DEVICE FOR CREATING A PATTERNED EVANESCENT FIELD ON A SURFACE AND METHOD THEREFOR

Abstract

The invention relates to a device (1) for creating a patterned evanescent field on the surface (S) of a dioptre comprising an objective lens (O), in an image focal plane of which the surface (S) of the dioptre is arranged, a light injection element (2) emitting a collimated light beam (B), an optical assembly (4) between the element (2) and the objective lens (O) by which the object plane of the objective lens (O) is optically conjugate with the image plane of the element (2), the assembly (4) being configured so that a collimated light beam (B) from the element (4) is emitted towards the objective lens (O) to be reflected towards the surface (S) of the dioptre with an angle of incidence greater than or equal to the critical angle of the dioptre, an optical device (FS1) for forming patterns is in the object plane of the element (2), so that the pattern formed by the optical device for forming patterns in transmitted light on the light beam in the object plane of the element (2) is found on the surface of the dioptre.

Claims

1. Device for creating a patterned evanescent field on the surface of a diopter separating two media of respective refractive indices n1 and n2, wherein the device comprises: an objective lens, the surface of the diopter being arranged in an image focal plane of the objective lens, a light injection element emitting a collimated light beam of diameter, an optical assembly between the light injection element and the objective lens whereby the object plane of the objective lens is optically conjugate with the image plane of the light injection element, the optical assembly being configured so that a collimated light beam from the light injection element is emitted towards the objective lens to be reflected towards the diopter surface with a minimum angle of incidence θmin greater than or equal to the critical angle θc so that the light beam undergoes total internal reflection on the diopter surface to generate an evanescent wave on the diopter surface, an optical device for forming patterns in the object plane of the light injection element, which is mounted off-axis with respect to the optical axis of the objective lens, in such a way that the pattern formed by the optical device for forming patterns in transmitted light on the light beam in the object plane of the light injection element is found on the surface of the diopter, the light beam reflected by the surface being reflected towards the objective lens to be focused in the back focal plane of the objective lens.

2. The device according to claim 1, further comprising at least one optical element between the object plane of the light injection element and the optical assembly allowing to adjust one of the improvement of the resolution of the formed pattern and the filtering of the angle of incidence of the light beam on the diopter.

3. The device according to claim 1, wherein the at least one optical element is one of a spatial diaphragm and a filter.

4. The device according to claim 1, wherein forming patterns is at least one of a diaphragm, an amplitude mask, a spatial light modulator, and a micro-mirror array.

5. The device according to claim 1, further comprising a laser light source for generating the collimated light beam to be injected into the light injection element.

6. The device according to claim 1, wherein the optical assembly is constituted by a first intermediate lens, the object plane of the first intermediate lens being optically conjugate with the image plane of the light injection element and the image plane of the first intermediate lens being optically conjugate with the object plane of the objective lens.

7. The device according to claim 6, wherein the first intermediate lens is optically conjugated with the light injection element by a second intermediate lens, the object plane of the second intermediate lens corresponding to the image plane of the light injection element and the image plane of the second intermediate lens corresponding to the object plane of the first intermediate lens.

8. The device according to claim 1, wherein the device is a total internal reflection fluorescence microscope, the sample corresponding to the diopter and being placed in the image plane of the microscope objective lens, the optical assembly corresponding to the microscope optics, the light injection element being arranged upstream of the microscope, the optical device for forming patterns being arranged between a light source and the light injection element.

9. Method of creating a patterned evanescent field on a surface using a device according to claim 1, wherein the method comprises: arranging the surface on the image plane of the objective lens; forming the desired transmitted light amplitude pattern in the object plane of the light injection element using the pattern forming device; injecting a collimated light beam into the light injection element.

10. The method according to claim 9, wherein the surface evanescent field is created on a biological sample in order to selectively excite by photoactivation, according to the created evanescent field, specific surface regions of the biological sample.

11. The device of claim 1, wherein the device is a total internal reflection fluorescence microscope, the sample corresponding to the diopter and being placed in the image plane of the microscope objective lens, the optical assembly corresponding to the microscope optics, the light injection element being integrated into the microscope optics, the optical device for forming patterns being integrated into the microscope optics

12. The device according to claim 2, wherein the at least one optical element is one of a spatial diaphragm and a filter.

13. The device according to claim 2, wherein forming patterns is at least one of a diaphragm, an amplitude mask, a spatial light modulator, and a micro-mirror array.

14. The device according to claim 3, wherein forming patterns is at least one of a diaphragm, an amplitude mask, a spatial light modulator, and a micro-mirror array.

15. The device according to claim 2, further comprising a laser light source for generating the collimated light beam to be injected into the light injection element.

16. The device according to claim 3, further comprising a laser light source for generating the collimated light beam to be injected into the light injection element.

17. The device according to claim 4, further comprising a laser light source for generating the collimated light beam to be injected into the light injection element.

18. The device according to claim 2, wherein the optical assembly is constituted by a first intermediate lens, the object plane of the first intermediate lens being optically conjugate with the image plane of the light injection element and the image plane of the first intermediate lens being optically conjugate with the object plane of the objective lens.

19. The device according to claim 3, wherein the optical assembly is constituted by a first intermediate lens, the object plane of the first intermediate lens being optically conjugate with the image plane of the light injection element and the image plane of the first intermediate lens being optically conjugate with the object plane of the objective lens.

20. The device according to claim 4, wherein the optical assembly is constituted by a first intermediate lens, the object plane of the first intermediate lens being optically conjugate with the image plane of the light injection element and the image plane of the first intermediate lens being optically conjugate with the object plane of the objective lens.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] To better illustrate the object of the present invention, particular embodiments are described below, by way of illustration and not limitation, with reference to the attached drawings.

[0043] On these drawings:

[0044] FIG. 1 is a schematic diagram of a device according to a first embodiment of the present invention;

[0045] FIG. 2 is a schematic diagram of a device according to a first variant of a second embodiment of the present invention; and

[0046] FIG. 3 is a schematic diagram of a device according to a second variant of the second embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Referring to FIG. 1, we can see that a device 1 for creating a patterned evanescent field on a surface S is shown.

[0048] The surface S is a diopter separating two media of different refractive indices n1 and n2.

[0049] In the non-limiting embodiment shown, a sample E has been placed on the surface S, the sample E here being a cell whose surface proteins are desired to be activated by evanescent wave formation on the surface S.

[0050] In this first embodiment of the device 1 according to the invention, a light injection element 2, constituted by a light source 3 generating a collimated, preferably coherent, light beam and a lens L1 of focal length f1, creates a collimated light beam B towards an optical assembly 4, constituted by a doublet of lenses L2 and L3, of respective focal lengths f2 and f3, the image plane of the lens L2 corresponding to the object plane of the lens L3. The image focal plane of the light injection element 2 corresponds to the object focal plane of the lens L2.

[0051] This beam of light B is directed at the output of the optical assembly 4 towards an objective lens O of focal length fO, the object focal plane of the objective lens O corresponding to the image focal plane of the lens L3.

[0052] The collimated light beam B is injected into the optical assembly 4 off-axis with respect to the optical axis of the optical assembly 4.

[0053] The surface S is placed in the image focal plane of the objective lens O.

[0054] Furthermore, the optical assembly 4 and the objective lens O are configured so that the light beam B emitted from the optical assembly 4 towards the objective lens O is incident on the surface S with an angle greater than the critical angle θc=sin.sup.−1(n1/n2) of the diopter constituted by the surface S in order to have a total internal reflection and to generate an evanescent wave in the sample E. The injection of the light beam B in an off-axis way with respect to the optical axis of the optical assembly 4 allows an incidence of the latter at the output of the optical assembly 4 on the peripheral part of the objective lens O. The light beam B reflected by the surface S is reflected to the objective lens O to be focused in the back focal plane BFP of the objective lens O as a divergent and non-parallel beam, the size of the Fourier image of the pattern, formed in the periphery of the back focal plane BFP, not being punctual, but limited by the thickness of the available supercritical margin. Thus, the light beam B from the optical assembly 4 is not incident on the objective lens O along its optical axis, but is incident on the objective lens O at its periphery, allowing reflection on the surface S at an angle greater than the critical angle θc. Thus what is projected onto the objective lens O and reflected on the surface S is directly the pattern we are trying to form, already formed in the light beam B.

[0055] Contrary to the previous state of the art, the pattern to be formed on the surface S is formed upstream of the objective lens, and does not result from an optical interference formed on the objective lens by one or more incident beams.

[0056] An element FS1 for forming patterns is placed in the object focal plane of the lens L1 and is used to form, from the light from the source 3, a collimated light beam B with a pattern formed therein which, via the optical assembly 4 and the objective lens, will be formed on the surface S in order to generate a patterned evanescent wave thereon.

[0057] The element FS1 for forming patterns may in particular be a field diaphragm, but could also be, without departing from the scope of the present invention, a diaphragm, an amplitude mask, a spatial light modulator, a micro-mirror array.

[0058] The aperture diaphragm AS in the image focal plane of the L1 lens serves to filter the light reflected from the edges of the field diaphragm FS1, to ensure that the light arrives on the surface S at an angle greater than or equal to the critical angle θc, so as not to produce far-field excitation. Even if the sharpness of the pattern edges in the plane of the sample E decreases with decreasing aperture of the aperture diaphragm AS, it was found experimentally that it was possible to form patterns for regions of dimensions larger than the wavelength of the light beam, including regions of dimensions 5-10 times the wavelength of the light beam B (regions of size 3-5 μm with excitation at 515 nm).

[0059] In this first embodiment, the optical assembly and objective lens may, for example, be integrated into an existing microscope. The invention then allows, from an existing microscope, to form a pattern on the surface S, without having to touch the optics of the microscope, from only the outside of the microscope. This embodiment avoids any occlusion of the optical path and therefore maintains the aperture, sensitivity and resolution of the microscope. This avoids any reduction in the detection channel. Referring to FIG. 2, it can be seen that a device 10 according to a first variant of a second embodiment of the invention has been shown.

[0060] In this first variant, the light source 13 emits a collimated beam of light towards the mirror M, the mirror M reflecting the beam towards a field diaphragm FS. In this variant, the light injection element is constituted by the mirror M. It should be noted that the light could also be collimated at the output of an optical fiber directly in the microscope stand, or introduced and collimated by any other suitable optical means.

[0061] Through a lens L′, of focal length f′, constituting the optical assembly 14, the light beam B coming from the field diaphragm FS is sent towards the objective lens O, always to arrive on the surface S with an angle of incidence O greater than the critical angle θc to generate a total internal reflection on the surface S and an evanescent wave therein, the beam reflected by the surface S towards the objective lens O being focused on the back focal plane BFP of the objective lens O. As in the first embodiment, the aperture diaphragm AS allows to filter the light reflected from the edges of the aperture diaphragm FS to allow only rays with an angle of incidence greater than the critical angle θc to pass. Also as in the first embodiment, the light beam is off-axis with respect to the optical axis of the objective lens O, to be incident on the objective lens O at its periphery and to be reflected by the surface S with an angle greater than or equal to the critical angle θc. This assembly could, for example, be integrated directly into new types of microscopes.

[0062] FIG. 3 is a variant of FIG. 2 of a device 20 according to a second variant of the second embodiment, and the elements bearing the same reference will not be described further. The difference lies in the means of forming a pattern upstream of the optical assembly 24, the pattern being formed here by reflection of collimated light from the light source 23 onto a digital micromirror DMD, said digital micromirror DMD reflecting the light to a device BS for forming patterns reflecting only a portion of the light received with a pattern to the digital micromirror DMD, which injects it into the lens L′ in the same manner as described with reference to FIG. 2. Also as in the first embodiment, the light beam is off-axis with respect to the optical axis of the objective lens O, to be incident on the objective lens O at the periphery thereof and to be reflected on the surface S at an angle greater than or equal to the critical angle θc. The invention can therefore be implemented with an existing microscope, for example in the case of the first embodiment, or be integrated into a microscope, as in the case of the second embodiment.

[0063] The invention can be applied to photolithography, surface photochemistry, microscopy and regiospecific photostimulation of nanoparticles, creation of nano-films and nano-objects between liquid phases forming the diopter, etching of barcodes, grids or other micrometric patterns on photosensitive surfaces, regiospecific evolutions of surface plasmon resonance technologies, development of regiospecific activation in evanescent wave sensors, regionalization of evanescent wave production, confinement and manipulation of light by photonic crystals, integration of photonic patterns on silicon chips, development of artificial composite materials to realize new optical functions, optoelectronics.

[0064] The present invention also has a particularly interesting application in biology, e.g. for the optogenetic addressing of subcellular perimembrane structures or their formation (e.g. focal adhesion, podosome, lamellipod, endo or exocytosis vesicle, cytoskeletal anchoring), for the fine local control of cell geometry, polarity and movement, for the local activation of plasma membrane receptors (EGFR, IGFR, . . . ) or transcription factors (Stat3, 5, . . . ) without modifying their nuclear reserve (essential for analyses by Fluorescence Correlation Spectroscopy (FCS) for example), for the quantification of the subcellular signaling rate between separated membrane structures, for the structuring of cellular substrates by photolithography processes with micrometric lateral and nanometric axial resolution.