holder with possible linear polarization of the incident light beams represented on its faces and

-Fig.9 comprises two diagrams showing the calculated light distribution in an oil immersion irradiated in the cubic sample holder of Fig.8, the left diagram showing the light distribution in the direction of the diagonal of the cubic sample holder and the right diagram showing the light distribution in the two mutually orthogonal directions perpendicular to said diagonal direction.

Detailed description of the embodiments An optical device according to the invention comprises at least three objectives, here three objective lenses, for irradiating light on a sample or collecting light from the sample, for imaging, analyzing or manipulating the sample. Analyzing the sample may cover several analysis applications, where a light beam is interacted with a sample in order to gather information on this sample; this interaction may be, for instance, reflection, transmission, scattering, diffraction, etc.

The objective lenses may be used to irradiate the sample, for excitation or detection processes, the light coming back being collected in the same or other lenses. One particular objective lens may be adapted to focus light for irradiating the sample, to collect light coming back from the sample, or both; the light coming back from the sample may be light irradiated through another objective lens and collected by this particular objective lens or light irradiated and collected by the same objective lens. All arrangements can be contemplated. For instance, only some lenses may be used to irradiate the sample, or all of them. Opposing lenses may work in pairs: one lens (or both) irradiates the sample and the opposite one (or both) detects light coming back. The optical axes of the objective lenses are coplanar or not.

The device comprises a polyhedron- shaped sample holder which is adapted to the arrangement of objective lenses of the device. The sample holder is formed so as to contain at least as many planar faces as objective lenses, the device being arranged so that each face of the sample holder corresponding to an objective lens is substantially perpendicular to the optical axis of this objective lens and is at least partially transparent to the light beams, passing through said face, irradiating on or collected from the sample. The sample device may comprise further faces which do not correspond to a particular objective lens; thus, a sample holder with m faces may be used in an optical device comprising m objective lenses or less. In other words, the sample holder comprises at least the same number of faces as the number of objective lenses, each objective lens being coupled to a face, of the sample holder, in order to face it with its optical axis perpendicular to said face, and the sample holder may comprise other faces not coupled to any objective lens. Those further faces may be curved or opaque since they are not used for transmitting light between the sample and an objective lens; according to another embodiment, they also are transparent or partially transparent to the light beams involved. The polyhedron should comprise faces which permit that the optical axes of the at least three corresponding objective lenses substantially intersect in the sample, preferably into a single spot (surrounding the focal point).

Here are some examples of polyhedron shapes that can be used for the sample holder of the invention: a tetrahedron, a cube, an octahedron, a dodecahedron, a triangular bipyramid, a pentagonal bipyramid, a square pyramid, a pentagonal pyramid, a triangular cupola, a square cupola, a pentagonal cupola, an octagonal prism, a cuboctahedron, a truncated octahedron, etc.

An optical device for imaging a sample, will now be described with reference to Fig.3. The optical device 23 is a 4π-microscope, comprising four objective lenses 24, 25, 26, 27, having respective optical axes 24', 25', 26', 27'. The optical axes 24', 26' of the first and third objective lenses 24, 26 are aligned and the optical axes 25', 27' of the second and fourth objective lenses 25, 27 are aligned. In other words, the objective lenses are coupled in pairs (24, 26), (25, 27) with collinear optical axes (24', 26'), (25', 2T).

The 4π-microscope 23 comprises a sample holder 28, in the form of an octahedron, that is to say, a polyhedron comprising eight planar faces. According to the described embodiment, the sample holder is a regular octahedron. The apexes of the octahedron are denominated A, B, C, D, E, F. The faces will be designated by the apexes which define them; for instance, the face which is between the apex A, the apex B and the apex E will be designated as the face ABE.

The octahedron 28 and the objective lenses 24, 25, 26, 27 are arranged so that each objective lens 24, 25, 26, 27 faces a face of the octahedron 28 the surface of which is substantially perpendicular to the optical axis of said lens. In the described embodiment, lens 24 is coupled to face ABE, lens 25 is coupled to face BEF, lens 26 is coupled to face CDF and lens 27 is coupled to face ACD.

It may be noted that, in this embodiment, four faces (ABC, ADE, BCF and DEF) are not coupled to any objective lens. According to an alternative embodiment, not represented, eight objective lenses are provided, each one in front of a corresponding face of the octahedron 28.

In any case, each objective lens is facing the nearest face of the octahedron 28 with its optical axis substantially perpendicular to said nearest face, therefore permitting to obtain an aberration-free microscope.

The faces of the sample holder 28 define a volume, which is the interior volume of the sample holder 28. A sample is placed and positioned within the volume of the sample holder 28, as will be explained below. According to an embodiment, the sample to be imaged, analyzed or manipulated is placed right at the center of the octahedron 28.

The functioning of the 4π-microscope of Fig.3 is classical and does not need to be described in details. Light beams are irradiated on the sample through one or more of the four lenses, the light interacted with the sample being collected from the sample into one or more of the four lenses, usually in the four lenses.

The structure of the octahedron sample holder 28 will now be described in more details. The following embodiments of the octahedron 28 will all be described with reference to Fig.4, regardless their differences, since they all have the same general shape.

According to a first embodiment, the octahedron 28 is formed of two pyramids ABCDE and BCDEF which are pressed the one against the other to sandwich the sample there between. Each pyramid ABCDE, BCDEF is made of a homogeneous solid material transparent to the light beams irradiating on or collected from the sample; for instance, the pyramids are made of glass or plastic. Each pyramid ABCDE, BCDEF comprises five faces, including four triangles (forming four faces of the octahedron- shaped sample holder 28) and a square basis BCDE, adapted to be pressed against the corresponding square basis BCDE of the other pyramid; for simplification reasons, those two basis faces have been designated with the same letters, but they in fact are distinct surfaces. The two square bases of the pyramids ABCDE, BCDEF, pressed the one against the other, hold the sample between them, forming a thin plane comprising the sample. What is important is that the light beams impact or get out from the sample holder 28 through faces perpendicular to the optical axes of the objective lenses 24, 25, 26, 27, while the gap between the two pyramids ABCDE, BCDEF can be neglected. Such a plane contact between the two pyramids is represented on Fig. 3 and designated by reference P.

According to a second embodiment, the octahedron 28 is a hollow element 28, comprising eight faces of a solid material, transparent to the light beams, for instance, glass or plastic. The hollow octahedron 28 may be filled with a liquid or a solid forming or containing the sample to be imaged.

According to a first embodiment in that case, the octahedron 28 comprises one face that is removable so as to fill the volume of the octahedron, this face being closed afterwards.

According to a second embodiment in that case, the octahedron 28 comprises an inlet for filling the volume of the octahedron 28, for instance a hole located at an apex that may be connected to filling means. The octahedron 28 may also comprise an outlet, which may be or not the same hole as the inlet. According to a third embodiment in that case, the octahedron 28 is composed of two open hollow pyramids ABCDE and BCDEF, each pyramid being open on its square basis BCDE. Both pyramids ABCDE, BCDEF are filled with a liquid containing or forming the sample and then fixed the one to the other; this filling of two hollow pyramids may be performed, for instance, by submerging the two pyramids within the liquid they should be filled with or by filling them with a solid sample.

In case of a hollow octahedron 28 filled with a liquid, the sample may be formed of particles submerged within the liquid that do not need to be fixed in position in the volume of the octahedron 28.

The sample may be a particular particle or group of particles that need to be fixed in position in the volume of the octahedron 28, for instance at the center of this volume. Then, the sample may be fixed in position by mechanical means, for instance by a very tiny and sharp point holding the sample. The sample may otherwise be fixed in position by electrical means imposing an electrical field to the sample. According to a particular embodiment, the sample is positioned within the volume of the octahedron 28 by means of an optical tweezers. The use of an optical tweezers as a positioning device for another optical device such as a 4π-microscope will be detailed below and will therefore not be explained in details here.

With reference to Fig.5, a 4π-microscope 29 will now be described, which comprises six objective lenses 30, 31, 32, 33, 34, 35, which are in this embodiment planar-convex lenses, surrounding a cubic sample holder 36. The lenses are grouped in pairs, each pair comprising two facing lenses having collinear optical axes: two lenses 30, 31 with an axis x, two lenses 32, 33 with an axis y and two lenses 34, 35 with an axis z. The optical axes x, y, z are perpendicular to the corresponding faces of the cube 36.

Again, the lenses 30-35 are used to irradiate light on or collect light from a sample positioned within the volume of the cubic holder 36, the faces of which are at least partially transparent, herein transparent, to the light beams. According to an embodiment, the sample is positioned at the center 37 of the cube 36.

Again, the cube 36 may be hollow or may comprise two parallelepipeds, made of a homogeneous solid material, pressed the one against the other to sandwich the sample between them. According to a particular embodiment represented on Fig.6, a cubic sample holder 38 is adapted to define a channel 39 extending through the volume of the sample holder 38. This permits to have the lenses 30-35 focus a light beam or light beams on a fluid flowing through the channel 39, for instance for imaging or analyzing a biological fluid containing cells or molecules of interest, for instance for diagnostic purposes.

According to the described embodiment, the cubic sample holder 38 is made of a full material, that is to say, a homogeneous solid material, herein a transparent material such as glass or plastic, the material being recessed so as to define a rectilinear channel 39 going from an apex 40 to the opposite apex 41 in the volume of the sample holder 38. The channel 39 is connected, at the apexes 40, 41, to an inlet hose 42 and an outlet hose 43 respectively carrying a fluid in and out the channel 39.

For instance, the lenses 30-35 may focus on the center of the cube 38, which the channel 39 crosses, and therefore image or analyze the fluid flowing in the channel 39 when it passes at this center. In an embodiment, the fluid may be imaged or analyzed in a continuous manner. In another embodiment, the fluid flow may be stopped for imaging or analyzing; in a particular embodiment in that case, the fluid may be flowed again after imaging or analyzing and stopped regularly for further imaging or analyzing.

According to an embodiment of the invention, whatever the shape of the sample holder, the optical device comprises a plurality of objective lenses with a low numerical aperture, for instance inferior to 1.0, preferably in the range 0.4 to 0.9.

In particular in that case, cheap objective lenses may be used.

In particular, according to the invention singlet lenses used for CD (Compact Disc; NA between 0.4-0.55) or BD (Blu-ray Disc; NA between 0.75-0.9), or preferably for DVD (Digital Versatile Disc; NA between 0.55-0.75) may advantageously be used. The advantage is that, for a given image resolution, the microscope with cheap lenses will be less expensive, the objective lenses being an important part of the cost of a microscope.

According to an embodiment, an even number of lenses are provided, that are all aligned in pairs. In case of lens pairs the sample holder necessarily has opposing parallel faces, each corresponding to one of the opposing objective lenses of the pair.

In such a case, the lenses may be aligned in pairs thanks to positioning means such as electro-mechanical means (actuators), adapted to position each pair of lenses in the directions perpendicular and parallel to the common axis of the lenses. For performing the positioning of the lenses, a quadrant photo-detector (i.e. a photo-detector subdivided into four quadrants) may be added to each objective lens. During the positioning procedure, light focused by one lens is captured by the photo-detector coupled to the opposite objective lens; if the lenses are well aligned an equal amount of light falls on the four segments (quadrants) of the quadrant photo-detector; if not, the difference signals between the four segments of the photo-detector are used to control the position of the lenses, with the actuators. According to an embodiment, an actuator may position a lens in the axial direction and one of the two transverse directions, while the actuator of the opposite lens will be adapted to position the lens at least in the other transverse position.

A few examples of objective lenses arrangements will now be given. With the octahedron configuration of Fig.3, objective lenses known from Compact Disc readout may be used, which have a numerical aperture in the range 0.45-0.55. With the cube configuration of Fig.5, objective lenses known from Digital Versatile Disc readout may be used, which have a numerical aperture in the range 0.60-0.65.

The performance of a microscope with low numerical aperture objective lenses will now be described in more details with reference to Figs.7-9.

A 4π-microscope according to the described embodiment, namely with a plurality of objective lenses with a low numerical aperture surrounding a polyhedron- shaped sample holder, the objective lenses preferably being grouped in pairs, gives a scanning spot that is comparable in size to the scanning spot of a conventional (expensive) 4π-microscope consisting of two high-NA immersion lenses.

Fig.7 shows the lateral (left diagrams) and axial (right diagrams) light distribution in an oil immersion irradiated through an objective lens with a numerical aperture NA=I.4, at a wavelength λ=0.650μm (the focal region in the medium is an oil having a refractive index n=1.5). The top diagrams are relative to a conventional microscope with a unique objective lens and the bottom diagrams are relative to a 4π- microscope, comprising two facing objective lenses. Lateral light distribution should be understood as the light distribution in the focal plane (transversally to the optical axis) and axial light distribution should be understood as the distribution along the optical axis. It can be seen on Fig.7 that, going from a conventional single objective to a double 4π-objective decreases the width of the central peak of the axial intensity distribution by a factor of about 4 (see the change of scale by a factor two between the top right diagram and the bottom right diagram). The resolution of the 4π-microscope is therefore higher. The goal of the described embodiment is to obtain a similar resolution with cheap lenses.

According to a particular embodiment described above, the microscope of the invention may comprise six objective lenses, arranged around a cubic sample holder 36 so that the optical axes of the six lenses are perpendicular to the faces of the cube 36 (see Fig.5). In such a case, and as seen above, the lenses can be cheap plastic objective lenses known from the readout of Digital Versatile Discs (DVDs). Such lenses are optimized for a wavelength λ=0.650μm, have a numerical aperture NA=O.60, and are designed for focusing into a layer of material with refractive index n=1.5 (the disc).

According to an embodiment, the entrance polarization for each of the six objective lenses may be chosen so as to obtain a small spot irradiated on the sample. According to an embodiment, and with reference to Fig.8, the six incident light beams comprise a linear polarization 44 parallel to the diagonals of the faces of the cube 36, such that the polarization directions of three adjacent faces point to the corner of the cube which is common to (shared by) the three faces, as indicated by the double arrows 44 on Fig.8. The body diagonal of the cube 36, indicated by the dashed line 45 on Fig.8, is then a threefold symmetry axis. The light distribution close to focus then also has this threefold rotation symmetry. In particular, the light distributions along the transverse and axial directions are the same.

Fig.9 shows the calculated light distribution in the focal region of this embodiment, with the left diagram corresponding to the (axial) light distribution in the direction of the body diagonal 45 of the cube 36 and the two graphs of the right diagram corresponding to the (lateral) light distribution in the two mutually orthogonal directions perpendicular to said diagonal direction 45; the calculations have been made for a numerical aperture of the lenses NA=O.60, optimized for a wavelength λ=0.65μm and for a focus in a medium with refractive index n=1.5 (oil immersion); the scale of the left diagram is the same as the scale of the bottom right diagram of Fig.7. Clearly, the resulting spot size is quite similar to the 4π-microscope of the prior art case described above with reference to Fig.7, bottom diagrams. This confirms that a good resolution can be obtained with a cheap microscope, comprising low numerical aperture objective lenses around a polyhedron shaped sample holder.

According to another embodiment, the microscope of the invention may comprise eight objective lenses, arranged around an octahedron sample holder 28 similar to the one of Fig.4, so that the optical axes of the eight lenses are perpendicular to the faces of the octahedron 28. In such a case, and as seen above, the lenses can be cheap plastic objective lenses known from the readouts of Compact Discs (CDs).

According to an embodiment, the entrance polarization for each of the eight objective lenses may be chosen so as to obtain a small spot irradiated on the sample. According to an embodiment, the eight objective lenses are adapted to irradiate light beams with a linear polarization, the polarization of the light beams irradiated on four adjacent faces ABC, ACD, ADE, ABE pointing towards the apex A common to (shared by) those four faces ABC, ACD, ADE, ABE and the polarization of the light beams irradiated on the four other adjacent faces BCF, CDF, DEF, BEF pointing towards the apex F common to (share by) those four faces, which is opposite the other apex A.

The optical 4π-microscope of the invention may be used in a bio-sensing application based on imaging.

The mechanism observed to analyze or image the sample volume can be diffraction and/or absorption at the wavelength of the incident light or related to a change in wavelength. For example, fluorescence, or non-linear processes such as second harmonic generation or two-photon fluorescence may be used. Non-linear techniques are particularly suitable because the relatively large side lobes of the axial intensity distribution are relatively unimportant in that case. These non-linear techniques may permit to obtain a resolution below 100 nm. This is particularly relevant for molecular diagnostics and molecular medicine (protein complexes in the cells typically have that size). According to an embodiment, the observed mechanism may be angle-resolved scattering, in which an object containing scattering elements (e.g. markers attached to bio-molecules) is illuminated and the amount of scattered light is measured as a function of the direction of scattering, which may be indicative for the type of scatterer. According to an embodiment, the microscope may be an imaging microscope in which the plurality of lenses allows for a tomographic approach to 3D-imaging. The invention allows for 4π-microscopes with a nearly isotropic resolution, that is to say, a resolution that is comparable in all directions. According to an embodiment, the optical device is a scanning microscope in which the plurality of lenses makes a focal spot that is scanned through a 3D sample volume.

According to a first embodiment in that case, the focused spot is scanned by moving the sample holder, for instance by rotating it, the objective lenses and the light beams being fixed; even if the movement of the sample holder changes slightly the angles between the light beams and the faces of the sample holder, those angles stay substantially perpendicular to the surfaces of the sample holder and the aberrations resulting therefrom may be neglected; indeed, scanning angles are angles which are not as important as the angles between the light beams and the surfaces of a sample holder of the prior art (see Fig.2). Light in that case is collected from the field of view of the objective, which area can be imaged without significant aberrations.

According to a second embodiment in that case, the focused spot is scanned by changing the position and/or angle of the objective lenses of the microscope, by means of appropriate driving means; this is a traditional way to scan a sample, by moving some of the optical elements.

According to a third embodiment in that case, the focused spot is scanned by changing the angle between the light beams and the optical axis of the objective lenses; again, this is a way known in the art to scan a focused spot within a sample. Again, in the two last embodiments, the aberrations induced by the angle changes between the light beams and the faces of the sample holder produce aberration which are negligible.

According to an embodiment of the invention, the optical device which comprises a polyhedron sample holder comprises an optical tweezers device. Optical tweezers are well known in the art. The principle is optical trapping: a laser beam is incident on a particle through an objective lens so as to trap the particle in the focal region of the lens, provided that the so-called gradient force (pointing towards the maximum intensity at the focal point) can overcome the so-called scattering force (pointing away from the incident beam). Those gradient forces are induced by the laser beam. If the numerical aperture of the lens is high enough, the force may be so high as to grab and move a particle or a group of particles. The typical range of size of particles that may be moved thanks to optical tweezers is nowadays from a few nanometers to a few tens of micrometers.

An optical tweezers device comprising two opposing objective lenses has the advantage that the scattering forces due to the two counter-propagating incident light beams cancel, whereas the gradient forces add up. This greatly enhances the stability of the trap. More than two lenses may be used.

An optical tweezers device with at least three objective lenses may comprise a sample holder according to the invention, comprising faces perpendicular to the optical axes of the lenses.

The optical device comprising the sample holder of the invention may be optical tweezers as such or, according to a particular embodiment, the device may be a combination of another type of optical device with optical tweezers, the sample being positioned within the sample holder by means of the optical tweezers and imaged (or analyzed) by means of the other optical system, which may for instance be a scanning microscope. Therefore, the optical device comprises at least one objective lens provided for a tweezers function and at least one objective lens provided for an imaging or analysis function, the total number of lenses being at least three. Therefore, at least one light beam is a laser beam focused on at least a particle or a group of particles to be manipulated by the laser beam for positioning the particle in the volume of the sample holder for it to be imaged by the 4π-microscope. In particular, the sample holder may be a hollow element, such as the hollow octahedron or cube as described above, filled with a liquid containing a particle or a group of particles to be imaged. Some faces of the sample holder may be facing objective lenses used for imaging the particles and other faces of the sample holder may be facing objective lenses focusing laser beams used for trapping the particles within a focal point according to the principles of optical tweezers. Such an optical device is therefore a combined microscope and optical tweezers, the sample holder being adapted to all the objective lenses involved in this optical device. When applicable, all what has been presented with relation to an octahedron or a cube may be applied or adapted to any polyhedron.

In case the sample holder is made of plastic, it may be manufactured by injection molding. While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments.

Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. A computer program may be stored/distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems. Any reference signs in the claims should not be construed as limiting the scope.

Claims

1- Optical device which comprises at least three objectives (24, 25, 26, 27), each objective (24, 25, 26, 27) comprising an optical axis (24', 25', 26', 27'), the device further comprising a polyhedron-shaped sample holder (28, 36, 38), comprising a plurality of faces defining a volume within which the sample is to be placed, each objective (24, 25, 26, 27) facing one corresponding face of the polyhedron-shaped sample holder (28, 36, 38), said face being substantially perpendicular to the optical axis (24', 25', 26', 27') of the corresponding objective (24, 25, 26, 27).

2- Optical device according to claim 1, wherein at least two objectives ((24, 25, 26, 27) are aligned in pairs with collinear optical axes ((24', 25', 26', 27').

3- Optical device according to claim 2, comprising a plurality of singlet lenses as objectives (24, 25, 26, 27), with a numerical aperture in the range of 0.4-0.55 or 0.75- 0.9 and preferably 0.55-0.75.

4- Optical device according to any of claims 1-3, wherein it comprises six objective lenses, the sample holder (28) being cubic, and it further comprises means for generating six light beams arranged to irradiate respective faces of said holder, the generating means and this holder being arranged so that each light beam has a linear polarization (44) parallel to a diagonal of the respective face.

5- Optical device according to any of claims 1-3, wherein it comprises eight objective lenses, the sample holder (28) being an octahedron, and it further comprises means for generating eight light beams arranged to irradiate respective faces of said holder, the generating means and this holder being arranged such that each light beam has a linear polarization, and such that, on one hand, the polarization of light beams irradiated on four adjacent faces (ABC, ACD, ADE, ABE) points towards a first apex (A) common to those four faces, and on the other hand, the polarization of light beams irradiated on the four other adjacent faces (BCF, CDF, DEF, BEF) points towards a second apex (F) common to those four other faces, the first apex (A) being opposite to the second apex (F).

6- Optical device according to any of the preceding claims, wherein the sample holder (38) is made of a homogeneous solid material, with a recessed channel (39) extending in the volume of the sample holder (38).

7- Optical device according to any of the preceding claims, wherein it forms a

4π-microscope (23, 29) and/or an optical tweezers.

8- Sample holder for an optical device comprising at least three objectives ((24, 25, 26, 27), each objective (24, 25, 26, 27) having an optical axis (24', 25', 26', 27'), the sample holder being of a polyhedron shape and comprising a plurality of faces defining a volume within which the sample is to be placed, the faces being arranged so that each objective (24, 25, 26, 27) may face one corresponding face of the sample holder with its optical axis (24', 25', 26', 27') substantially perpendicular to said face.

9- Sample holder according to claim 8, wherein it forms a octahedron (28).

10- Sample holder according to claim 9, wherein the octahedron is formed by two pyramids of a homogeneous solid material, the pyramids being adapted to be pressed the one against the other so as to sandwich the sample therebetween.

11- Sample holder according to claim 8, wherein it forms a cube (36, 38).

12- Sample holder according to any of claims 8-11, wherein it is hollow and may be filled with the sample.

13- Sample holder according to any of claims 8-12, comprising a recessed channel (39) extending in the volume for enabling a fluid to flow through said channel (39).