Device for creating an evanescent field

Abstract

The invention relates to a device (1, 1A, 1B, 1C, 1D) for creating an evanescent field (3) at the surface of a dioptre (4) separating two media that have different refractive indices, the device comprising: - a light source (6A, 6B, 6C, 6D); - a lens (9A, 9B, 9C, 9D) comprising an object focal plane positioned at the surface of the dioptre, and a pupil plane (PP) comprising a subcritical region (R2) and a supercritical region (R1); - an active optical means (7A, 7B, 70, 7D) comprising an active surface (10A, 10B, 10C, 10D) extending at least through the entirety of the supercritical region (R1') of a conjugate plane of the pupil plane or extending through a conjugate plane of the object focal plane of the lens, the active optical means comprising a set of individually controllable microelements configured to illuminate the entire supercritical region of the lens.

Description

[0001] DESCRIPTION

[0002] Device for creating an evanescent field

[0003] Technical field of the invention

[0004] The invention relates to a device for creating an evanescent field on the surface of a diopter separating two media with different refractive indices. The invention also relates to a method of using such a device.

[0005] State of the prior art

[0006] Fluorescence microscopy is a technique using an optical device that takes advantage of the phenomenon of fluorescence, instead of, or in addition to, conventional observation by reflection or transmission of natural or artificial visible light. This technique allows the observation of various objects and substances (organic or inorganic), including living cells. This technique also allows the excitation of photosensitive molecules in biological objects. It is desirable to localize the excitation in all three spatial directions, so as to reach a very precise area of ​​the object to be observed. Such excitation is particularly sought after for the optogenetic activation of proteins likely to join the basal membrane of living cells.

[0007] There are several types of fluorescence microscopes, including total internal reflection fluorescence microscopes (commonly referred to as TIRFM). These microscopes are configured to illuminate the entire field of view of an objective with an evanescent exciting wave of homogeneous or periodic transverse structure. An evanescent wave, or in other words an evanescent field, is obtained by illuminating the supercritical region of the pupil plane of a high numerical aperture objective. This allows an exciting wave to be projected onto a plane diopter separating two media with different refractive indices with an angle of incidence greater than or equal to the value of the critical angle defined by these two refractive indices. The exciting wave thus undergoes total internal reflection and causes the appearance of an evanescent wave on the surface of the diopter.This evanescent wave interacts with the surface of the object to be observed over a very limited depth, for example of the order of a hundred nanometers. This type of microscope has three main variants.

[0008] The first approach, which is also the simplest and most common, consists of focusing an exciting laser beam into a single spot in the supercritical region of the pupil plane of the objective. This generates a homogeneous evanescent wave on the surface of the diopter. Document EP3969954A1 discloses, for example, a device for creating an evanescent field based on this approach. This simple configuration, however, has the disadvantage of anisotropic illumination in a direction predefined by the position of the single spot in the supercritical region, which is detrimental to the quality of the imaging.

[0009] A second approach consists of successively positioning a laser spot at different points in the supercritical region of the pupil plane of the objective and averaging the corresponding fluorescence images. This allows for better homogeneity of the diopter illumination. This second approach is generally referred to by the generic name "azimuthal beam spinning". To sequentially move the laser spot in the pupil plane, a galvanometric scanner system can be used. Alternatively, a micromirror array (commonly referred to by the Anglicism DMD) can be used, which is optically conjugated to the object focal plane of the objective so that it is uniformly illuminated. A third approach consists of modifying the classic TIRFM configuration by analogy with structured illumination microscopy (SIM).To do this, an incident laser beam is split into two so as to simultaneously generate, via a liquid crystal spatial light modulator (commonly referred to as SLM) or a micro-mirror matrix, two diametrically opposed spots in the pupil plane of the objective. The object focal plane remains illuminated over the entire field of view of the objective. The two corresponding evanescent waves interfere in the object focal plane, giving rise to a fringe system used as a carrier to access spatial frequencies of the sample twice as large as those resulting from the numerical aperture of the objective.

[0010] The total internal reflection fluorescence microscopes known from the state of the art all have drawbacks. In particular, they do not allow illumination to be obtained that is both isotropic and has a high spatial resolution in the transverse plane, i.e. in the plane of the diopter. Indeed, the evanescent wave generated by known devices illuminates an object to be observed over a surface of several tens to several hundred micrometers of lateral extension, which does not meet the need for precision of excitation in the transverse plane. From a practical point of view, the total internal reflection fluorescence microscopes known from the state of the art do not allow selective illumination of very well-defined areas of an object to be observed, such as a part of the basal membrane of a living cell.

[0011] Presentation of the invention

[0012] The aim of the invention is to provide a device for creating an evanescent field and a method of using such a device which overcomes the above drawbacks and improves the devices and methods known from the prior art.

[0013] More specifically, a first object of the invention is a device for creating an evanescent field making it possible to obtain a pattern of predetermined shape and position, uniformly illuminated, and with very high spatial resolution, in particular of the order of a micrometer.

[0014] Summary of the invention

[0015] The invention relates to a device for creating an evanescent field on the surface of a diopter separating two media of different refractive indices, the device comprising:

[0016] - a light source,

[0017] - an objective comprising an object focal plane positioned on the surface of the diopter, and a pupil plane comprising a subcritical region and a supercritical region,

[0018] - an active optical means configured to modulate the amplitude and / or phase of light rays emitted by the light source, the active optical means comprising an active surface extending at least in the entire supercritical region of a conjugate plane of the pupil plane or extending in a conjugate plane of the object focal plane of the objective, the active optical means comprising a set of individually controllable microelements configured to illuminate the entire supercritical region of the objective, and

[0019] - a means of controlling each microelement configured so as to produce a light pattern of predefined shape in the object focal plane of the lens.

[0020] According to one embodiment, the active optical means comprises a liquid crystal spatial light modulator or a micro-mirror matrix.

[0021] According to one embodiment, the light source is a coherent light source, in particular a laser light source, and the active surface extends at least throughout the supercritical region of a plane conjugate to the pupil plane.

[0022] According to one embodiment, the active optical means comprises a liquid crystal spatial light modulator, and the device comprises an optical mask extending throughout the subcritical region of a conjugate plane of the pupil plane.

[0023] According to one embodiment, the active optical means comprises an array of micro-mirrors, the array of micro-mirrors extending throughout the supercritical region and throughout the subcritical region of the conjugate plane of the pupil plane, each micro-mirror in the subcritical region of the conjugate plane of the pupil plane being controlled so as to deflect light rays out of the objective.

[0024] According to one embodiment, the active surface extends in a plane conjugated to the object focal plane, the control means being configured so as to reproduce said predefined shape pattern directly on the active surface of the active optical means, the device further comprising an optical mask extending throughout the subcritical region of a plane conjugated to the pupil plane.

[0025] According to one embodiment, the light source is an incoherent light source, in particular a light-emitting diode, the control means being configured so as to reproduce the entirety of said predefined shape pattern directly on the active surface of the active optical means. According to one embodiment, the light source is a coherent light source, in particular a laser, and the active optical means is controlled so as to modulate the phase and / or the amplitude of a wavefront reflected by the active surface by holography.

[0026] According to one embodiment, the device comprises at least one mask configured to mask a pattern of diffraction order 0.

[0027] The invention also relates to a method of operating a device as defined above, the method comprising:

[0028] - the placement of an object, in particular a living cell, to be observed on a transparent support, the object and the support having different refractive indices, then

[0029] - the definition of the shape and position of an area of ​​the object to be excited with an evanescent field, then

[0030] - the control of each microelement of the active optical means of the device so as to simultaneously illuminate the entire supercritical region of the objective, and so as to produce a light pattern in the object focal plane of the objective, the spatial resolution of said light pattern in the object focal plane being limited by diffraction, the shape and position of said light pattern corresponding to the predefined zone to be excited.

[0031] According to one embodiment, the light pattern is formed by scanning a light spot in the object focal plane of the lens, the entire supercritical region of the lens being illuminated at each instant.

[0032] Defining the shape and position of an area of ​​the object to be excited with an evanescent field may include:

[0033] - a preliminary observation of the object to be excited, then

[0034] - identification of the outline of an area of ​​the object to be excited based on prior observation.

[0035] The identification of the contour of an area of ​​the object to be excited can be carried out manually by means of a human-machine interface.

[0036] The operating method may comprise an iterative calculation, for example of the Gerchberg-Saxton type, of at least one configuration of the active surface making it possible to form said light pattern in the object focal plane of the objective.

[0037] The operating method may comprise the calculation of a first configuration and at least one second configuration of the active surface, each configuration of the active surface being calculated so as to obtain the same light pattern, the light patterns obtained with the first configuration and with the at least one second configuration comprising the same shape and the same position but a different brightness distribution, and in that each microelement of the active optical means is controlled successively according to the first configuration then according to the at least one second configuration.

[0038] Presentation of figures

[0039] These objects, characteristics and advantages of the present invention will be explained in detail in the following description of different particular embodiments in relation to the attached figures among which:

[0040] Figure 1 is a schematic sectional view of a living cell excited by an evanescent wave generated by a device for creating an evanescent field according to one embodiment of the invention.

[0041] Figure 2 is a schematic view of a device for creating an evanescent field according to a first embodiment of the invention. Figure 3 is a schematic sectional view of an active optical means of the device of Figure 2.

[0042] Figure 4 is a schematic front view of an active surface of the active optical means of Figure 3.

[0043] Figure 5 is a schematic view of the pupil plane of a lens of the device of Figure 2.

[0044] Figure 6 is a schematic view of a device for creating an evanescent field according to a second embodiment of the invention.

[0045] Figure 7 is an isometric view of two individually controllable microelements of a possible active optical means of the device of Figure 6.

[0046] Figure 8 is a schematic front view of an active surface of the active optical means of Figure 6.

[0047] Figure 9 is a schematic view of the pupil plane of a lens of the device of Figure 6.

[0048] Figure 10 is a schematic view of a device for creating an evanescent field according to a third embodiment of the invention.

[0049] Figure 11 is a schematic view of a device for creating an evanescent field according to a fourth embodiment of the invention.

[0050] Detailed description

[0051] Figure 1 schematically illustrates a living cell C whose basal membrane is intended to be excited by an evanescent wave generated by a device for creating an evanescent field 1 according to one embodiment of the invention. The cell C comprises in particular a nucleus N and various proteins P1, P2. The cell rests on a transparent support 2 extending parallel to a plane, called the transverse plane, defined by two axes X and Y. The support 2 can be, for example, a glass slide. The Z axis is defined as the axis perpendicular to the plane in which the support 2 extends. The Z axis is parallel to the vertical axis. The transverse plane is a horizontal plane. Among the proteins of the cell C, a group of proteins P1 is located in a well-defined zone of a basal membrane M of the cell C in contact with the support 2.As we will explain, the device for creating an evanescent field 1, more simply called device 1 hereinafter, is intended to generate an evanescent field 3, or in other words an evanescent wave, capable of exciting only the group of proteins P1, without exciting the other proteins P2 of the cell. The evanescent field 3 generated by the device 1 has a very high resolution in the three dimensions of space: as an evanescent field, it interacts with the cell C over a very small depth along the Z axis. In addition, the evanescent field 3 also has a very small spread in the transverse plane. The evanescent field can excite an area of ​​the cell having a diameter D in the transverse plane less than or equal to 1 pm, or even less than or equal to 500 nm, or even less than or equal to 200 nm.

[0052] The support 2 comprises a first refractive index n1 and the object to be observed, in this case the cell C, comprises a second refractive index n2 different from the first refractive index n1. The first refractive index n1 can be substantially equal to the refractive index of glass and the second refractive index n2 can be substantially equal to the refractive index of water. A diopter 4, extending in a plane, can be defined as the surface separating the support 2 from the cell C and its medium. The diopter 4 therefore extends in a plane parallel to the X and Y axes. A critical angle A1 can be defined by the formula: A1 = arcsin (n1 / n2). When the diopter 4 is illuminated by an incident field 5 having an angle of incidence A2 greater than or equal to the critical angle A1, a phenomenon of total reflection occurs: this propagating field is entirely reflected and an evanescent field 3 is produced.The evanescent field 3 interacts with the surface of the cell C over a very limited depth, for example of the order of a hundred nanometers.

[0053] The device 1 can be used to observe and / or excite a cell, and more generally any type of object. In the remainder of the description, this object is designated by the term "object to be observed" although it may also, or even exclusively, be intended to be excited by the evanescent field 3.

[0054] Such an evanescent field 3 is therefore obtained by means of a device 1 according to the invention. Several embodiments of the device 1 are envisaged. In particular, four embodiments are illustrated respectively in Figures 2, 6, 10 and 11 and will be described successively. In order to facilitate reading, the suffixes A, B, C and D are used respectively to describe identical or similar elements belonging respectively to the first embodiment, the second embodiment, the third embodiment, and the fourth embodiment. The characteristics of an element or its arrangement described in the context of a particular embodiment may be transposed to the other embodiments. To simplify the description, we will mainly focus on describing the differences of the second, third and fourth embodiments without repeating their common characteristics.

[0055] Figure 2 therefore illustrates a device for creating an evanescent field 1A according to a first embodiment of the invention. The device 1A comprises a light source 6A, an active optical means 7A, a means 8A for controlling the active optical means 7A, and an objective 9A.

[0056] According to this first embodiment, the light source 6A is a coherent light source such as a laser light source. The temporal coherence length of the light source is preferably at least a few centimeters. The light source 6A is arranged to illuminate the active optical means 7A. The active optical means 7A is configured to reflect light rays from the light source 6A towards the objective 9A.

[0057] The active optical means 7A is configured to modulate the phase of light rays emitted by the light source 6A. For this purpose, the active optical means 7A comprises a set of individually controllable microelements 15A. Each microelement 15A forms a pixel of an image projected by the active optical means 7A. These microelements 15A extend along an active surface 10A of the active optical means 7A.

[0058] More specifically, the active optical means 7A is preferably a liquid crystal spatial light modulator. A spatial light modulator is an electronically programmable device that can modulate a light output based on a specific fixed spatial pattern, essentially projecting light that is controlled, either only in amplitude or only in phase. Such an active optical means notably comprises a liquid crystal display. Each microelement 15A of the spatial light modulator corresponds to an area of ​​the spatial light modulator whose liquid crystals can be specifically oriented, depending on an electric field applied to this area. In this first embodiment, the spatial light modulator is configured to modulate the phase of a light beam at each microelement.

[0059] Figure 3 schematically illustrates the operation of the spatial light modulator. On the left part of Figure 3, an incident wave 01 is shown in the direction of the active surface 10A. This wave 01 is flat and incident in a direction parallel to an optical axis ZO. The phase is constant at all points of the wavefront. On the right part of Figure 3, a reflected wave 02 is shown by the active surface 10A. The reflected wavefront 02 is no longer flat but, on the contrary, is locally deformed as a function of the configuration of the microelement 15A on which the wave 01 is reflected, that is to say as a function of the local orientation of the liquid crystals.

[0060] The objective 9A is an optical element intended to focus incident light rays onto the surface of the diopter 4. The objective 9A is preferably a pre-assembled and non-modifiable component. The objective may be an oil immersion objective so that its refractive index n is as close as possible to the refractive index n1 of the support 2, which is preferably made of glass.

[0061] The diopter 4 extends in a plane coincident with an object focal plane of the lens 9A. In other words, the object focal plane of the lens 9A is positioned on the surface of the diopter 4.

[0062] The 9A lens comprises optical components capable of focusing light rays in the object focal plane of the lens with an angle of incidence greater than or equal to the critical angle A1.

[0063] The optical axis ZO can be defined as the axis parallel to the Z axis passing through a center of the objective 9A. The active surface 10A of the active optical means 7A can be oriented so that the light rays it returns are generally oriented parallel to the optical axis ZO.

[0064] Optical components such as optical lenses may be interposed between the active optical means 7A and the objective 9A. In this case, the device 1A comprises a first lens 11A and a second lens 12A interposed between the active optical means 7A and the objective 9A. The lenses 11A and 12A may be converging lenses. They may also extend perpendicular to the optical axis Z0 and be centered on the optical axis Z0. The first lens 11A and the second lens 12A have focal lengths f1 and f2 respectively. The active surface 10A extends in the object focal plane of the first lens 11A. An image focal plane PF1 of the first lens 11A coincides with an object focal plane of the second lens 12A. An image focal plane of the second lens 12A coincides with a pupil plane PP of the objective 9A.

[0065] The pupil plane PP of the lens is the same as the Fourier plane of the lens. Thus, this plane can be indistinctly called the pupil plane or the Fourier plane.

[0066] As a side note, the pupil (meaning entrance pupil) of a microscope objective is the opening at the rear of the objective, located on the side opposite the sample. By definition, this pupil is the image of the aperture diaphragm (through the optics located behind it), the most blocking with respect to the inclination of the light rays received by the objective (on the side of the object to be observed).

[0067] The diameter D of the entrance pupil is given by the following formula:

[0068] D = 2 x ON x FIM where:

[0069] - ON is the numerical aperture of the objective.

[0070] - FIM is its image focal length.

[0071] The numerical aperture ON can be given by the following formula:

[0072] ON = nx sin(0), where:

[0073] - n is the refractive index of the objective immersion medium

[0074] - 0 is the angle of the most inclined ray accepted by the aperture diaphragm.

[0075] The refractive index n2 of the object to be observed is less than the numerical aperture ON. Thus, there is a subcritical region in the central part of the entrance pupil of the objective which corresponds to rays propagating into the object to be observed. Conversely, rays passing into the peripheral zone of the entrance pupil, called the supercritical region, cannot propagate into the object to be observed.

[0076] Microscope objectives today are designed so that the entrance pupil is located in the rear focal plane of the objective, i.e., in the Fourier plane of the objective. Furthermore, these objectives are called "infinity" or "infinity-corrected" because they give an image at infinity of the object, so that a real image is produced when they are combined with a so-called tube lens. In the description of the present invention, reference is only made to an infinity objective. The tube lens is not involved anywhere as such.

[0077] The object focal plane of the first lens 11A, which coincides with the plane in which the active surface 10A extends, is a conjugate plane of the pupil plane PP. In other words, any point of the pupil plane PP of the objective 9A has an image in the plane in which the active surface 10A extends.

[0078] Alternatively, the optical device could be more complex and include, for example, more lenses and / or reflection means such as mirrors. In more complex versions of the device, several conjugate planes of the pupil plane could be defined.

[0079] As explained previously, the pupil plane PP of the objective 9A can be separated into two complementary regions: a supercritical region R1 and a subcritical region R2. The supercritical region R1 corresponds to the part of the pupil plane PP in which incident rays are focused by the objective 9A with an angle of incidence greater than or equal to the critical angle A1. The subcritical region R2 corresponds to the part of the pupil plane PP in which incident rays are focused by the objective 9A with an angle of incidence strictly less than the critical angle A1.

[0080] The subcritical region R2 has the shape of a disc centered on the optical axis Z0. The supercritical region R1 has an annular shape extending around the subcritical region R2. It is therefore understood that the precise dimensions of the subcritical region R2 and the supercritical region R1 depend on the refractive indices of the support 2 and the object to be observed as well as the focal length FIM of the objective and its numerical aperture ON.

[0081] Since the active surface 10A extends in a plane conjugate with the pupil plane, a supercritical region R1' and a subcritical region R2' can also be defined in this conjugate plane. The supercritical region R1' and the subcritical region R2' may have homothetic shapes respectively to the supercritical region R1 and to the subcritical region R2. The active surface 10A extends at least in the entire supercritical region R1' of the conjugate plane of the pupil plane PP.

[0082] The active surface 10A may also extend into the subcritical region R2'. However, in order to obtain an evanescent wave at the surface of the diopter 4, it is necessary that no light ray illuminates the diopter 4 with an angle of incidence strictly less than the critical angle A1. This is why the device 1A is configured to emit no light ray in the subcritical region R2 of the pupil plane PP. To do this, the active optical means 7A may be controlled so that the light rays coming from the light source 6A incident at the subcritical region R2' are reflected out of an optical path directed towards the objective 9A. Alternatively, it is also possible to provide an optical mask 13A extending throughout the subcritical region of a conjugate plane of the pupil plane.The mask 13A may extend in the plane in which the active surface 10A extends, or in any other plane conjugate to the pupil plane, or even directly in the pupil plane PP. However, the integration of the mask 13A directly in the pupil plane PP may be difficult to achieve and would require an optomechanical modification of the lens. In any case, the mask 13A prevents any light ray from reaching the subcritical region R2 of the pupil plane, and therefore the formation of a propagating wave on the surface of the diopter 4.

[0083] According to the invention, to obtain a pattern of predefined shape on the surface of the diopter 4, the active optical means 7A is controlled so as to illuminate the entire supercritical zone R1 of the objective. Thus, the entire ring forming the supercritical zone R1 receives light rays, and not only a point or an area of ​​this ring. It is therefore understood that the device 1A is configured to illuminate the entire supercritical region R1 of the objective, and not only a part of this supercritical region. No element of the device 1A can prevent the illumination of the entire supercritical region R1 of the objective.

[0084] The control means 8A is configured to individually control each microelement of the active optical means 7A which extends into the supercritical region RT. The control means 8A is thus configured to modify the phase of a wavefront in the entire supercritical region RT.

[0085] The control means 8A may in particular comprise a computer provided with a human-machine interface. The control means 8A is programmed so as to produce a light pattern ML of predefined shape in the object focal plane of the objective, that is to say on the surface of the diopter 4. Conventionally, the computer may comprise display means such as a screen and control means such as a keyboard and a computer mouse. The display means are intended to display the object studied with the device 1A, in particular said cell C. This allows a user to identify a particular area of ​​interest on this object. The control means are intended to define the desired light pattern ML.

[0086] For example, if the user seeks to excite the P1 protein group of cell C, he can manually draw, with said control means (for example with a computer mouse), a geometric shape which surrounds the P1 protein group of cell C. This geometric shape therefore has a shape and a position which depend on a prior observation of the object to be observed. Alternatively, the shape and the position of said geometric shape could be determined automatically, in particular thanks to automatic processing of an image resulting from a prior observation of said object.

[0087] The control means 8A comprises a memory in which the shape and position of the light pattern ML previously specified by the user are stored. The control means 8A also comprises a computer program in which a transfer function is coded for calculating an activation state of each microelement of the active optical means as a function of the shape and position of the desired light pattern ML.

[0088] Each 15A microelement is a small-scale structure that radiates light waves. The spatial resolution of the ML light pattern in the object focal plane of the objective is limited by diffraction due to the numerical aperture of the objective. Thus, the incident wave at the pupil plane PP has: i) a controlled phase at any point in the supercritical region R1 of the entrance pupil of the objective, and ii) a uniformly zero amplitude throughout the subcritical region R2 of the entrance pupil of the objective.

[0089] The phase of the wavefront at any point in the supercritical region R1 of the pupil plane PP is optimized to produce the desired intensity pattern in the object focal plane of the objective 9A.

[0090] The shape of the ML light pattern can be freely chosen. For example, a light pattern of circular, ovoid, elliptical, polygonal, in particular triangular, square, rectangular shape or any other desirable geometric shape can be obtained. Advantageously, the shape of the light pattern is defined according to an observation or a prior deduction of the position of photosensitive zones of the observed object. In Figure 2, the ML light pattern has an arbitrary shape which can therefore be freely adapted according to requirements. As a note, the mask 13A previously described makes it possible to filter the low frequencies of the wavefront in the Fourier space. The presence of this mask does not prevent the formation of the ML light pattern, but it may possibly alter the lighting homogeneity of this light pattern.

[0091] As illustrated in Figure 2, this light pattern could be observed by positioning a screen in the image focal plane PF1. Of course, in practice, the device 1A does not include such a screen since it would block or limit the passage of light rays. The image focal plane PF1 corresponds to a Fourier plane of the pupil plane PP. In other words, the pattern projected onto the pupil plane PP corresponds to a Fourier transform of the light pattern ML. Figure 4 schematically illustrates the active surface 10A for producing the light pattern ML as shown in Figure 2. Each microelement of the active surface is represented with a certain gray level depending on the local phase shift it induces. By convention, the white color indicates a zero phase shift and the black color indicates a phase shift equal to 2TT. Macroscopically, the active surface 10A appears gray.However, the phase shift produced by the active surface 10A is not at all uniform. Indeed, if we observe the active surface 10A at the microscopic scale, as shown on the right part of Figure 4, we see that each microelement 15A is in a particular configuration, producing a phase shift between 0 and 2TT. The phase pattern thus generated on the active surface 10A corresponds to the complex part of the Fourier transform of the desired pattern in the object focal plane of the objective.

[0092] Figure 5 schematically illustrates the pupil plane PP of the objective. The supercritical zone R1 of the pupil plane PP is entirely illuminated by a pattern substantially identical to the pattern generated on the active surface 10A. Similarly, the pattern formed on the pupil plane PP of the objective corresponds to a Fourier transform of the desired pattern in the object focal plane of the objective.

[0093] According to an alternative embodiment, it is possible to add, by means of the control means 8A, a linear phase gradient in the pupil plane PP, so as to move the position, in the object focal plane of the objective, of a super-resolved laser spot which reproduces by successive scanning the desired pattern. In other words, the active optical means 7A can be controlled so as to produce at each instant, in the object focal plane of the objective, not a particular geometric shape or pattern, but a single light spot whose size is limited by diffraction. Then the desired geometric shape is scanned with this light spot. To obtain a light spot, the active optical means is controlled in a suitable manner. If the wavefront 02 is perpendicular to the optical axis ZO, a light spot centered on the optical axis ZO is obtained. If the wavefront 02 is inclined relative to the optical axis ZO, a light spot offset relative to the optical axis ZO is obtained.In all cases, the entire supercritical zone R1 of the pupil plane of the objective is illuminated, even to obtain a bright spot.

[0094] Advantageously, the device 1 A may further comprise a means for polarizing the incident wave in the pupil plane PP of the objective. Indeed, the large numerical aperture of the objective generates very inclined light rays whose amplitudes may depend on the angle and plane of incidence. The most suitable polarization state and allowing the best spatial resolution is radial polarization. Radial polarization can be obtained, from linear polarization, using polarization plates, ideally placed close to the pupil plane of the objective.

[0095] To determine the phase shift of each microelement of the active surface 10A, an iterative calculation is carried out, for example of the Gerchberg-Saxton type, which, of course, depends on the shape and position of the predefined ML light pattern, but also on an initial condition. This initial condition may correspond to an initial phase condition in the image focal plane PF1. The phase shift of each microelement of the active surface 10A thus depends on an initial condition. For the same ML light pattern, there are several possible configurations of each microelement of the active surface 10A. The ML light patterns obtained with these different configurations comprise the same shape and the same position. However, the different light patterns all have a certain luminous inhomogeneity, that is to say that certain portions of each light pattern are more or less illuminated.The different light patterns are distinguished from each other by a different brightness distribution within the ML light pattern itself. To obtain an ML light pattern of predefined shape and with homogeneous brightness, it is advantageous to calculate for the same ML light pattern several configurations of the active surface 10A with different initial conditions of the iterative calculation. Then, the active optical means 7A can be controlled so that it successively and cyclically produces the different configurations of the active surface 10A. The greater the number of different configurations, the better the luminous homogeneity of the light pattern.

[0096] Figure 6 illustrates a device for creating an evanescent field 1 B according to a second embodiment of the invention. As for the first embodiment, the device 1 B comprises a light source 6B, an active optical means 7B, a means 8B for controlling the active optical means 7B, and an objective 9B. The active optical means 7B comprises a set of individually controllable microelements 15B. The device 1 B also comprises a first lens 11 B and a second lens 12B interposed between the active optical means 7B and the objective 9B. The light source 6B, the objective 9B, the first lens 11 B and the second lens 12B may have the same characteristics and the same arrangements as respectively the light source 6A, the objective 9A, the first lens 11A and the second lens 12B described previously.As for the first embodiment, the active optical means 7B comprises an active surface 10B which extends in a plane conjugate to the pupil plane PP, and which can be decomposed into a supercritical region R1' and a subcritical region R2'. The device 1B is also configured to illuminate the entire supercritical region R1 of the objective, and not only a part of this supercritical region. This second embodiment is distinguished from the first embodiment by its active optical means 7B and the associated control means 8B. Indeed, the active optical means 7B and the associated control means 8B are configured to produce a light pattern in the object focal plane by holography. More precisely, the active optical means 7B is used to encode a wavefront using a high-frequency carrier. A carrier is a periodic pattern of high spatial frequency applied to the microelements 15B.This periodic pattern typically consists of a series of parallel lines. This periodic pattern is illustrated in particular in Figure 8.

[0097] According to a first variant of the second embodiment, the high-frequency carrier may be a network of fringes of variable intensity, in other words of variable amplitude. Each microelement 15B of the active surface 10B is then configured either in the "ON" position so as to re-emit light rays capable of reaching the pupil plane of the objective of the same intensity as incident light rays, or in the "OFF" position so as not to re-emit any light rays capable of reaching the pupil plane of the objective. The position of each microelement of the active surface 10B may be obtained by an iterative calculation analogous to the iterative calculation described for the first embodiment.

[0098] The active optical means 7B is then preferably a matrix of micromirrors. Alternatively, the active optical means 7B could also be a spatial light modulator adapted to modulate the amplitude or phase of the incident wave.

[0099] A micro-mirror array is a micro-electromechanical system for projecting a digital image by reflection on micro-mirrors that can be switched to two different positions: in the "ON" position, a micro-mirror is oriented so that the light rays it reflects reach the lens 9B. In the "OFF" position, a micro-mirror is oriented so that the light rays it reflects do not reach the lens 9B. A micro-mirror array offers low cost and high display speed. Figure 7 illustrates, as an example, two micro-elements 15B of a micro-mirror array. One micro-element is in the "ON" position and the other micro-element is in the "OFF" position.

[0100] As illustrated in FIG. 8, the control means 8B is configured to individually control each micro-mirror such that the micro-mirrors of the supercritical region R1' are either in the OFF position or in the ON position, and all the positioned micro-mirrors of the subcritical region R2' are in the OFF position. The use of a micro-mirror array also makes it possible to dispense with the use of an optical mask extending over a conjugate plane of the subcritical region. Thus, the micro-mirror array can be used to control the amplitude of the incident wavefront in the pupil plane PP of the objective.

[0101] A microscopic observation of the supercritical region R1' of the active surface 10B makes it possible to visualize the network of fringes of variable intensity, which results in an arrangement of the micro-mirrors in the ON and OFF position substantially along parallel lines. In general, the control means 8B can encode on a carrier the phase and / or the amplitude of the wavefront which must be generated in the pupil plane of the objective, so as to obtain the desired light pattern ML in the object focal plane of the objective 9B.

[0102] In the case where only the phase of the supercritical region is controlled, this can be encoded through the fringe positions of a lattice displayed on the micromirror matrix following the Lee method, given by the following equation: Or :

[0103] - x and y denote the coordinates of each micro-mirror along the X and Y axes,

[0104] - c|)(x,y) denotes the phase sought, and

[0105] - p denotes the period of the network (the carrier frequency is therefore given by 1 / p).

[0106] Due to the diffraction generated by a fringe grating, a series of patterns with different diffraction orders are observed in the image focal plane PF1 of the first lens 11 B. Thus, by allowing only light coming from order 1 of the diffraction grating to pass through the image focal plane PF1, a wavefront is obtained in the pupil plane whose phase corresponds to <|)(x,y). Advantageously, a mask 14B is provided extending in the image focal plane PF1 to mask light coming from orders other than order 1, i.e. orders 0, -1, +2, -2 etc. The wavefront in the pupil plane PP is thus freed from its high-frequency component. In Figure 6, the light pattern of order 0 is indicated by the reference MLO and the light pattern of order 1 is indicated by ML1. Although represented as a transparent mask in Figure 6 (so as to visualize the light pattern MLO), the mask 14B is indeed an opaque mask.

[0107] The wavefront in the pupil plane PP is illustrated in Figure 9. The supercritical region R1 of the pupil plane PP is fully illuminated by a pattern corresponding to a Fourier transform of the desired pattern in the object focal plane. The spatial resolution of the intensity patterns obtained in the object focal plane of the lens depends on the optimization of the phase, and possibly also the amplitude, in the supercritical region of the pupil plane and its encoding via the carrier displayed on the micromirror array. The extension of the field of view (i.e., the region of the object focal plane of the lens where the intensity can be controlled), also depends on the encoding via the carrier displayed on the micromirror array.

[0108] A variant of Lee's holography also allows the amplitude of the wave to be encoded by varying the width of the fringes. It should be noted that since the pixels of a given period are used to control the amplitude in addition to the phase, the number of achievable phase levels is more limited.

[0109] The technique presented above makes it possible to form, with a single illumination, intensity patterns in the object focal plane of the lens. However, it is also possible to reconstruct patterns by scanning an evanescent spot, the position of which is then determined by the period and orientation of the carrier grating. Since the micro-mirror array can have a refresh rate of around 10 kHz, the scanning method makes it possible to reconstruct patterns in less than a second. The phase 4>(x,y) with constant gradient in the pupil plane can also be the result of a tip-tilt of the incident laser beam achieved using galvanometric mirrors or acousto-optic crystals, placed in a plane optically conjugate to the pupil plane (4f type assembly).

[0110] The deformations of the surface of the micromirror array, as well as the propagation of the light wave in the entire device 1 B are important sources of optical aberrations that can degrade the spatial resolution of the ML light pattern. It is possible to compensate for these aberrations by adaptively modifying the phase of the wavefront encoded on the micromirror array with the Lee method mentioned above.

[0111] According to a second variant of the second embodiment, the high-frequency carrier could be a variable phase fringe array instead of a variable intensity fringe array. In this case, the active optical means is then preferably a spatial light modulator. The ON and OFF positions of each microelement then correspond respectively to a zero phase shift and a phase shift equal to TT.

[0112] Figure 10 illustrates a device for creating an evanescent field 1 C according to a third embodiment of the invention. As previously, the device 1 C comprises a light source 6C, an active optical means 7C, a means 8C for controlling the active optical means 7C, and an objective 9C. The device 1 C is also configured to illuminate the entire supercritical region R1 of the objective, and not only a part of this supercritical region. The specificities of these elements will be described below. The device 1 C further comprises a first lens 11 C, a second lens 12C and a third lens 13C interposed between the active optical means 7C and the objective 9C.

[0113] The lenses 11C, 12C and 13C may be converging lenses. They may extend perpendicular to the optical axis Z0 and be centered on the optical axis Z0. The first lens 11C, the second lens 12C and the second lens 13C respectively comprise a focal length f1, f2 and f3. An active surface 10C of the active optical means 7C extends in the object focal plane of the first lens 11C. An image focal plane PF1 of the first lens 11C coincides with an object focal plane of the second lens 12C. An image focal plane PF2 of the second lens 12C coincides with an object focal plane of the third lens 13C. An image focal plane of the third lens 13C coincides with a pupil plane PP of the objective 9A.The various lenses 11C, 12C, and 13C and the objective 9C are arranged so that the active surface 10C extends in a conjugate plane of the object focal plane, that is to say that any point of the object focal plane of the objective 9C has an image in the plane of the active surface 10C. Alternatively, the optical device could be more complex and include in particular more lenses and / or reflection means such as mirrors.

[0114] According to this embodiment, the light pattern ML is directly reproduced by the active optical means 7C on the active surface 10C. A mask 14C is then provided extending into the subcritical region R2' of a plane conjugate to the pupil plane, so as to prevent light rays from reaching the subcritical region R2 of the pupil plane. As previously, the entire supercritical region R1 of the pupil plane is illuminated.

[0115] According to this third embodiment, the light source 6C may be an incoherent light source. The light source 6C may, for example, comprise a light-emitting diode, which simplifies the design of the device 1C. The active optical means 7C may comprise a liquid crystal spatial light modulator or a micro-mirror array. The active optical means 7C is configured to control the amplitude of the incident illumination. The pattern is reproduced with a set of micro-elements in the ON position, while the other micro-elements of the active optical means 7C are in the OFF position.

[0116] According to this embodiment, the active optical means 7C is easier to produce than in the first and second embodiments previously described because the microelements are activated according to the shape and position of the desired light pattern ML. It is therefore not useful to resort to iterative Fourier transform calculations to determine which microelements must be activated.

[0117] As a note, when using a monochromatic and spatially coherent incident wave, the transfer function of the imaging system is given by the supercritical region of the entrance pupil of the objective. It therefore excludes the central, low-frequency part of the Fourier plane, which makes it a priori difficult to reproduce illumination patterns comprising homogeneous regions, except by using phase control by holography. Conversely, the production of a central spot is achievable directly, which can also be scanned by modifying the parameters of the fringe network as seen previously.

[0118] Conversely, if the light source is spatially incoherent, the transfer function of the imaging system is given by the autocorrelation of the supercritical region of the entrance pupil of the objective. This corresponds to a more homogeneous distribution of the transmitted frequencies and contains, in addition to the high frequencies, the central, low-frequency part of the Fourier plane. This makes it possible to reproduce illumination patterns comprising homogeneous regions limited by very narrow boundaries.

[0119] Figure 11 illustrates a device for creating a 1 D evanescent field according to a fourth embodiment of the invention. The device 1 D comprises a light source 6D, an active optical means 7D, a means 8D for controlling the active optical means 7D, an objective 9D, a first lens 11 D, a second lens 12D and a third lens 13D interposed between the active optical means 7D and the objective 9D. The device 1 D is also configured to illuminate the entire supercritical region R1 of the objective, and not only a part of this supercritical region. The characteristics and arrangements of these elements may be identical to those of the device 1 C according to the third embodiment.

[0120] The device 1D is distinguished from the device 1C on the one hand by the nature of the light source 6D which is now a coherent light source. On the other hand, the active optical means 7C and the associated control means 8C are configured to produce a pattern in the object focal plane by holography. The active optical means 7D can be configured to modulate the phase and / or the amplitude of the incident wavefront in the pupil plane PP of the objective by holography. By analogy with the second embodiment, the active optical means 7D can comprise a spatial light modulator or a micro-mirror matrix. The active optical means 7C is used to encode a wavefront using a high-frequency carrier. The high-frequency carrier can be a fringe array of variable intensity and / or a fringe array of variable phase.The position of each microelement of the 10D active surface can be obtained by an iterative calculation analogous to the iterative calculation described for the first embodiment.

[0121] The contour of the desired ML light pattern is directly reproduced on the 10D active surface. The microelements positioned inside this contour are controlled to produce a network of fringes.

[0122] Furthermore, the device 1 D may comprise a first mask 14D1 extending into the subcritical region R2' of a plane conjugate to the pupil plane, so as to prevent light rays from reaching the subcritical region R2 of the pupil plane. The device 1 D must necessarily comprise a second mask 14D2 to mask the light coming from the order 0 of the diffraction grating. Although represented as a transparent mask in Figure 11 (so as to visualize the MLO light pattern), the mask 14D2 is indeed an opaque mask. Finally, to use any of the devices 1A, 1 B, 1C, 1 D previously described, one can proceed as follows. First of all, one places an object to be observed on a transparent support, the object and the support having different refractive indices, that of the object being lower than that of the support. For example, one positions a living cell on a glass slide.

[0123] Next, the shape and position of an area of ​​the object to be excited with an evanescent field are defined. This definition may result from prior observation of the object and / or calculations and / or hypotheses relating to the object. For example, in the case of a cell, a portion of the cell's basal membrane containing proteins of a desired type can be defined.

[0124] Once this area has been defined, each micro-element of the active optical means of the device is controlled so as to produce a light pattern in the object focal plane of the lens that corresponds to the predefined shape and position. For this purpose, the control means of the device calculates, if necessary with the support of a predefined transfer function and / or iteratively, how each of the micro-elements is to be controlled. The active control means directs the light rays from the light source so as to illuminate the entire supercritical region R1 of the pupil plane of the lens. The active control means (or, if necessary, correctly dimensioned and positioned masks) blocks the incident light rays at the subcritical region of the pupil plane of the lens. This produces an evanescent wave whose shape and position in the transverse plane correspond to the predefined area.

[0125] Since the entire supercritical region R1 of the pupil plane of the objective is illuminated, the amount of light passing through the objective is maximized and isotropic illumination is produced. These advantages are achieved while producing a light pattern of a predetermined shape.

[0126] A light pattern is obtained on the surface of the diopter whose shape and position correspond to the predefined area to be excited. This results in circumscribed excitation of the object in the three spatial dimensions. The invention thus makes it possible to obtain uniform illumination with very high spatial resolution. The spatial resolution of said light pattern in the object focal plane of the objective is limited by diffraction.

Claims

CLAIMS 1. Device (1, 1 A, 1 B, 1 C, 1 D) for creating an evanescent field (3) on the surface of a diopter (4) separating two media of different refractive indices, comprising: - a light source (6A, 6B, 6C, 6D), - an objective (9A, 9B, 9C, 9D) comprising an object focal plane positioned on the surface of the diopter, and a pupil plane (PP) comprising a subcritical region (R2) and a supercritical region (R1), - an active optical means (7A, 7B, 7C, 7D) configured to modulate the amplitude and / or the phase of light rays emitted by the light source, the active optical means comprising an active surface (10A, 10B, 10C, 10D) extending at least in the entire supercritical region (RT) of a conjugate plane of the pupil plane or extending in a conjugate plane of the object focal plane of the objective, the active optical means comprising a set of individually controllable microelements configured to illuminate the entire supercritical region of the objective, and - a means (8A, 8B, 8C, 8D) for controlling each microelement configured so as to produce a light pattern of predefined shape in the object focal plane of the lens.

2. Device (1, 1A, 1B, 1C, 1D) according to the preceding claim, characterized in that the active optical means (7A, 7B, 7C, 7D) comprises a liquid crystal spatial light modulator or a matrix of micro-mirrors.

3. Device (1 A, 1 B) according to claim 1, characterized in that the light source (6A, 6B) is a coherent light source, in particular a laser light source, and in that the active surface (10A, 10B) extends at least throughout the supercritical region. (R1 ') of a plane conjugate to the pupil plane.

4. Device (1A, 1B) according to the preceding claim, characterized in that the active optical means (7A, 7B) comprises a liquid crystal spatial light modulator, and in that it comprises an optical mask (13A) extending throughout the subcritical region (R2 1 ) of a plane conjugate to the pupil plane.

5. Device (1 B) according to claim 3, characterized in that the active optical means (7B) comprises a matrix of micro-mirrors, the matrix of micro-mirrors extending throughout the supercritical region (R1 ') and throughout the subcritical region (R2 1) of the conjugate plane of the pupil plane, each micro-mirror in the subcritical region of the conjugate plane of the pupil plane being controlled so as to deflect the light rays out of the objective.

6. Device (1 C, 1 D) according to claim 1 or 2, characterized in that the active surface (10C, 10D) extends in a plane conjugate with the object focal plane, the control means (7C, 7D) being configured so as to reproduce said predefined shape pattern directly on the active surface of the active optical means, the device further comprising an optical mask (14C, 14D1) extending throughout the subcritical region (R2 1 ) of a plane conjugate to the pupil plane.

7. Device (1 C) according to the preceding claim, characterized in that the light source is an incoherent light source, in particular a light-emitting diode, the control means (7C) being configured so as to reproduce the whole of said predefined shape pattern directly on the active surface of the active optical means.

8. Device (1 B, 1 D) according to one of the preceding claims, characterized in that the light source (6B, 6D) is a coherent light source, in particular a laser, and in that the active optical means (7B, 7D) is controlled so as to modulate the phase and / or the amplitude of a wavefront reflected by the active surface (10B, 10D) by holography.

9. Device (1 B, 1 D) according to the preceding claim, characterized in that it comprises at least one mask (14B, 14D2) configured to mask a pattern of diffraction order 0.

10. Method of operating a device (1, 1A, 1B, 1C, 1D) according to one of claims 1 to 9, characterized in that it comprises: - the placement of an object, in particular a living cell (C), to be observed on a transparent support (2), the object and the support having different refractive indices, then - the definition of the shape and position of an area of the object to be excited with an evanescent field, then - controlling each microelement of the active optical means (7A, 7B, 7C, 7D) of the device so as to simultaneously illuminate the entire supercritical region of the objective, and so as to produce a light pattern in the object focal plane of the objective, the spatial resolution of said light pattern in the object focal plane being limited by diffraction, the shape and position of said light pattern corresponding to the predefined zone to be excited.

11. Operating method according to the preceding claim, characterized in that the light pattern is formed by scanning a light spot in the object focal plane of the objective, the entire supercritical region of the objective being illuminated at each moment.

12. Operating method according to claim 10 or 11, characterized in that the definition of the shape and position of an area of the object to be excited with an evanescent field comprises: - a preliminary observation of the object to be excited, then - identification of the outline of an area of the object to be excited based on prior observation.

13. Operating method according to the preceding claim, characterized in that the identification of the outline of an area of the object to be excited is carried out manually by means of a human-machine interface.

14. Operating method according to one of claims 10 to 13, characterized in that it comprises an iterative calculation, for example of the Gerchberg-Saxton type, of at least one configuration of the active surface making it possible to form said light pattern (ML) in the object focal plane of the objective.

15. Operating method according to one of claims 10 to 14, characterized in that it comprises the calculation of a first configuration and at least one second configuration of the active surface, each configuration of the active surface being calculated so as to obtain the same light pattern (ML), the light patterns obtained with the first configuration and with the at least one second configuration comprising the same shape and the same position but a different brightness distribution, and in that each microelement of the active optical means (7A, 7B, 7C, 7D) is controlled successively according to the first configuration then according to at least one second configuration.

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