Engineered microscope slide for structured illumination

Abstract

Microscope slide (100, 101, 102, 200, 300, 400) comprising: a transparent planar substrate (10, 30) for holding a sample (52), the substrate (10, 30) having at least one input port configured for receiving a light beam from a fiber (11, 21, 31) external to the substrate (10, 30); at least one optical circuit (12, 13, 14, 15, 16, 17, 18, 19, 20, 20', 22, 23, 23', 23'', 24, 24', 24'', 25, 25', 25'', 26, 26', 26'', 27, 27', 28, 29, 29', 29'', 31, 31', 32, 32', 33, 33', 41, 41', 42, 42', 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 170, 171, 172, 173, 174, 175) housed in the substrate (10, 30); said at least one optical circuit (12, 13, 14, 15, 16, 17, 18, 19, 20, 20', 22, 23, 23', 23'', 24, 24', 24'', 25, 25', 25'', 26, 26', 26'', 27, 27', 28, 29, 29', 29'', 31, 31', 32, 32', 33, 33', 41, 41', 42, 42', 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 170, 171, 172, 173, 174, 175) is configured for being coupled with the input port and for transforming the light beam in a structured illumination pattern outside the substrate (10, 30), the pattern having a cartesian component orthogonal to the substrate (10, 30).

Description

[0001] ENGINEERED MICROSCOPE SLIDE FOR STRUCTURED ILLUMINATION

[0002] DESCRIPTION

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present invention relates to an engineered slide for structured illumination microscopy. Generally speaking, a microscope glass slide is a transparent object holder used to hold samples for examination under a microscope. The slide of the present invention consists in a glass substrate wherein all the necessary optical components for structured light generation are integrated on the glass chip. The light is modulated by multiple beams interference and translated on demand over the sample, which will be positioned directly on the glass slide. To this aim, optical waveguides, beam splitters, thermal phase-shifters and mirrors are integrated on chip. The light is delivered to the chip through an optical fiber coupled to the optical circuit and glued to the substrate to ensure stable alignment and facilitating the device handling. When in use the engineered glass-slide will be positioned on the stage of a standard microscope which will be used for fluorescence signal collection. In this way, it will be possible to upgrade the standard microscope in structured illumination microscope, without complicating the end-user experience.

[0005] STATE OF THE ART

[0006] Structured illumination microscopy (SIM) enables increasing image resolution, overcoming diffraction limit. In this fluorescence-based imaging technique the sample is illuminated by a patterned light, whose frequency determines the image resolution improvement. At the current state of the art, this technique is characterized by complex implementations, cumbersome and expensive setups and often unstable component alignments, limiting its widespread use.

[0007] In literature, some devices are known, allowing the sample to be illuminated with modulated light and the illumination to be shifted by exploiting integrated optical circuits to split the incoming light, gratings to deflect the light and integrated thermal phase shifters to control the relative phase of the beams. These types of devices cannot operate in contact with the sample to allow for the overlapping of the beams that occurs after some free-field propagation. Therefore, the sample must be deposited on a microscope slide kept separate from the photonic device, limiting the stability of the system [Lin, Chupao, et al. "UV photonic integrated circuits for far-field structured illumination autofluorescence microscopy." Nature Communications 13.1 (2022)], [Liu, Yong, et al. "Structured illumination chip based on integrated optics." Chinese Physics Letters 33.5 (2016)]. Devices are also known with integrated gratings to illuminate the sample with modulated light [Tang, Mingwei, et al. "High-Refractive-Index Chip with Periodically Fine-Tuning Gratings for Tunable Virtual-Wavevector Spatial Frequency Shift Universal Super-Resolution Imaging." Advanced Science 9.9 (2022)]. However, in such devices, it is necessary to use a Spatial Light Modulator (SLM) to illuminate the gratings appropriately and to move the modulated light. The complexity of the standard SIM setup is, thus reduced, but not eliminated and, in addition, diffractive elements are characterised by high signal losses. Thus, the need arises to overcome these limitations by integrating the necessary parts directly on the chip on which the sample is mounted. At this regard, glass slides are known in the art, that comprise integrated optical components that are not able to generate and move modulated light and, therefore, are not suitable for SIM microscopy (EP 3 599541 Al, EP 3 832201 Al, EP 3 876 004 Al, EP 3 926 233 Al, EP 4 036 465 Al, EP 4 124 033 Al). Recently, some integrated devices for SIM microscopy were introduced wherein the interference of evanescent waves is used to generate the light pattern used to illuminate the sample (US 11454791 B2). However, these devices only illuminate a few tens of nanometres of sample and, importantly, they are not able to acquire volumetric information because, they are designed so that the light does not have an orthogonal component to the plane of the slide, but remains confined in it. [Helle, 0ystein Ivar, et al. "Structured illumination microscopy using a photonic chip. " Nature photonics 14.7 (2020): 431-438], [Villegas-Hernandez, Luis E., et al. "Chip-based multimodal super-resolution microscopy for histological investigations of cryopreserved tissue sections." Light: Science & Applications 11.1 (2022)], [Priyadarshi, Anish, et al. "A transparent waveguide chip for versatile total internal reflection fluorescence-based microscopy and nanoscopy." Communications Materials 2.1 (2021): 85].

[0008] OBJECTS AND SUMMARY OF THE INVENTION

[0009] A first object of the present invention is, therefore, to provide a slide for structured illumination microscopy that allows to decrease the complexity and handling of optical setups necessary for structured light generation and that, at the same time, allows to acquire volumetric information about the sample, thus improving the subcellular features observation.

[0010] A second object of the present invention is also to provide a slide for structured illumination microscopy that is easy to use and does not require additional expertise from the end-user.

[0011] These objects are achieved by the present invention by means of a microscope slide comprising: a transparent planar substrate for holding a sample, the substrate having external to the substrate;

[0012] - at least one optical circuit housed in the substrate;

[0013] The least one optical circuit is configured for being coupled with the input port and for transforming the light beam in a structured illumination pattern outside the substrate, the pattern having a cartesian component orthogonal to the substrate.

[0014] More particularly, the optical circuit comprises:

[0015] • at least two optical waveguides configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides being configured for being coupled to the input port;

[0016] • at least one coupler for each couple of the waveguides, said at least one coupler being configured for splitting the light beam between the two waveguides of the couple;

[0017] • at least one phase shifter for each couple of the waveguides, said at least one phase shifter being configured for creating a phase difference between the two waveguides of the couples; and

[0018] • at least one planar mirror for each of the optical waveguides and a collimating mirror, for each of the optical waveguides.

[0019] The transparent planar substrate comprises a first face configured for the sample being positioned thereon and a second face opposite to the first face, the at least one planar mirror being positioned on the first face and the collimating mirror, being positioned on the second face. For each of the optical waveguides, the at least one planar mirror is configured for reflecting the transmitted light beam towards the collimating mirror thus generating a reflected light beam; the collimating mirror is configured for reflecting the reflected light beam out of the substrate, thus generating a final light beam not coplanar with the substrate; and the final light beam generated by the collimating mirror of one of the two waveguides interferes with the final light beam generated by the collimating mirror of the other of the two waveguides, thus generating the structured illumination pattern. The waveguides are configured in such a way that the transmitted light beams are three-dimensional. The projections of the transmitted light beams in the direction orthogonal (z) to the plane (xy) of the substrate can have all the same magnitude or, alternatively, the projection of at least one of the transmitted light beams in the direction (z) can have a magnitude different from that one of the projection in the same direction (z) of the other transmitted light beams. In the first case, i.e. the projections of the transmitted light beams in the direction (z) have the same magnitude, the resulting pattern illuminating the sample will be modulated along the axis (x) and (y), that are the axis defining the plane of the substrate, whereas in the second case, i.e. the projections of the transmitted light beams in the direction (z) have different magnitude, the resulting pattern illuminating the sample will be modulated along the axis (z), that is the axis orthogonal to the plane of the substrate. A third object of the present invention is to provide a slide for structured illumination microscopy that is compatible with large field of view, thus allowing for rapid large tissue observation.

[0020] To this aim, the microscope slide of the present invention can comprise more than one planar mirror on which the light beams strikes before striking on the collimating mirror. For example, the slide can comprise a first planar mirror, a second planar mirror (51) and a third planar mirror for each one of the waveguides, the first planar mirror being configured for reflecting the transmitted light beam towards the second planar mirror thus generating a first reflected light beam; the second planar mirror being configured for reflecting the first reflected light beam towards the third planar mirror thus generating a second reflected light beam; the third planar mirror being configured for reflecting the second reflected light beam towards the collimating mirror thus generating a third reflected light beam and the collimating mirror being configured for reflecting the third reflected light beam, thus generating the final light beam. By varying the number of planar mirrors, it is possible to enlarge the field of view, thus increasing the dimension of the pattern illuminating the sample without changing the thickness of the slide.

[0021] A fourth object of the present invention is, then, to provide a microscope slide that enables different pattern generation, so that the user can choose the pattern properties depending on the specific analysis he / she wants to perform.

[0022] To this aim, the substrate of the microscope slide according to the present invention can have a first input port configured for receiving a light beam from a first fiber external to the substrate and a second input port configured for receiving a light beam from a second fiber external to the substrate. In this case, in the substrate, a first optical circuit and a second optical circuit are located. The first optical circuit is configured for generating by interference a first structured illumination pattern outside the substrate. The first pattern having a cartesian component orthogonal to the substrate; and a second optical circuit is configured for generating by interference a second structured illumination pattern outside the substrate, the second pattern having a cartesian component orthogonal to the substrate. In this way, according to the input fiber used, it is possible to illuminate the sample with different pattern with different morphology and different frequency.

[0023] A fifth object of the present invention is also to provide a microscope slide that allows to illuminate the sample with structured light with different wavelengths. For this purpose, when the slide is in use, different laser beams with different wavelengths have to be sent into the input fiber. In order to make it possible, the components of the slide can be optimized. More particularly, waveguides should be single mode waveguides for more than one wavelength, the couplers should be configured for having the same working mode for all the wavelengths and the mirrors.

[0024] Last but not least, a further object of the present invention is to provide a slide for structured illumination microscopy that is compatible with standard microscopes in use in biological laboratories. The microscope slide of the present invention achieves this object because it can work in the transmission mode or in the reflection mode. More particularly, it is configured for both receiving a light source illuminating the sample and for transmitting the pattern to an objective lens, the slide being interposed between the light source and the objective lens (transmission mode); that for receiving a light source illuminating the sample and for transmitting the pattern to an objective lens, the light source being positioned at the same side with respect to the slide in which the objective lens is positioned (reflection mode).

[0025] These and further features of the present invention will be made clearer by reading the following detailed description, relating to some preferred embodiments of the present invention, to be considered by way of a non-limiting example of the more general concepts claimed.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following description refers to the accompanying drawings, in which:

[0028] - Figure 1 is a schematic perspective view of the glass slide of the first embodiment of the present invention;

[0029] - Figure 2 is a schematic representation of the use of the glass slide of the present invention together with a microscope;

[0030] - Figure 3a is a schematic representation of a first illumination mode of the glass slide of the present invention;

[0031] - Figure 3b is a schematic representation of a second illumination mode of the glass slide of the present invention;

[0032] - Figure 4 is a top view of a schematic layout of the glass slide of a first embodiment of the present invention; - Figure 5a is a side view of a particular of the glass slide of a first embodiment of the present invention relating to one couple of a planar mirror and a collimating mirror;

[0033] - Figure 5b is a side view of a particular of a second embodiment of the glass slide of the present invention showing an example of multimirror integration relating to a couple of a planar mirror and a collimating mirror;

[0034] - Figure 6a is a schematic planar representation of the beam configuration of the first embodiment, or a first option of the beam configuration of the fifth, or of the sixth, embodiment of the glass slide of the present invention;

[0035] - Figure 6b is a bidimensional image showing the pattern generated in the first embodiment of the glass slide of the present invention or when the beams configuration showed in figure 6a is used in the fifth, or in the sixth, embodiment of the glass slide of the present invention;

[0036] - Figure 7 is a top view of a schematic layout of the glass slide of a third embodiment of the present invention;

[0037] - Figure 8a is a schematic planar representation of the beams configuration of the third embodiment of the glass slide of the present invention;

[0038] - Figure 8b is a bidimensional image showing the pattern generated in the third embodiment of the glass slide of the present invention;

[0039] - Figure 9 is a top view of a schematic layout of the glass slide of a fourth embodiment of the present invention;

[0040] - Figure 10a is a schematic planar representation of the beams configuration of the fourth embodiment of the glass slide of the present invention;

[0041] - Figure 10b is a bidimensional image showing the pattern generated in the fourth embodiment of the glass slide of the present invention;

[0042] - Figure 11 is a top view of a schematic layout of the glass slide of a fifth embodiment of the present invention;

[0043] - Figure 12a is a schematic planar representation of a second option of the beams configuration of the fifth embodiment of the glass slide of the present invention;

[0044] - Figure 12b is a bidimensional image showing the pattern generated when the beams configuration showed in Fig. 12a is used in the fifth embodiment of the glass slide of the present invention;

[0045] - Figure 13 is a top view of a schematic layout of the glass slide of a sixth embodiment of the present invention;

[0046] - Figure 14a is a schematic planar representation a second option of the bemas configurations of the sixth embodiment of the glass slide of the present invention;

[0047] - Figure 14b is a bidimensional image showing the pattern generated when the beams configuration showed in figure 14a is used in the sixth embodiment of the glass slide of the present invention;

[0048] - Figure 15 is a schematic perspective view of the glass slide of the seventh embodiment the present invention;

[0049] - Figure 16 a schematic representation of the use of the seventh embodiment glass slide of the present invention together with a microscope;

[0050] - Figure 17a is a schematic planar representation of one of the possible beams configurations of the seventh embodiment of the glass slide of the present invention; and

[0051] - Figure 17b is a schematic planar representation of one of the possible beams configurations of an eighth embodiment of the glass slide of the present invention.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] Referring to Figures 1, 2, 3a, 3b, 4, 5a, 6a, 6b, a first embodiment of the microscope slide (100) of the present invention comprises:

[0054] - a transparent planar substrate (10) for holding a sample (52), the substrate (10) having one input port configured for receiving a light beam from a fiber (11) external to the substrate (10); and one optical circuit (12, 13, 14, 27, 27', 41, 42, 43, 170, 171) housed in the substrate (10) configured for being coupled with the input port and for transforming the light beam in a structured illumination pattern outside the substrate (10), the pattern having a cartesian component orthogonal to the substrate (10).

[0055] The optical circuit (12, 13, 14, 27, 27', 41, 42, 43, 170, 171) comprises:

[0056] • three optical waveguides (12, 13, 14) configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides (12) being configured for being coupled to the input port;

[0057] • one coupler (27, 27') for each couple of waveguides (12, 13, 14); more particularly, a first coupler (27) configured for splitting, by means of evanescent coupling, the light beam between a first (12) and a second (13) waveguide and a second coupler (27') configured for splitting the light beam between the second (13) and a third (14) waveguide;

[0058] • one phase shifter (171, 172) for each couple of waveguides (12, 13, 14); more particularly, a first phase shifter (171) configured for creating a phase difference between the first (12) and the second (13) waveguide and a second phase shifter (172) configured for creating a phase difference between the second (13) and the third (14) waveguide; and

[0059] • one planar mirror (41, 42, 43) for each of the optical waveguides (12, 13, 14) and a collimating mirror (41'), for each of the optical waveguides (12, 13, 14).

[0060] The transparent planar substrate (10) comprises a first face configured for the sample (52) being positioned thereon and a second face opposite to the first face, the planar mirrors (41, 42, 43) being positioned on the first face and the corresponding collimating mirrors (41'), being positioned on the second face. The planar mirrors (41, 42, 43) are locally metallised.

[0061] The three waveguides (12, 13, 14) are configured in such a way that each one of the transmitted light beams (71, 72, 73) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate (10), said third projections having the same for all the transmitted light beams (71, 72, 73). The first and the second projections of the transmitted light beams (71, 72, 73) of the three waveguides (12, 13, 14) form an angle of 90°, with each other.

[0062] The planar mirrors (41, 42, 43) are configured for reflecting the transmitted light beams towards the collimating mirrors (41') thus generating reflected light beams; the collimating mirrors (41') are configured for reflecting the reflected light beams out of the substrate (10), thus generating final light beams not coplanar with the substrate (10); and the final light beam generated by the collimating mirror (41') of one of the three waveguides (12) interferes with the final light beam generated by the collimating mirror of the other two waveguides (13, 14), thus generating the structured illumination pattern.

[0063] The phase shifters (171, 172) are thermal phase shifters, i.e. they are resistors that heat up, when an electric current passed through them, and modify the local glass index leap, thus allowing a phase alteration of the light beam passing through the waveguide wich the shifter is coupled with.

[0064] The thermal phase shifters are manufactured in correspondence to the waveguides and allow for translation of the structured light necessary for acquiring the superresolved images. The number of thermal phase shifters should be equal to the number of the interfering final light beams (i.e. the number of waveguides) minus one, as already mentioned above.

[0065] Referring to Figures 1, 2, 3a, 3b, 4, 5b, 6a, 6b, a second embodiment of the microscope slide of the present invention has the same components and structure of the first embodiment (100) with the only exception that the microscope slide comprises a first planar mirror (141), a second planar mirror (51) and a third planar mirror (142) for at each one of the waveguides (12, 13, 14), the first planar mirror (141) being configured for reflecting the transmitted light beam towards the second planar mirror (51) thus generating a first reflected light beam; the second planar mirror (51) being configured for reflecting the first reflected light beam towards the third planar mirror (142) thus generating a second reflected light beam; the third planar mirror (142) being configured for reflecting the second reflected light beam towards the collimating mirror (42') thus generating a third reflected light beam and the collimating mirror (42') being configured for reflecting the third reflected light beam, thus generating the final light beam. As already mentioned above, the number of plane mirrors (41, 42, 43, 141) on which the light beams strike before reaching the collimating mirror (41', 42'), allows to define the final beam path length. The longer the path, the larger the region illuminated by the final light beam became, thus increasing the dimension of the pattern illuminating the sample without modifying the thickness of the microscope slide.

[0066] Referring to Figures 1, 2, 3a, 3b, 7, 8a, 8b, a third embodiment of the microscope slide (101) of the present invention comprises:

[0067] - a transparent planar substrate (10) for holding a sample (52), the substrate (10) having one input port configured for receiving a light beam from a fiber (11) external to the substrate (10);

[0068] - one optical circuit (15, 16, 28, 44, 45, 172) housed in the substrate (10) and configured for being coupled with the input port and for transforming the light beam in a structured illumination pattern outside the substrate (10), the pattern having a cartesian component orthogonal to the substrate (10).

[0069] The optical circuit (15, 16, 28, 44, 45, 172) comprises:

[0070] • two optical waveguides (15, 16) configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides (15) being configured for being coupled to the input port;

[0071] • one coupler (28) configured for splitting, by means of evanescent coupling, the light beam between a first (15) and a second (16) waveguide;

[0072] • one phase shifter (172) configured for creating a phase difference between the first (15) and the second (16) waveguide; and

[0073] • one planar mirror (44, 45) for each of the optical waveguides (15, 16) and a collimating mirror, for each of the optical waveguides (15, 16).

[0074] The transparent planar substrate (10) comprising a first face configured for the sample (52) being positioned thereon and a second face opposite to the first face, the planar mirrors (44, 45) being positioned on the first face and the corresponding collimating mirrors, being positioned on the second face. The mirrors (44, 45) are locally metallised.

[0075] The two waveguides (15, 16) are configured in such a way that each one of the transmitted light beams (74, 75) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate (10), said third projections having the same for all the transmitted light beams (74, 75). The first and the second projections of the transmitted light beams (74, 75) of the three waveguides (15, 16) form an angle of 180°, with each other.

[0076] The planar mirrors (44, 45) are configured for reflecting the transmitted light beams towards the corresponding collimating mirror thus generating reflected light beam; the collimating mirrors are, then, configured for reflecting the reflected light beams out of the substrate (10), thus generating final light beams not coplanar with the substrate (10); and the final light beam generated by the collimating mirror of one of the two waveguides (15, 16) interferes with the final light beam generated by the collimating mirror of the other waveguide (15, 16), thus generating the structured illumination pattern.

[0077] Even in the third embodiment of the microscope slide (101) of the present invention it is possible to include a so-called "multi-mirror" configuration like that of the second embodiment. More particularly, a first planar mirror, a second planar mirror and a third planar mirror for each one of the waveguides (15, 16) can be provided. In this case, the first planar mirror will be configured for reflecting the transmitted light beam towards the second planar mirror thus generating a first reflected light beam; the second planar mirror will be configured for reflecting the first reflected light beam towards the third planar mirror thus generating a second reflected light beam. Finally, the third planar mirror will be configured for reflecting the second reflected light beam towards the collimating mirror thus generating a third reflected light beam and the collimating mirror will be configured for reflecting the third reflected light beam, thus generating the final light beam.

[0078] Referring to Figures 1, 2, 3a, 3b, 9, 10a, 10b, a fourth embodiment of the microscope slide (102) of the present invention comprises:

[0079] - a transparent planar substrate (10) for holding a sample (52), the substrate (10) having one input port configured for receiving a light beam from a fiber (11) external to the substrate (10);

[0080] - one optical circuit (17, 18, 19, 22, 29, 29', 29", 46, 47, 48, 49, 173, 174, 175) housed in the substrate (10) and configured for being coupled with the input port and for transforming the light beam in a structured illumination pattern outside the substrate (10), the pattern having a cartesian component orthogonal to the substrate (10).

[0081] The optical circuit (17, 18, 19, 22, 29, 29', 29", 46, 47, 48, 49, 173, 174, 175) comprises:

[0082] • four optical waveguides (17, 18, 19, 22) configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides (19) being configured for being coupled to the input port;

[0083] • one coupler (29, 29', 29") for each couple of waveguides (17, 18, 19); more particularly, a first coupler (29") configured for splitting, by means of evanescent coupling, the light beam between a first (22) and a second (19) waveguide, a second coupler (29) configured for splitting the light beam between the second (19) and a third (18) waveguide, and a third coupler (29') configured for splitting the light beam between the third (18) and a fourth (17) waveguide;

[0084] • one phase shifter (173, 174, 175) for each couple of waveguides (17, 18, 19, 22); more particularly, a first phase shifter (174) configured for creating a phase difference between the first (22) and the second (19) waveguide; a second phase shifter (173) configured for creating a phase difference between the second (19) and the third (18) waveguide; and a third phase shifter (175) configured for creating a phase difference between the third (18) and the fourth (17) waveguide; and

[0085] • one planar mirror (46, 47, 48, 49) and a collimating mirror, for each of the optical waveguides (17, 18, 19, 22).

[0086] The transparent planar substrate (10) comprising a first face configured for the sample (52) being positioned thereon and a second face opposite to the first face, the planar mirrors (46, 47, 48, 49) being positioned on the first face and the corresponding collimating mirrors, being positioned on the second face. The mirrors (46, 47, 48, 49) are locally metallised.

[0087] The four waveguides (17, 18, 19, 22) are configured in such a way that each one of the transmitted light beams (76, 77, 78, 79) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate (10), said third projections having the same for all the transmitted light beams (76, 77, 78, 79). The first and the second projections of the transmitted light beams (76, 77, 78, 79) of the four waveguides (17, 18, 19, 22) form an angle of 90°, with each other. The planar mirrors (46, 47, 48, 49) are configured for reflecting the transmitted light beams towards the corresponding collimating mirrors thus generating reflected light beams; the collimating mirrors are configured for reflecting the reflected light beams out of the substrate (10), thus generating final light beams not coplanar with the substrate (10); and the final light beam generated by the collimating mirror of one of the four waveguides (17, 18, 19, 22) interferes with the final light beam generated by the collimating mirror of the other three waveguides (17, 18, 19, 22), thus generating the structured illuminating pattern.

[0088] Even in the fourth embodiment of the microscope slide (102) of the present invention it is possible to include a so-called "multi-mirror" configuration like that of the second embodiment. More particularly, a first planar mirror, a second planar mirror and a third planar mirror for each one of the four waveguides (17, 81, 91, 22) can be provided. In this case, the first planar mirror will be configured for reflecting the transmitted light beam towards the second planar mirror thus generating a first reflected light beam; the second planar mirror will be configured for reflecting the first reflected light beam towards the third planar mirror thus generating a second reflected light beam. Finally, the third planar mirror will be configured for reflecting the second reflected light beam towards the collimating mirror thus generating a third reflected light beam and the collimating mirror will be configured for reflecting the third reflected light beam, thus generating the final light beam.

[0089] Referring to Figures 1, 2, 3a, 3b, 6a, 6b, 11, 12a, 12b, a fifth embodiment of the microscope slide (300) of the present invention comprises:

[0090] - a transparent planar substrate for holding a sample (52), the substrate having a first input port configured for receiving a light beam from a first fiber (11) external to the substrate and a second input port configured for receiving a light beam from a second fiber (21) external to the substrate;

[0091] - a first optical circuit (20, 20, 23, 23', 23", 52, 53, 54) housed in the substrate and configured for generating by interference a first structured illumination pattern outside the substrate, the first pattern having a cartesian component orthogonal to the substrate;

[0092] - a second optical circuit (24, 24', 24", 31, 31', 55, 56, 57) housed in the substrate and configured for generating by interference a second structured illumination pattern outside the substrate, the second pattern having a cartesian component orthogonal to the substrate.

[0093] The first optical circuit (20, 20, 23, 23', 23", 52, 53, 54) comprises:

[0094] • three optical waveguides (23, 23', 23") configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides (23) being configured for being coupled to the first input port ;

[0095] • one coupler (20, 20') for each couple of waveguides (23, 23', 23"); more particularly, a first coupler (20) configured for splitting ,by means of evanescent coupling, the light beam between a first (23) and a second (23') waveguide and a second coupler (20') configured for splitting the light beam between the second (23') and a third (23") waveguide; and

[0096] • one planar mirror (54, 52, 43) for each of the optical waveguides (23, 23', 23") and a collimating mirror, for each of the optical waveguides (23, 23', 23").

[0097] The second optical circuit (24, 24', 24", 31, 31', 55, 56, 57) comprises:

[0098] • three optical waveguides (24, 24', 24") configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides (24) being configured for being coupled to the second input port ;

[0099] • one coupler (31, 31') for each couple of waveguides (24, 24', 24"); more particularly, a first coupler (31) configured for splitting ,by means of evanescent coupling, the light beam between a first (24) and a second (24') waveguide and a second coupler (31') configured for splitting the light beam between the second (24') and a third (24") waveguide; and

[0100] • one planar mirror (57, 56, 55) for each of the optical waveguides (24, 24', 24") and a collimating mirror, for each of the optical waveguides (24, 24', 24").

[0101] The transparent planar substrate comprises a first face configured for the sample (52) being positioned thereon and a second face opposite to the first face, the planar mirrors (54, 52, 53, 57, 56, 55) being positioned on the first face and the corresponding collimating mirrors, being positioned on the second face. The mirrors (54, 52, 53, 57, 56, 55) are locally metallised.

[0102] The three waveguides (23, 23', 23") of the first optical circuit (20, 20, 23, 23', 23", 52, 53, 54) are configured in such a way that each one of the transmitted light beams (71, 72, 73) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate (10), said third projections having the same for all the transmitted light beams (71, 72, 73). The first and the second projections of the transmitted light beams (71, 72, 73) of the three waveguides (23, 23', 23") of the first optical circuit (20, 20', 23, 23', 23", 52, 53, 54) form an angle of 90°, with each other.

[0103] The planar mirrors (52, 53, 54) of the first optical circuit (20, 20', 23, 23', 23", 52, 53, 54) are configured for reflecting the transmitted light beams towards the collimating mirrors thus generating reflected light beams; the collimating mirrors are configured for reflecting the reflected light beams out of the substrate, thus generating final light beams not coplanar with the substrate; and the final light beam generated by the collimating mirror of one of the three waveguides (52) interferes with the final light beam generated by the collimating mirror of the other two waveguides (53, 54), thus generating the structured illumination pattern.

[0104] The three waveguides (24, 24', 24") of the second optical circuit (24, 24', 24", 31, 31', 55, 56, 57) are configured in such a way that each one of the transmitted light beams (80, 81, 82) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate, said third projections having the same for all the transmitted light beams (80, 81, 82). The first and the second projections of the transmitted light beams (80, 81, 82) of the three waveguides (24, 24', 24") of the second optical circuit (24, 24', 24", 31, 31', 55, 56, 57) form an angle of 120°, with each other.

[0105] The planar mirrors (55, 56, 57) of the second optical circuit (24, 24', 24", 31, 31', 55, 56, 57) are configured for reflecting the transmitted light beams towards the collimating mirrors thus generating reflected light beams; the collimating mirrors are configured for reflecting the reflected light beams out of the substrate, thus generating final light beams not coplanar with the substrate; and the final light beam generated by the collimating mirror of one of the three waveguides (55) interferes with the final light beam generated by the collimating mirror of the other two waveguides (56, 57), thus generating the structured illumination pattern.

[0106] In this way, according to the input port (11, 12) receiving a light beam from a fiber external to the substrate, it is possible to illuminate the sample with different pattern. If the light beam comes from the first input port (11), the generated pattern is determined by the interference of the three final light beams generated by the collimating mirrors of the three waveguides (23, 23, 23") of the first optical circuit (23, 23', 23", 52, 53, 54). Conversely, if the light beam comes from the second input port (12), the generated pattern is determined by the interference of the three final light beams generated by the collimating mirrors of the three waveguides (24, 24', 24") of the second optical circuit (24, 24', 24", 31, 31', 55, 56, 57).

[0107] Referring to Figures 1, 2, 3a, 3b, 6a, 6b, 13, 14a, 14b, a sixth embodiment of the microscope slide (400) of the present invention comprises:

[0108] - a transparent planar substrate for holding a sample (52), the substrate having a first input port configured for receiving a light beam from a first fiber (11) external to the substrate and a second input port configured for receiving a light beam from a second fiber (21) external to the substrate;

[0109] - a first optical circuit (25, 25', 25", 32, 32', 58, 59, 60) housed in the substrate and configured for generating by interference a first structured illumination pattern outside the substrate, the first pattern having a cartesian component orthogonal to the substrate;

[0110] - a second optical circuit (26, 26', 26", 33, 33', 61, 62, 63) housed in the substrate and configured for generating by interference a second structured illumination pattern outside the substrate, the second pattern having a cartesian component orthogonal to the substrate.

[0111] The first optical circuit (25, 25', 25", 32, 32', 58, 59, 60) comprises:

[0112] • three optical waveguides (25, 25', 25") configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides (25) being configured for being coupled to the first input port;

[0113] • one coupler (32, 32') for each couple of waveguides (58, 59, 60); more particularly, a first coupler (32) configured for splitting ,by means of evanescent coupling, the light beam between a first (25) and a second (25') waveguide and a second coupler (32') configured for splitting the light beam between the second (25') and a third (25") waveguide; and

[0114] • one planar mirror (58, 59, 60) and a collimating mirror, for each of the optical waveguides (58, 59, 60).

[0115] The second optical circuit (26, 26', 26", 33, 33', 61, 62, 63) comprises:

[0116] • three optical waveguides (26, 26', 26") configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides (26) being configured for being coupled to the second input port;

[0117] • one coupler (33, 33') for each couple of waveguides (26, 26', 26"); more particularly, a first coupler (31) configured for splitting ,by means of evanescent coupling, the light beam between a first (26) and a second (26') waveguide and a second coupler (33') configured for splitting the light beam between the second (26') and a third (26") waveguide; and

[0118] • one planar mirror (61, 62, 63) and a collimating mirror, for each of the optical waveguides (26, 26', 26").

[0119] The transparent planar substrate comprises a first face configured for the sample (52) being positioned thereon and a second face opposite to the first face, the planar mirrors (58, 59, 60, 61, 62, 63) being positioned on the first face and the corresponding collimating mirrors, being positioned on the second face. The mirrors (58, 59, 60, 61, 62, 63) are locally metallised.

[0120] The three waveguides (25, 25', 25") of the first optical circuit (25, 25', 25", 32, 32', 58, 59, 60) are configured in such a way that each one of the transmitted light beams (71, 72, 73) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate, said third projections having the same for all the transmitted light beams (71, 72, 73). The first and the second projections of the transmitted light beams (71, 72, 73) of the three waveguides (25, 25', 25") of the first optical circuit (25, 25', 25", 32, 32', 58, 59, 60) form an angle of 90°, with each other.

[0121] The planar mirrors (58, 59, 60) of the first optical circuit (25, 25', 25", 32, 32', 58, 59, 60) are configured for reflecting the transmitted light beams towards the collimating mirrors thus generating reflected light beams; the collimating mirrors are configured for reflecting the reflected light beams out of the substrate, thus generating final light beams not coplanar with the substrate; and the final light beam generated by the collimating mirror of one of the three waveguides (58) interferes with the final light beam generated by the collimating mirror of the other two waveguides (59, 60), thus generating the structured illumination pattern.

[0122] The three waveguides (26, 26', 26") of the second optical circuit (26, 26', 26", 33, 33', 61, 62, 63) are configured in such a way that each one of the transmitted light beams (83, 84, 85) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate, said third projections having the same for all the transmitted light beams (83, 84, 85). The first and the second projections of the transmitted light beams (83, 84, 85) of the three waveguides (26, 26', 26") of the second optical circuit (26, 26', 26", 33, 33', 61, 62, 63) form an angle of 60°, with each other.

[0123] The planar mirrors (61, 62, 63) of the second optical circuit (26, 26', 26", 33, 33', 61, 62, 63) are configured for reflecting the transmitted light beams towards the collimating mirrors thus generating reflected light beams; the collimating mirrors are configured for reflecting the reflected light beams out of the substrate, thus generating final light beams not coplanar with the substrate; and the final light beam generated by the collimating mirror of one of the three waveguides (61) interferes with the final light beam generated by the collimating mirror of the other two waveguides (62, 63), thus generating the structured illumination patterns.

[0124] In this way, according to the input port (11, 12) receiving a light beam from a fiber external to the substrate, it is possible to illuminate the sample with different pattern. If the light beam comes from the first input port (11), the generated pattern is determined by the interference of the three final light beams generated by the collimating mirrors of the three waveguides (25, 25, 25") of the first optical circuit (25, 25', 25", 32, 32', 58, 59, 60). Conversely, if the light beam comes from the second input port (12), the generated pattern is determined by the interference of the three final light beams generated by the collimating mirrors of the three waveguides (26, 26', 26") of the second optical circuit (26, 26', 26", 33, 33', 61, 62, 63).

[0125] Referring to Figures 15, 16, 17a, in a seventh embodiment of the microscope slide (200) of the present invention, each one of the three transmitted light beams (86, 87, 88) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate (30), and the third projection of at least one (87) of the transmitted light beams (86, 87, 88) has a magnitude different from that one of the third projection of the other transmitted light beams (86, 88). This allows for a modulation of the illuminating pattern also along the axis (z) orthogonal to the plane (xy) of the substrate, and not only in the plane (xy) of the substrate like in the previous embodiments. In this way, it becomes possible to acquire super-resolved images along all the three axes (x, y, z) and not only in the plane (xy) of the substrate. All the volume of the sample, is, thus, illuminated, and, by means of an appropriate translation of the slide, it is possible to acquire three-dimensional images of the sample. More particularly, a first one (87) of the three transmitted light beams (86, 87, 88) is parallel to axis (z) (i.e. orthogonal to the plane (xy) of the substrate), while the other two transmitted light beams (86, 88) have a component different from zero also in the direction (y) of the plane (xy). In this case, from the interference of the three final light beams derived from the collimation of said transmitted light beams (86, 87, 88), it is obtained a pattern that is modulated along the axis (y) and axis (z) but not along the axis (x).

[0126] Referring to Figures 15, 16, 17b, also in a eighth embodiment of the microscope slide (200) of the present invention, each one of the four transmitted light beams (89, 90, 91, 92) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate (30), and the third projection of at least one (92) of the transmitted light beams (89, 90, 91, 92) has a magnitude different from that one of the third projection of the other transmitted light beams (89, 90, 91). More particularly, three (89, 90, 91) of the four transmitted light beams (89, 90, 91, 92) are parallel to axis (z) (i.e. orthogonal to the plane (xy) of the substrate), while the other three transmitted light beams (89, 90, 91) have components along the axis (z) with the same magnitude, but components in the plane (xy) with different magnitude. In this case, from the interference of the four final light beams derived from the collimation of said transmitted light beams (89, 90, 91, 92), it is obtained a pattern that is modulated in all the volume (xyz).

[0127] Referring to the Figures 1, 2, 3a, 3b, 4, 7, 11, 13, 15 and 16, a ninth embodiment of the present invention, can have the same structure of whichever previous described embodiments, from the first to the eight, but comprises waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26") that are single mode waveguides for more than one wavelength. In the ninth embodiment of the present invention, the couplers (20, 20', 27, 27', 28, 29, 29', 29", 31, 31', 32, 32', 33, 33') are configured for having the same working mode for all the wavelengths. The mirrors (41, 41', 141, 142, 42, 42', 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 141, 142) are also configured for having the same working mode for all the wavelengths, and, more particularly, the chromatic dependencies of the collimating mirrors will be designed so to be reduced and so that their profile is optimized.

[0128] In this way, it is possible to illuminate the sample with a structured pattern with several wavelengths. To achieve such result, during the use of the slide, light beams of several wavelengths will be transmitted to the input ports through the fibers connected to the latter, and during the manufacturing process broadband optical components will be realized.

[0129] Referring to Figures 2, 3a, 16, the microscope slide (100, 200) according to any of the embodiment described above, can be configured for receiving a light source illuminating the sample (52) and for transmitting the pattern to an objective lens (50), the slide (100, 200) being interposed between the light source and the objective lens (50). More particularly, the sample (52) is interposed between the slide (100, 200) and a cover slip (51) that is, in its turn, positioned between the objective lens (50) and the slide (100, 200). In other words, the microscope slide (100) according to the present invention can work in the so-called transmission mode.

[0130] Referring to Figure 3b, the microscope slide (100) according to any of the embodiment described above, can be configured for receiving a light source illuminating the sample (52) and for transmitting the pattern to an objective lens (50), the light source being positioned at the same side with respect to the slide (100) in which the objective lens (50) is positioned. More particularly, the sample (52) is interposed between the slide (100, 200) and a cover slip (51) that is, in its turn, positioned at the opposite side with respect to the slide (100) in which the objective lens (50) is positioned. In other word the microscope slide (100) according to the present invention can work in the so-called reflection mode.

Claims

CLAIMS1. Microscope slide (100, 101, 102, 200, 300, 400) comprising:- a transparent planar substrate (10, 30) for holding a sample (52), the substrate (10, 30) having at least one input port configured for receiving a light beam from a fiber (11, 21, 31) external to the substrate (10, 30); at least one optical circuit (12, 13, 14, 15, 16, 17, 18, 19, 20, 20', 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26", 27, 27', 28, 29, 29', 29", 31, 31', 32, 32', 33, 33', 41, 41', 42, 42', 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 170, 171, 172, 173, 174, 175) housed in the substrate (10, 30); the at least one optical circuit (12, 13, 14, 15, 16, 17, 18, 19, 20, 20', 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26", 27, 27', 28, 29, 29', 29", 31, 31', 32, 32', 33, 33', 41, 41', 42, 42', 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 170, 171, 172, 173, 174, 175) being configured for being coupled with the input port and for transforming the light beam in a structured illumination pattern outside the substrate (10, 30), the pattern having a cartesian component orthogonal to the substrate (10, 30); wherein the at least one optical circuit (12, 13, 14, 15, 16, 17, 18, 19, 20, 20', 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26", 27, 27', 28, 29, 29', 29", 31, 31', 32, 32', 33, 33', 41, 41', 42, 42', 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 170, 171, 172, 173, 174, 175) comprises: at least a first and a second optical waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26") configured to transmit a light beam to their ends thus generating a transmitted light beam, at least one of said waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26") being configured for being coupled to the input port;- at least one coupler (20, 20', 27, 27', 28, 29, 29', 29", 31, 31', 32, 32', 33, 33') for each couple of the waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23',23" , 24, 24', 24", 25, 25', 25", 26, 26', 26"), said at least one coupler (20, 20', 27, 27', 28, 29, 29', 29", 31, 31', 32, 32', 33, 33') being configured for splitting the light beam between the waveguides of the couple (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26"); at least one phase shifter (170, 171,172, 173, 174, 175) for each couple of waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26"), said at least one phase shifter (170, 171,172, 173, 174, 175) being configured for creating a phase difference between the two waveguides of the couples (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26"); at least one planar mirror (41, 43, 44, 45, 46, 47, 48, 49, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 141, 142) and a collimating mirror (41', 42'), for each of the optical waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26"); wherein, for each of the optical waveguides, the at least one planar mirror (43, 44, 45, 46, 46, 48, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 141, 142) is configured for reflecting the transmitted light beam towards the collimating mirror (41', 42') thus generating a reflected light beam; the collimating mirror (41', 42') is configured for reflecting the reflected light beam out of the substrate (10, 30), thus generating a final light beam not coplanar with the substrate (10, 30); and wherein the final light beam generated by the collimating mirror (41', 42') of one of the two waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26") interferes with the final light beam generated by the collimating mirror (41', 42') of the other of the two waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26"), thus generating the structured illumination pattern.

2. Microscope slide (100, 101, 102, 200) according to claim 1 wherein the transparent planar substrate (10, 30) comprising a first face configured for the sample (52) being positioned thereon and a second face opposite to the first face, the at least a planar mirror (41, 42, 141, 142, 43, 44, 45, 46, 46, 48, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 141, 142) being positioned on the first face and the collimating mirror (41', 42'), being positioned on the second face.

3. Microscope slide (100, 101, 102, 200) according to claim 1 or 2, wherein the coupler (20, 20', 27, 27', 28, 29, 29', 29", 31, 31', 32, 32', 33, 33') is configured for splitting the light beam between the two waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26") by means of evanescent coupling.

4. Microscope slide (200) according to any claims from 1 to 3 wherein the waveguides (12, 13, 14, 17, 18, 19, 22) are configured in such a way that each one of the transmitted light beams (71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate (10), said third projections having the same magnitude for all the transmitted light beams (71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85).

5. Microscope slide (101) according to claim 4, wherein the two waveguides (15, 16) are configured in such a way that the first and the second projections of the transmitted light beams (74, 75) of the two waveguides (15, 16) form an angle of 180° with each other.

6. Microscope slide (100, 200) according to claim 4 comprising three waveguides (12, 13, 14) configured in such a way that the first and the second projections of the transmitted light beams (71, 72, 73) of the three waveguides (12, 13, 14) form an angle of 90°, with each other.

7. Microscope slide (100, 200) according to claim 4 comprising three waveguides (12, 13, 14) configured in such a way that the first and the second projections of the transmitted light beams (80, 81, 82) of the three waveguides (12, 13, 14) form an angle of 120°, with each other.

8. Microscope slide (102) according to claim 4 comprising four waveguides (17, 18,19, 22) configured in such a way that the first and the second projections of the transmitted light beams (76, 77, 78, 79) of the four waveguides (17, 18, 19, 22) form an angle of 90° with each other.

9. Microscope slide (200) according to claim 1 wherein the waveguides are configured in such a way that each one of the transmitted light beams (86, 87, 88, 89, 90, 91, 92) has a first and a second projection in the plane of (xy) of the substrate and a third projection in a direction orthogonal (z) to a plane (xy) parallel to the substrate (30), and the third projection of at least one (87, 92) of the transmitted light beams (86, 87, 88, 89, 90, 91, 92) has a magnitude different from that one of the third projection of the other transmitted light beams (86, 88, 89, 90, 91).

10. Microscope slide (100, 101, 102, 200) according to any of the preceding claims, comprising a first planar mirror (141), a second planar mirror (51) and a third planar mirror (142) for at each one of the waveguides (12, 13, 14, 15, 16, 17, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26"), the first planar mirror (141) being configured for reflecting the transmitted light beam towards the second planar mirror (51) thus generating a first reflected light beam; the second planar mirror (51) being configured for reflecting the first reflected light beam towards the third planar mirror (142) thus generating a second reflected light beam; the third planar mirror (142) being configured for reflecting the second reflected light beam towards the collimating mirror (42') thus generating a third reflected light beam and the collimating mirror (42') being configured for reflecting the third reflected light beam, thus generating the final light beam.

11. Microscope slide (100, 101, 102, 200) according to any of the claims from 4 to 10 wherein the waveguides (12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 23', 23", 24, 24', 24", 25, 25', 25", 26, 26', 26") are single mode waveguides for more than one wavelength, the couplers (20, 20', 27, 27', 28, 29, 29', 29", 31, 31', 32, 32', 33, 33') and the mirrors (41, 41', 42, 42', 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 141, 142)are configured for having the same working mode for all the wavelengths.

12. Microscope slide (100, 101, 102, 200, 300, 400) according to any of the preceding claims configured for receiving a light source illuminating the sample (52) and for transmitting the pattern to an objective lens (50), the slide (100, 101, 102, 103, 104, 105, 106, 200, 300, 400) being interposed between the light source and the objective lens (50).

13. Microscope slide (100, 101, 102, 200, 300, 400) according to any of the claims from 1 to 14 configured for receiving a light source illuminating the sample (52) and for transmitting the pattern to an objective lens (50), the light source being positioned at the same side with respect to the slide (100, 101, 102, 103, 104, 105, 106, 200, 300, 400) in which the objective lens (50) is positioned.

Images