Device for forming an evanescent field

Abstract

The present invention relates to an apparatus (1, 1A, 1B, 1C, 1D) that generates an evanescent field (3) on the surface of a diopter (4) separating two media of different refractive indices, the apparatus comprising a light source (6A, 6B, 6C, 6D), an object focal plane positioned on the surface of the diopter, and a lens (9A, 9B, 9C, 9D) comprising a pupil plane (PP) including a subcritical region (R2) and a supercritical region (R1). Active optical means (7A, 7B, 7C, 7D) comprising an active surface (10A, 10B, 10C, 10D) extending over the entire supercritical region (R1') of at least the conjugate plane of the pupil plane or extending over the 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

Technical Field

[0001] The present invention relates to an apparatus for forming an evanescent field on the surface of a diopter separating two media with different refractive indices. The present invention also relates to a method of using the apparatus.

Background Art

[0002] Fluorescence microscopy is a technique that uses an optical device that utilizes the fluorescence phenomenon instead of or in addition to conventional observations by reflection or transmission of natural light or artificial visible light. This technique enables the observation of various objects and substances (organic or inorganic), particularly living cells. This technique also makes it possible to excite photosensitive molecules in a biological sample. In order to reach a very precise area of the observation object, it is desirable to localize the excitation in all three spatial directions. Such excitation is particularly required in the optogenetic activation of proteins that can bind to the basement membrane of living cells.

[0003] There are several types of fluorescence microscopes, including total internal reflection fluorescence microscopes (commonly abbreviated as TIRFM). These microscopes are configured to illuminate the entire field of view of the objective lens with an evanescent excitation wave having a uniform or periodic lateral structure. The evanescent wave, or evanescent field, is obtained by irradiating the supercritical region of the pupil plane of a high numerical aperture objective lens. This makes it possible to project an excitation wave onto a planar diopter that separates two media with different refractive indices at an angle greater than the critical angle defined by these two refractive indices. The excitation wave undergoes total internal reflection, and an evanescent wave appears on the surface of the diopter. This evanescent wave interacts with the surface of the observation object in a very limited depth range, for example, on the order of 100 nanometers. There are mainly three types of this type of microscope.

[0004] The first approach, the simplest and most common, involves focusing an excitation laser beam onto a single spot in the supercritical region of the pupil surface of the objective lens. This generates a uniform evanescent wave on the surface of the diopter. For example, reference EP3969954A1 discloses an apparatus for generating an evanescent field based on this method. However, this simple configuration has the drawback of generating anisotropic illumination in a direction predetermined by the position of the single spot in the supercritical region, which negatively impacts image quality.

[0005] The second approach involves sequentially positioning laser spots at various points within the supercritical region of the objective pupil plane and averaging the corresponding fluorescence images. This results in more uniform diopter illumination. This second approach is commonly called "azimuthal beam spinning." A galvanometric scanner system can be used to continuously move the laser spot within the pupil plane. Alternatively, a micromirror array (often called a DMD in the UK) that is optically conjugate to the object focal plane of the objective lens and illuminates uniformly can be used.

[0006] A third approach involves modifying the conventional TIRFM configuration, similar to structured illumination microscopy (SIM). To do this, the incident laser beam is split into two, and a liquid crystal spatial light modulator (commonly acronym SLM) or a micromirror array is used to simultaneously generate two opposite spots on the pupil plane of the objective lens. The object's focal plane remains illuminated across the entire field of view of the objective lens. The two corresponding evanescent waves interfere at the object's focal plane, generating a fringe pattern that is used as carriers to access the sample's spatial frequency, which is twice as large as the frequency resulting from the objective lens's numerical aperture.

[0007] All conventional total internal reflection fluorescence microscopes have drawbacks. In particular, they cannot achieve isotropic illumination with high spatial resolution in the cross-sectional plane, i.e., the diopter plane. This is because the evanescent waves generated by known devices illuminate the object over a surface area of ​​tens to hundreds of micrometers in the transverse direction, thus failing to meet the need for precise excitation in the cross-sectional plane. From a practical standpoint, conventional total internal reflection fluorescence microscopes cannot selectively illuminate very clearly defined areas of the object, such as a portion of the basement membrane of a living cell. [Overview of the Initiative]

[0008] The object of the present invention is to provide an apparatus for generating an evanescent field and a method for using the apparatus, which overcomes the aforementioned drawbacks and improves upon apparatuses and methods known in the prior art.

[0009] More specifically, the first subject of the present invention is an apparatus for generating an evanescent field that enables obtaining a uniformly illuminated pattern of a predetermined shape and position, with a very high spatial resolution, particularly on the order of micrometers.

[0010] The present invention relates to an apparatus for generating an evanescent field on the surface of a diopter that separates two media with different refractive indices. The apparatus comprises the following: ·light source, An objective lens comprising an object focal plane positioned on the surface of a diopter, and a pupil plane including subcritical and supercritical regions. Active optical means configured to modulate the amplitude and / or phase of light rays emitted from a light source, comprising an active surface extending at least across the entire supercritical region of the conjugate plane of the pupil or across the conjugate plane of the object focal plane of the objective lens, wherein the active optical means comprises a set of individually controllable microelements configured to illuminate the entire supercritical region of the objective lens, • Means for controlling each minute element, configured to generate a light pattern of a predetermined shape at the object focal plane of the objective lens.

[0011] According to one embodiment, the active optical means includes a liquid crystal spatial light modulator or a micromirror array.

[0012] According to one embodiment, the light source is a coherent light source, in particular a laser light source, and the active surface extends over the entire supercritical region of at least the conjugate surface of the pupil.

[0013] According to one embodiment, the active optical means comprises a liquid crystal spatial light modulator, and the device comprises an optical mask extending across the entire critical region of the conjugate plane of the pupil.

[0014] According to one embodiment, the active optical means comprises a micromirror array extending across the entire supercritical and subcritical regions of the conjugate plane of the pupil, and each micromirror within the subcritical region of the conjugate plane of the pupil is controlled to deflect light rays from the objective lens.

[0015] According to one embodiment, the active surface extends through the conjugate surface of the object's focal plane, the control means is configured to directly reproduce the predetermined pattern on the active surface of the active optical means, and the device further comprises an optical mask extending through the entire subcritical region of the conjugate surface of the pupil plane.

[0016] According to one embodiment, the light source is a non-coherent light source, in particular an LED, and the control means is configured to directly reproduce the entire predetermined shape pattern on the active surface of the active optical means.

[0017] According to one embodiment, the light source is a coherent light source, in particular a laser, and the active optical means is controlled to modulate the phase and / or amplitude of the wavefront reflected by the active surface by holography.

[0018] According to one embodiment, the device comprises at least one mask configured to mask a diffraction order of 0 pattern.

[0019] The present invention also relates to a method for operating the above-mentioned apparatus. The method comprises: - A step in which the object to be observed, especially living cells, is placed on a transparent support so that the refractive index of the object and the support are different. - A step of defining the shape and location of the region of the object excited by the evanescent field. The process involves controlling each minute element of the active optical means of the apparatus to simultaneously illuminate the entire supercritical region of the objective lens, generating a light pattern at the object focal plane of the objective lens, the spatial resolution of the light pattern at the object focal plane being limited by diffraction, and the shape and position of the light pattern corresponding to a predetermined region being excited.

[0020] According to one embodiment, the light pattern is formed by scanning a light spot within the object focal plane of the objective lens, so that the entire supercritical region of the objective lens is always illuminated.

[0021] The definition of the shape and location of the region of an object excited by the evanescent field is carried out as follows: • Pre-observation of the excited object, • Identify the contour of the region of the excited object based on prior observations.

[0022] Identifying the contours of the region of the excited object can be done manually using a human-machine interface.

[0023] The operation method may include, for example, a Gerchberg-Saxton type iterative calculation, i.e., an iterative calculation of at least one configuration of an active surface that allows a light pattern to be formed at the focal plane of the objective lens.

[0024] The operating method may include calculating a first configuration and at least one second configuration of the active surface, each configuration of the active surface being calculated to obtain the same light pattern, and the light patterns obtained with the first configuration and the at least one second configuration having the same shape and the same position but different luminance distributions, and each microelement of the active optical means being continuously controlled according to the first configuration and then according to the at least one second configuration.

Brief Description of the Drawings

[0025] These subjects, features, and advantages of the present invention will be described in detail in the following descriptions of various specific embodiments in relation to the accompanying drawings. [Figure 1] It is a schematic cross-sectional view of a living cell excited by an evanescent wave generated by an evanescent field generating device according to an embodiment of the present invention. [Figure 2] It is a schematic diagram of a device for generating an evanescent field according to a first embodiment of the present invention. [Figure 3] It is a schematic cross-sectional view of the active optical means of the device of FIG. 2. [Figure 4] It is a schematic front view of the active surface of the active optical means of FIG. 3. [Figure 5] It is a schematic diagram of the pupil plane of the objective lens of the device of FIG. 2. [Figure 6] It is a schematic diagram of a device for generating an evanescent field according to a second embodiment of the present invention. [Figure 7] It is an isometric view of two individually controllable microelements of the possible active optical means of the device of FIG. 6. [Figure 8] It is a schematic front view of the active surface of the active optical means of FIG. 6. [Figure 9] [ It is a schematic diagram of the pupil plane of the objective lens of the device of FIG. 6. <000011{0> [Figure 10] It is a schematic diagram of a device for generating an evanescent field according to a third embodiment of the present invention. [Figure 11] [[ID= It is a schematic diagram of a device for generating an evanescent field according to a fourth embodiment of the present invention. [Modes for carrying out the invention]

[0026] Figure 1 schematically shows a living cell C in which the basement membrane is intended to be excited by evanescent waves generated by an evanescent field generator 1 according to one embodiment of the present invention. Cell C consists, in particular, of a nucleus N and various proteins P1, P2. Cell C is placed on a transparent support 2 extending parallel to a plane called a cross-section, defined by the X and Y axes. The support 2 is, for example, a microscope slide. The Z axis is defined as an axis perpendicular to the plane in which the support 2 extends. The Z axis is parallel to the vertical axis. The cross-section is a horizontal plane.

[0027] Among the proteins within cell C, protein group P1 is located within a clearly defined region of the basement membrane M of cell C that is in contact with the support 2. As will be described later, the evanescent field generator 1 (hereinafter simply referred to as "device 1") is designed to generate an evanescent field 3, or evanescent wave, that can excite only protein group P1 without exciting other intracellular proteins P2. The evanescent field 3 generated by device 1 has very high resolution in all three dimensions of space: as an evanescent field, it interacts with cell C at a very shallow depth along the Z axis. Furthermore, the evanescent field 3 has very little spread even in cross-section. The evanescent field can excite cellular regions with a diameter D of 1 μm or less in cross-section, and even 500 nm or less, and even 200 nm or less.

[0028] The support 2 has a first refractive index n1, and the object being observed (in this case, cell C) has a second refractive index n2 that is different from the first refractive index n1. The first refractive index n1 may be substantially equal to the refractive index of glass, and the second refractive index n2 may be substantially equal to the refractive index of water. The diopter 4, which extends in a planar direction, can be defined as the plane separating the support 2 from the cell C and its medium. Thus, the diopter 4 extends in a planar direction parallel to the X and Y axes. The critical angle A1 can be defined by the following equation: A1 = arcsin(n1 / n2). When the diopter 4 is illuminated by an incident field 5 with an incident angle A2 greater than or equal to the critical angle A1, total internal reflection occurs: this propagated field is completely reflected, and an evanescent field 3 is generated. The evanescent field 3 interacts with the surface of cell C at a very limited depth, for example, about 100 nanometers.

[0029] Apparatus 1 can be used to observe and / or excite cells, more generally, any kind of object. In the following description, this object will be referred to as the “object to be observed,” but may be intended to be excited by the evanescent field 3, or may be intended to be excited by the evanescent field 3 alone.

[0030] Such an evanescent field 3 is obtained by the apparatus 1 according to the present invention. Several embodiments of the apparatus 1 are envisioned. In particular, four embodiments are shown in Figures 2, 6, 10, and 11, respectively, and will be described in order. For readability, the suffixes A, B, C, and D are used to represent identical or similar elements belonging to the first, second, third, and fourth embodiments, respectively. Features of elements or their arrangement described in the context of a particular embodiment may be transferred to other embodiments. To simplify the description, the differences between the second, third, and fourth embodiments will be described mainly, without repeating common features.

[0031] Figure 2 shows an evanescent field generating apparatus 1A according to a first embodiment of the present invention. The apparatus 1A comprises a light source 6A, an active optical means 7A, a means 8A for controlling the active optical means 7A, and an objective lens 9A.

[0032] In 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 several centimeters. The light source 6A is positioned to illuminate an active optical medium 7A. The active optical medium 7A is configured to reflect the light rays from the light source 6A toward the objective lens 9A.

[0033] The active optical means 7A is configured to modulate the phase of light rays emitted from 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 the image projected by the active optical means 7A. These microelements 15A extend across the active surface 10A of the active optical means 7A.

[0034] 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 modulates an optical output based on a specific fixed spatial pattern and can project light that is controlled in amplitude only or in phase only. Such active optical means include, in particular, liquid crystal displays. Each microelement 15A of the spatial light modulator corresponds to a region of the spatial light modulator to which the liquid crystal can be oriented in a specific direction in response to an electric field applied to that region. In this first embodiment, the spatial light modulator is configured to modulate the phase of the optical beam in each microelement.

[0035] Figure 3 schematically illustrates the operation of a spatial light modulator. The left side of Figure 3 shows the incident wave O1 propagating toward the active surface 10A. This wave O1 is a plane wave and is incident in a direction parallel to the optical axis Z0. The phase is constant at all points on the wavefront. The right side of Figure 3 shows the wave O2 reflected by the active surface 10A. The reflected wavefront O2 is no longer plane and, conversely, is locally deformed according to the configuration of the micro-element 15A from which wave O1 was reflected (i.e., the local orientation of the liquid crystal).

[0036] The objective lens 9A is an optical element designed to focus light rays incident on the surface of the diopter 4. The objective lens 9A is preferably a pre-assembled and immutable component. The objective lens may be an oil immersion objective lens, and its refractive index n is preferably as close as possible to the refractive index n1 of the support 2, which is preferably made of glass.

[0037] The diopter 4 extends along a plane that coincides with the object focal plane of the objective lens 9A. In other words, the object focal plane of the objective lens 9A is located on the surface of the diopter 4.

[0038] The objective lens 9A is equipped with an optical component that can focus light rays to the focal plane of the objective lens at an incident angle greater than or equal to the critical angle A1.

[0039] The optical axis Z0 can be defined as an axis parallel to the Z-axis passing through the center of the objective lens 9A. The active surface 10A of the active optical means 7A can be oriented so that the reflected light rays are approximately parallel to the optical axis Z0.

[0040] An optical component, such as an optical lens, may be interposed between the active optical means 7A and the objective lens 9A. In this case, the device 1A includes a first lens 11A and a second lens 12A interposed between the active optical means 7A and the objective lens 9A. Lenses 11A and 12A may be condensing 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 to the object focal plane of the first lens 11A. The image focal plane PF1 of the first lens 11A coincides with the object focal plane of the second lens 12A. The image focal plane of the second lens 12A coincides with the pupil plane PP of the objective lens 9A.

[0041] The pupil plane PP of a lens coincides with the lens's Fourier plane. Therefore, this plane is also called the pupil plane or Fourier plane.

[0042] Note that the pupil (entry pupil) of a microscope's objective lens is the opening located behind the objective lens, opposite the sample. By definition, this pupil is the image of the aperture diaphragm (through the optics behind it) and most effectively blocks the inclination of light rays received by the objective lens (on the side of the object being observed).

[0043] The entrance pupil diameter D is expressed by the following formula: D = 2 × ON × FIM ON is the numerical aperture of the objective lens. FIM is the focal length of the objective lens.

[0044] The numerical aperture ON is expressed by the following formula: ON = n × sin(θ) n is the refractive index of the immersion medium of the objective lens. θ is the angle of the most inclined light ray received by the aperture diaphragm.

[0045] The refractive index n² of the object being observed is lower than the numerical aperture ON. Therefore, a subcritical region exists at the center of the entrance pupil of the objective lens, corresponding to the light rays propagating through the object. Conversely, light rays passing through the peripheral region of the entrance pupil (called the supercritical region) cannot propagate to the object being observed.

[0046] Currently, microscope objective lenses are designed so that the entrance pupil is located at the rear focal plane of the objective lens, i.e., the Fourier plane of the objective lens. Furthermore, these objective lenses give an image of an object at infinity and are therefore called "infinity-corrected," which, when combined with a so-called tube lens, produces a real image. In this description, only infinity-corrected objective lenses are mentioned. Tube lenses are not involved at any point.

[0047] The focal plane of the first lens 11A coincides with the plane from which the active plane 10A extends, and is the conjugate plane of the pupil plane PP. In other words, all points on the pupil plane PP of the objective lens 9A form an image within the plane from which the active plane 10A extends.

[0048] Alternatively, the optical apparatus can become more complex, and in particular, it may be equipped with more reflective means such as lenses and mirrors. In more complex versions of the apparatus, multiple conjugate planes of the pupil can be defined.

[0049] As explained above, the pupil surface PP of the objective lens 9A can be divided into two complementary regions: the supercritical region R1 and the subcritical region R2. The supercritical region R1 corresponds to the portion of the pupil surface PP where incident light rays are focused by the objective lens 9A at an incident angle greater than or equal to the critical angle A1. The subcritical region R2 corresponds to the portion of the pupil surface PP where incident light rays are focused by the objective lens 9A at an incident angle strictly less than the critical angle A1.

[0050] The subcritical region R2 has a disk shape centered on the optical axis Z0, and the supercritical region R1 has an annular shape extending around the subcritical region R2. Therefore, the precise dimensions of the subcritical region R2 and the supercritical region R1 are determined by the refractive index of the support 2 and the object being observed, as well as the focal length FIM and numerical aperture ON of the objective lens.

[0051] Since the active surface 10A extends through the conjugate surface of the pupil surface, a supercritical region R1' and a subcritical region R2' can also be defined within this conjugate surface. The supercritical region R1' and the subcritical region R2' may have shapes similar to the supercritical region R1 and the subcritical region R2, respectively. The active surface 10A extends over at least the entirety of the supercritical region R1' of the conjugate surface of the pupil surface PP.

[0052] The active surface 10A can also extend to the subcritical region R2'. However, in order to obtain evanescent waves on the surface of the diopter 4, it is necessary that the light rays do not irradiate the diopter 4 at an incident angle strictly smaller than the critical angle A1. For this reason, the apparatus 1A is configured not to radiate light rays into the subcritical region R2 of the pupil surface PP. To achieve this, the active optical means 7A can be controlled so that light rays incident from the light source 6A into the subcritical region R2' are reflected from the optical path toward the objective lens 9A. Alternatively, an optical mask 13A can be provided that extends across the entire subcritical region of the conjugate plane of the pupil surface. The mask 13A can extend directly within the plane on which the active surface 10A extends, within other conjugate planes of the pupil surface, or within the pupil surface PP. However, directly incorporating the mask 13A into the pupil surface PP can be difficult and may require optical-mechanical modifications to the objective lens. In either case, the mask 13A prevents the light rays from reaching the subcritical region R2 on the pupillary surface, thereby preventing the formation of propagating waves on the surface of the diopter 4.

[0053] According to the present invention, in order to obtain a predetermined pattern on the surface of the diopter 4, the active optical means 7A is controlled to illuminate the entire supercritical region R1 of the objective lens. Thus, the entire ring forming the supercritical region R1 receives the light rays. Not just a point or region of this ring receives the light rays. Therefore, it is understood that the device 1A is configured to illuminate the entire supercritical region R1 of the objective lens, not just a part of it. No element of the device 1A can obstruct the illumination of the entire supercritical region R1 of the objective lens.

[0054] The control means 8A is configured to individually control each minute element of the active optical means 7A that extends into the supercritical region R1'. Therefore, the control means 8A is configured to change the phase of the wavefront over the entire supercritical region R1'.

[0055] The control means 8A may consist of a computer equipped with a human-machine interface. The control means 8A is programmed to generate a light pattern ML of a predetermined shape on the object focal plane of the objective lens, i.e., on the surface of the diopter 4.

[0056] Conventionally, a computer can be equipped with display means such as a screen and control means such as a keyboard or mouse. The display means is intended to display the object being studied together with the device 1A, in particular the cell C. This allows the user to identify a specific region of interest on the object. The control means is intended to define a desired light pattern ML.

[0057] For example, if a user wants to stimulate the P1 protein group in cell C, they can use a control means (such as a computer mouse) to manually draw a geometric shape surrounding the P1 protein group in cell C. This geometric shape then has a shape and position that depends on prior observation of the object being observed. Alternatively, the shape and position of the geometric shape can be automatically determined, particularly by automatically processing images obtained from prior observation of the object.

[0058] The control means 8A includes a memory that stores the shape and position of the light pattern ML specified in advance by the user. The control means 8A also includes a computer program in which a transfer function for calculating the activation state of each micro-element of the active optical means as a function of the shape and position of the desired light pattern ML is encoded.

[0059] Each micro-element 15A is a microstructure that emits light waves. The spatial resolution of the light pattern ML at the object focal plane of the objective lens is limited by diffraction due to the numerical aperture of the objective lens.

[0060] Therefore, the incident wave to the pupil plane PP is i) The objective lens has a controlled phase at all points in the supercritical region R1 of the entrance pupil, ii) The amplitude is uniformly zero across the entire subcritical region R2 of the objective lens's entrance pupil.

[0061] The wavefront phases at all points within the supercritical region R1 of the pupil plane PP are optimized to generate a desired intensity pattern within the object focal plane of the objective lens 9A.

[0062] The shape of the light pattern ML can be freely selected. For example, circular, oval, elliptical, polygonal, especially triangular, square, rectangular, or any other arbitrary geometric shape can be realized. It is advantageous that the shape of the light pattern is defined based on prior observation or inference of the location of the photosensitive region of the object being observed. In Figure 2, the light pattern ML has an arbitrary shape and can be freely adjusted as needed. It should be noted that the mask 13A described above allows for filtering of the low-frequency components of the wavefront in Fourier space. The presence of this mask does not prevent the formation of the light pattern ML, but it may alter the uniformity of illumination of this light pattern.

[0063] As shown in Figure 2, this light pattern can be observed by placing a screen at the image focal plane PF1. Of course, in reality, the apparatus 1A does not have such a screen, because a screen would block or restrict the passage of light rays. The image focal plane PF1 corresponds to the Fourier plane of the pupil plane PP. In other words, the pattern projected onto the pupil plane PP corresponds to the Fourier transform of the light pattern ML.

[0064] Figure 4 schematically shows the active surface 10A used to generate the optical pattern ML shown in Figure 2. Each microelement of the active surface is represented by a specific gray level depending on the local phase shift it induces. Conventionally, white indicates a phase shift of zero, and black indicates a phase shift of 2π. Macroscopically, the active surface 10A appears gray. However, the phase shift produced by the active surface 10A is not at all uniform. In fact, as shown on the right side of Figure 4, when the active surface 10A is observed at a microscopic scale, it can be seen that each microelement 15A has a specific configuration, producing a phase shift between 0 and 2π. The phase pattern thus generated on the active surface 10A corresponds to the complex part of the Fourier transform of the desired pattern at the focal plane of the objective lens.

[0065] Figure 5 schematically shows the pupillary surface PP of the objective lens. The supercritical region R1 of the pupillary surface PP is illuminated entirely by a pattern substantially identical to the pattern generated on the active surface 10A. Similarly, the pattern formed on the pupillary surface PP of the objective lens corresponds to the Fourier transform of the desired pattern at the object focal plane of the objective lens.

[0066] According to one embodiment, a linear phase gradient can be added to the pupil plane PP via the control means 8A to shift the position of the super-resolution laser spot that reproduces a desired pattern by continuous scanning within the object focal plane of the objective lens. In other words, the active optical means 7A can be controlled to always generate a single point of light whose size is limited by diffraction, rather than a specific geometric shape or pattern, within the object focal plane of the objective lens. This point of light is then used to scan for the desired geometric shape.

[0067] To obtain a light spot, the active optical means are appropriately controlled. If the wavefront O2 is perpendicular to the optical axis Z0, a light spot centered on the optical axis Z0 is obtained. If the wavefront O2 is inclined with respect to the optical axis Z0, a light spot offset from the optical axis Z0 is obtained. In either case, the entire supercritical region R1 of the pupil surface of the objective lens is illuminated to obtain the light spot.

[0068] Apparatus 1A also includes means for polarizing the incident wave at the pupil plane PP of the objective lens. In fact, a large numerical aperture of the objective lens generates a highly tilted ray whose amplitude changes depending on the angle and plane of incidence. The most suitable polarization state for achieving the best spatial resolution is radial polarization. Radial polarization can ideally be obtained from linear polarization by using a polarizer placed near the pupil plane of the objective lens.

[0069] To determine the phase shift of each microelement of the active surface 10A, an iterative calculation of the Gerchberg-Saxton type, for example, is performed. This calculation, of course, depends not only on the shape and position of the predefined light pattern ML, but also on the initial conditions. These initial conditions may correspond to the initial phase conditions at the image focal plane PF1. Therefore, the phase shift of each minute element of the active surface 10A depends on the initial conditions. For a given light pattern ML, there are multiple possible arrangements of each minute element of the active surface 10A. The light patterns ML obtained from these different arrangements have the same shape and position. However, all different light patterns exhibit some degree of optical non-uniformity; that is, certain parts of each light pattern are illuminated to a greater or lesser extent. Different light patterns are distinguished from each other by different luminance distributions within the light pattern ML itself. To obtain a light pattern ML with a given shape and uniform brightness, it is useful to calculate multiple configurations of the active surface 10A for the same light pattern ML by changing the initial conditions of the iterative calculation. The active optical means 7A can be controlled to continuously and periodically generate different configurations of the active surface 10A. The more different configurations there are, the better the uniformity of the light emission in the light pattern.

[0070] Figure 6 shows an evanescent field generating apparatus 1B according to a second embodiment of the present invention. Similar to the first embodiment, the apparatus 1B comprises a light source 6B, an active optical means 7B, means 8B for controlling the active optical means 7B, and an objective lens 9B. The active optical means 7B consists of a set of individually controllable microelements 15B. The apparatus 1B also comprises a first lens 11B and a second lens 12B positioned between the active optical means 7B and the objective lens 9B. The light source 6B, objective lens 9B, first lens 11B, and second lens 12B may each have the same features and arrangement as the light source 6A, objective lens 9A, first lens 11A, and second lens 12B described above. Similar to the first embodiment, the active optical means 7B comprises an active surface 10B that extends through the conjugate surface of the pupil surface PP and can be divided into a supercritical region R1' and a subcritical region R2'. Device 1B is configured to irradiate not only a portion of the supercritical region R1 of the objective lens, but the entire supercritical region R1.

[0071] This second embodiment differs from the first embodiment in the active optical means 7B and associated control means 8B. The active optical means 7B and associated control means 8B are configured to generate an optical pattern on the focal plane of an object by holography. More specifically, the active optical means 7B is used to encode the wavefront using a high-frequency carrier wave. The carrier wave is a high spatial frequency periodic pattern applied to the microelement 15B. This periodic pattern typically consists of a series of parallel lines. This periodic pattern is shown in Figure 8.

[0072] According to the first modification of the second embodiment, the high-frequency carrier wave may be a network of fringes of variable intensity, in other words, variable amplitude. Each microelement 15B of the active surface 10B is configured in an "on" position to re-emit rays that can reach the pupil of the objective lens with the same intensity as the incident rays, or in an "off" position not to re-emit rays that can reach the pupil of the objective lens. The position of each microelement of the active surface 10B can be obtained by iterative calculations similar to those described in the first embodiment.

[0073] The active optical means 7B is preferably a micromirror array. Alternatively, the active optical means 7B may be a spatial light modulator configured to modulate the amplitude or phase of the incident wave.

[0074] A micromirror array is an electromechanical microsystem that can project a digital image by the reflection of micromirrors that can switch between two different positions: in the "on" position, the micromirrors are oriented so that the reflected light rays reach the objective lens 9B. In the "off" position, the micromirrors are oriented so that the reflected light rays do not reach the objective lens 9B. Micromirror arrays are low-cost and have a fast display speed. Figure 7 shows two microelements 15B of a micromirror array as an example. The first microelement is in the "on" position, and the second microelement is in the "off" position.

[0075] As shown in Figure 8, the control means 8B is configured to individually control each micromirror so that the micromirrors in the supercritical region R1' are either in the off or on position, and all micromirrors located in the subcritical region R2' are in the off position. The use of a micromirror array eliminates the need for an optical mask extending to the conjugate plane of the subcritical region. Therefore, the micromirror array can be used to control the amplitude of the incident wavefront at the pupil plane PP of the objective lens.

[0076] When the supercritical region R1' of the active surface 10B is observed under a microscope, a network of variable intensity fringes can be visualized, resulting in the on and off position micromirrors being positioned along nearly parallel lines. Generally, the control means 8B can encode the phase and / or amplitude of the wavefront generated at the pupil plane of the objective lens onto the carrier so that a desired light pattern ML is obtained at the object focal plane of the objective lens 9B.

[0077] If we only want to control the phase in the supercritical region, we can encode it through the position of the grid fringes displayed on a micromirror array according to the Lie method, which is expressed by the following equation:

[0078]

number

[0079] x and y represent the coordinates of each micromirror along the X and Y axes. ·Φ(x,y) represents the desired phase. • p represents the period of the lattice (therefore, the carrier frequency is given by 1 / p).

[0080] Due to the diffraction generated by the striped grating, a series of patterns with different diffraction orders are observed within the image focal plane PF1 of the first lens 11B. Therefore, by allowing only light of order 1 of the diffraction grating to pass through the image focal plane PF1, a wavefront corresponding to phase φ(x,y) is obtained at the pupil plane. A mask 14B extending into the image focal plane PF1 is provided to mask light of orders other than order 1, i.e., orders 0, -1, +2, -2, etc. This removes high-frequency components from the wavefront of the pupil plane PP. In Figure 6, the 0th order light pattern is shown with reference number ML0, and the 1st order light pattern is shown with ML1. In Figure 6, the light pattern ML0 is shown as a transparent mask for visualization, but the mask 14B is actually an opaque mask.

[0081] The wavefront of the PP pupil surface is shown in Figure 9. The supercritical region R1 of the PP pupil surface is completely illuminated by a pattern corresponding to the Fourier transform of the desired pattern at the object's focal plane.

[0082] The spatial resolution of the intensity pattern obtained within the object's focal plane of the objective lens is determined by the optimization of the phase and amplitude in the supercritical region of the pupil plane, and by encoding via carriers displayed on a micromirror array. The expansion of the field of view (i.e., the region of the object's focal plane of the objective lens where intensity can be controlled) is also determined by encoding via carriers displayed on a micromirror array.

[0083] According to a variation of Lee's holography, it is also possible to encode the amplitude of a wave by adjusting the width of the fringes. However, it should be noted that since both the phase and amplitude are controlled using pixels of a fixed period, the number of achievable phase levels is more limited.

[0084] The above technology makes it possible to form an intensity pattern on the focal plane of the objective lens with a single illumination. However, it is also possible to reconstruct the pattern by scanning the evanescent spot, the position of which is determined by the period and direction of the carrier grating. Since the refresh rate of the micromirror array is approximately 10 kHz, the pattern can be reconstructed in less than one second with this scanning method. A constant gradient phase φ(x,y) in the pupil plane can be obtained by tip-tilting the incident laser beam using a galvanometer mirror or acousto-optic crystal positioned in a plane optically conjugate to the pupil plane (4f setup).

[0085] Surface deformation of the micromirror array and the propagation of light waves throughout device 1B are major sources of optical aberrations that can degrade the spatial resolution of the optical pattern ML. These aberrations can be compensated for by appropriately changing the phase of the encoded wavefront on the micromirror array using Lee's method described above.

[0086] In a second modification of the second embodiment, the high-frequency carrier wave may be a variable-phase fringe network instead of a variable-intensity fringe network. In this case, the active optical means is preferably a spatial light modulator. The on and off positions of each micro-element correspond to a phase shift of zero and a phase shift of π, respectively.

[0087] Figure 10 shows an evanescent field generating apparatus 1C according to a third embodiment of the present invention. As described above, apparatus 1C comprises a light source 6C, an active optical medium 7C, a control means 8C for the active optical medium 7C, and an objective lens 9C. Apparatus 1C is configured to irradiate not only a portion of the supercritical region but the entire supercritical region R1 of the objective lens. The specific features of these elements will be described later. Apparatus 1C further comprises a first lens 11C, a second lens 12C, and a third lens 13C interposed between the active optical means 7C and the objective lens 9C.

[0088] Lenses 11C, 12C, and 13C may be condensing lenses. These lenses extend perpendicular to the optical axis Z0 and may be centered on the optical axis Z0. The focal lengths of the first lens 11C, the second lens 12C, and the second lens 13C are f1, f2, and f3, respectively. The active surface 10C of the active optical means 7C extends to the object focal plane of the first lens 11C. The image focal plane PF1 of the first lens 11C coincides with the object focal plane of the second lens 12C. The image focal plane PF2 of the second lens 12C coincides with the object focal plane of the third lens 13C. The image focal plane of the third lens 13C coincides with the pupil plane PP of the objective lens 9A. The various lenses 11C, 12C, 13C and the objective lens 9C are arranged such that the active surface 10C extends through the conjugate plane of the object's focal plane, that is, all points on the object's focal plane of the objective lens 9C have an image in the plane of the active surface 10C. Alternatively, the optical device may become more complex and, in particular, include more lenses and / or reflective means such as mirrors.

[0089] According to this embodiment, the light pattern ML is reproduced directly on the active surface 10C by the active optical means 7C. Next, a mask 14C is provided that extends to the subcritical region R2' of the conjugate surface of the pupil surface to prevent light rays from reaching the subcritical region R2 of the pupil surface. As described above, the entire supercritical region R1 of the pupil surface is illuminated.

[0090] In this third embodiment, the light source 6C may be a non-coherent light source. The light source 6C is composed of, for example, light-emitting diodes, which simplifies the design of the device 1C. The active optical means 7C may consist of a liquid crystal spatial light modulator or a micromirror array. The active optical means 7C is configured to control the amplitude of the incident light. The pattern is reproduced with one set of microelements in the ON position and the other microelements of the active optical means 7C in the OFF position.

[0091] According to this embodiment, since the microelements are activated according to the shape and position of the desired light pattern ML, the active optical means 7C is easier to implement than in the first and second embodiments described above. Therefore, it is not necessary to rely on iterative Fourier transform calculations to determine which microelements should be activated.

[0092] When using a monochromatic and spatially coherent incident wave, it should be noted that the transfer function of the imaging system is given by the supercritical region of the objective lens's entrance pupil. Therefore, the low-frequency portion in the center of the Fourier plane is excluded, making it difficult to pre-prepare the illumination pattern in a homogeneous region unless holographic phase control is used. Conversely, the central spot can be generated directly, or scanned by changing the fringe parameters as described above.

[0093] Conversely, if the light source is spatially non-coherent, the transfer function of the imaging system is given by the autocorrelation of the entrance pupil of the objective lens in the supercritical region. This corresponds to a more uniform distribution of transmission frequencies, including not only high frequencies but also the low-frequency portion in the center of the Fourier plane. This makes it possible to reproduce illumination patterns with a uniform region enclosed by a very narrow boundary.

[0094] Figure 11 shows an apparatus for generating a 1D evanescent field according to a fourth embodiment of the present invention. Apparatus 1D comprises a light source 6D, an active optical means 7D, means 8D for controlling the active optical means 7D, an objective lens 9D, a first lens 11D, a second lens 12D, and a third lens 13D positioned between the active optical means 7D and the objective lens 9D. Apparatus 1D is configured to illuminate not only a portion but the entire supercritical region R1 of the objective lens. The features and arrangement of these elements may be identical to those of apparatus 1C in the third embodiment.

[0095] Apparatus 1D differs from apparatus 1C in that the light source 6D is a coherent light source. Furthermore, the active optical means 7C and associated control means 8C are configured to generate a pattern on the focal plane of an object by holography. The active optical means 7D can be configured to modulate the phase and / or amplitude of the incident wavefront at the pupil plane PP of the objective lens by holography. Similar to the second embodiment, the active optical means 7D may consist of a spatial light modulator or a micromirror array. The active optical means 7C is used to encode the wavefront using a high-frequency carrier wave. The high-frequency carrier wave may be a variable intensity fringe pattern and / or a variable phase fringe pattern. The position of each minute element of the active plane 10D can be obtained by iterative calculations similar to those described in the first embodiment.

[0096] The contour of the desired light pattern ML is directly reproduced on the active surface 10D. Micro-elements placed within this contour are controlled to form a striped network.

[0097] Furthermore, apparatus 1D includes a first mask 14D1 that extends to the subcritical region R2' of the conjugate plane of the pupillary surface, preventing light rays from reaching the subcritical region R2 of the pupillary surface. Apparatus 1D always includes a second mask 14D2 for masking light coming from the 0th order of the diffraction grating. Although shown as a transparent mask in Figure 11 (to visualize the light pattern ML0), mask 14D2 is actually an opaque mask.

[0098] Finally, the following procedure can be used to use any of the above devices 1A, 1B, 1C, or 1D. First, place the object to be observed on a transparent support. The refractive indices of the object and the support are different, with the refractive index of the object being lower than that of the support. For example, place a living cell on a glass slide.

[0099] Next, we define the shape and location of the region of the object excited by the evanescent field. This definition can be based on prior observation of the object, calculations, and assumptions about the object. For example, in the case of a cell, we can define the portion of the cell's basement membrane containing the desired type of protein.

[0100] Once this region is defined, each micro-element of the device's active optical medium is controlled to generate a light pattern on the object focal plane of the objective lens that corresponds to a predefined shape and position. For this purpose, the device's control means calculates how each micro-element needs to be controlled, using a predefined transfer function where applicable, and / or iteratively. The active control means guides rays from a light source to illuminate the entire supercritical region R1 of the pupil plane of the objective lens. The active control means (or a mask positioned of appropriate size and location) blocks incident rays outside the critical region of the pupil plane of the objective lens. This generates evanescent waves whose shape and position in cross-section correspond to a predetermined region.

[0101] Since the entire supercritical region R1 of the pupil surface of the objective lens is illuminated, the amount of light passing through the objective lens is maximized, resulting in isotropic illumination. These advantages are obtained while generating a light pattern of a predetermined shape.

[0102] A light pattern with a shape and position corresponding to a predetermined excitation region is formed on the surface of the diopter. This excites an object enclosed in three-dimensional space. In this way, the present invention makes it possible to obtain uniform illumination with very high spatial resolution. The spatial resolution of the light pattern at the focal plane of the objective lens is limited by diffraction.

Claims

1. An apparatus (1, 1A, 1B, 1C, 1D) that forms an evanescent field (3) on the surface of a diopter (4) that separates two media with different refractive indices, ・Light source (6A, 6B, 6C, 6D), - Objective lenses (9A, 9B, 9C, 9D) comprising an object focal plane disposed on the surface of the diopter and a pupil plane (PP) including a subcritical region (R2) and a supercritical region (R1), Active optical means (7A, 7B, 7C, 7D) configured to modulate the amplitude and / or phase of light rays emitted from the light source, comprising active surfaces (10A, 10B, 10C, 10D) extending at least over the entire supercritical region (R1') of the conjugate plane of the pupil or over the conjugate plane of the object focal plane of the objective lens, wherein the active optical means comprises a set of individually controllable microelements configured to illuminate the entire supercritical region of the objective lens, - Control means (8A, 8B, 8C, 8D) for controlling each of the microelements, configured to generate a light pattern of a predetermined shape at the object focal plane of the objective lens, A device equipped with the following features.

2. The apparatus according to claim 1, wherein the active optical means comprises a liquid crystal spatial light modulator or a micromirror array.

3. The apparatus according to claim 1, wherein the light source is a coherent light source, particularly a laser light source, and the active surface extends over the entire supercritical region of at least the conjugate surface of the pupil surface.

4. The apparatus according to claim 3, wherein the active optical means comprises a liquid crystal spatial light modulator, and the apparatus comprises an optical mask (13A) extending over the entire critical region of the conjugate surface of the pupil surface.

5. The apparatus according to claim 3, wherein the active optical means comprises a micromirror array, the micromirror array extending over the entire supercritical region and the entire subcritical region of the conjugate surface of the pupil surface, and each micromirror in the subcritical region of the conjugate surface of the pupil surface is controlled to deflect the light rays from the objective lens.

6. The apparatus according to claim 1 or 2, wherein the active surface extends through the conjugate surface of the object focal plane, the control means is configured to directly reproduce the predetermined shape of the pattern on the active surface of the active optical means, and the apparatus further comprises an optical mask (14C, 14D1) extending over the entire subcritical region of the conjugate surface of the pupil plane.

7. The apparatus according to claim 6, wherein the light source is a non-coherent light source, in particular a light-emitting diode, and the control means is configured to directly reproduce the entire predetermined shape pattern on the active surface of the active optical means.

8. The apparatus according to any one of claims 1 to 7, wherein the light source is a coherent light source, in particular a laser, and the active optical means is controlled to modulate the phase and / or amplitude of the wavefront reflected by the active surface by holography.

9. The apparatus according to claim 8, further comprising at least one mask (14B, 14D2) configured to mask a diffraction order of 0.

10. A method for operating the apparatus according to any one of claims 1 to 9, - A step of placing the object to be observed, particularly living cells (C), on a transparent support (2) so that the refractive index of the object and the support are different. - A step of defining the shape and location of the region of the object excited by the evanescent field. - Controlling each of the microelements of the active optical means of the apparatus to simultaneously irradiate the entire supercritical region of the objective lens and generate a light pattern on the object focal plane of the objective lens, wherein the spatial resolution of the light pattern on the object focal plane is limited by diffraction, and the shape and position of the light pattern correspond to a predetermined region to be excited, in steps, A method of having.

11. The method according to claim 10, wherein the light pattern is formed by scanning a light spot in the focal plane of the objective lens, and the entire supercritical region of the objective lens is always illuminated.

12. The definition of the shape and position of the region of the object excited by the evanescent field is: - Pre-observation of the object to be excited, - Identify the contour of the region of the object that is excited based on the prior observation. The method according to claim 10 or 11, as carried out by...

13. The method according to claim 12, wherein the contour of the region of the excited object is determined manually using a human-machine interface.

14. The method according to any one of claims 10 to 13, further comprising, for example, calculating the configuration of at least one of the active surfaces by an iterative calculation of the Gerchberg-Saxton type, such that the light pattern (ML) is formed on the focal surface of the objective lens.

15. The method according to any one of claims 10 to 14, comprising the step of calculating a first configuration and at least one second configuration of the active surface, each configuration of the active surface being calculated to obtain the same light pattern, the light patterns obtained from the first configuration and the at least one second configuration having the same shape and position but different luminance distributions, and each of the microelements of the active optical means being controlled sequentially according to the first configuration and then according to the at least one second configuration.

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