FR3146214A1 - Optical slide for total internal reflection microscopy, total internal reflection microscopy device comprising such a slide and method of manufacturing such a slide - Google Patents

Abstract

Optical slide (LO) intended to receive a biological sample (E) in a total internal reflection fluorescence microscopy device, the optical slide comprising: a so-called substrate layer (SB) transparent in a predetermined spectral region a so-called optimized stack arranged above the substrate (SB) and comprising a number of successive and alternating dielectric thin layers of a first dielectric material and a second dielectric material, each of the layers having a respective so-called optimized thickness, the optimized thicknesses and the number of dielectric thin layers being adapted so that said optical slide has at least: a first resonant absorption at a so-called illumination wavelength and at a first angle of incidence in total internal reflection regime, and a second resonant absorption at the illumination wavelength and at a second angle of incidence in total internal reflection regime, with and or . Fig. 3

Description

Optical slide for total internal reflection microscopy, total internal reflection microscopy device comprising such a slide and method of manufacturing such a slide

The invention relates to the field of optical microscopy. More particularly, the invention relates to a new concept of optical slide based on a multilayer stack as a support for enhancing the electromagnetic field suitable for total internal reflection microscopy.

The invention applies in particular, but not exclusively, to the field of imaging of biological samples by total internal reflection fluorescence microscopy or TIRF microscopy (for "Total Internal Reflection Fluorescence" ). Such an imaging technique is particularly well suited to the visualization, analysis and quantification of molecular events occurring in particular at the plasma membrane of biological cells.

Evanescent wave fluorescence microscopy is a fluorescence microscopy technique in which the excitation of fluorescent molecules contained in the observed sample is confined to a nanometer-thick region, located in the immediate vicinity of the sample slide. In particular, it allows selective observation of structures and processes located on a cell membrane, with a spatial resolution in an axial direction much better than the diffraction limit. In addition, compared to more conventional epifluorescence techniques, it allows for better contrast of the fluorescence image and reduces photobleaching effects and irradiation damage to cells.

The principle underlying evanescent wave fluorescence microscopy is illustrated in Figure 1A. We consider the case of a substrate SB with a refractive index n ₂ , having a surface Se in contact with an ambient medium MA with an index n ₁ <n ₂ . For example, the substrate SB can be constituted by a sample-holding slide, or by a glass element on which such a slide is placed, while the ambient medium MA can be an aqueous solution containing, in suspension, cells marked by fluorophores. A light beam FLI, coming from the substrate SB, is incident on the surface Se; its direction of propagation forms with the normal z _s to the surface an angle θ greater than a critical value θ _c (limit angle) (1).

Therefore the FLI beam undergoes a total internal reflection (RTI) forming a reflected beam FLR and producing an evanescent wave OE in the ambient medium MA. This evanescent wave has an intensity which decreases exponentially with the distance z from the surface S: , where the penetration length δ is given by (1bis), λ being the wavelength of the light radiation. The evanescent wave excites the fluorophores contained in the ambient medium, but only over a thickness of the order of δ, because beyond this its intensity quickly becomes negligible. For example, for λ=488 nm, n ₂ =1.514 (BK7 glass), n ₁ =1.33 (water) and we find 93 nm, which means that only fluorophores located in a layer about 100 nm thick are excited and contribute to the production of a fluorescence image.

There illustrates the most commonly used configuration in TIRF microscopy. In the case of the , the same microscope objective OBJ, located on the side of the substrate opposite to the MA medium, is used both to generate the evanescent waves by RTI and to collect the fluorescence radiation.

Thus, using this known technique, the images obtained have multiple qualities: first of all, they benefit from low background noise (because the fluorophores located in the deep layers of the sample (outside the evanescent field) are only very weakly excited) and from a relatively high axial resolution.

Microscope slides with complex structures, such as those based on surface metallization, have also been designed to locally enhance the evanescent electromagnetic field. Such optical slides, which are based on the principle of surface plasmon resonance, can improve the sensitivity of microscopy imaging. However, this known solution remains limited in terms of field enhancement value and in the choice of materials, i.e. noble metals, which limit the usable illumination conditions as well as biocompatibility, which is not optimal.

Another known technique, described in patent document US 2016/0238830, is based on a multilayer waveguide whose layer thicknesses and refractive indices are chosen to support a guided leakage mode. However, microscopy imaging experiments carried out with this technique remain limited in terms of sensitivity and resolution in particular.

Furthermore, it is known from application FR2108879 to produce microscopy slides covered with stacks of nanometric layers of dielectric materials designed to enhance the evanescent electromagnetic field at the interface between the slide and the biological objects deposited on it. This type of structure makes it possible to improve the sensitivity of total internal reflection microscopes which use a microscope objective to reach the critical angle of total reflection as illustrated in the .

The use of these dielectric stacks allows to generate this enhancement at all wavelengths of the visible/near infrared spectrum and at all illumination angles beyond the critical angle of RTI. However, this method is optimized for illuminations with plane waves, which is not the case in a microscope illuminating the object through the objective.

Indeed, one of the main limitations of resonant structures in RTI is their low angular tolerance, which is all the lower as the structure is resonant. However, unlike sensor applications which seek the most intense and finest spectral and angular enhancement of the evanescent field, in microscopy, the desired resonance is associated with a lower enhancement (from ten to a few hundred) with an angular tolerance at least of the order of magnitude of the divergence of the illumination by the microscope objective.

Accordingly, there is a need for an optical slide intended for a total internal reflection microscope allowing the evanescent field to be enhanced at the interface between the slide and the biological objects deposited on it with an increased angular tolerance compared to the slides of the prior art.

• la sélection d’un premier matériau diélectrique présentant un indice de réfraction et d’un deuxième matériau diélectrique présentant un indice de réfraction tel que
• la sélection d’une longueur d’onde dite d’illumination et d’un angle d’incidence dit optimal en fonction dudit dispositif de microscopie par réflexion totale interne auquel ladite lame optique est destinée
• la conception d’une première structure comprenant un premier empilement de couches minces diélectriques disposé au dessus d’une couche dite substrat (SB) transparente dans une région spectrale prédéterminée comprenant ladite longueur d’onde d’illumination ledit premier empilement comprenant de couches minces successives et alternées du premier matériau diélectrique et du deuxième matériau diélectrique, une première épaisseur respective de chaque couche mince diélectrique et ledit nombre de couches minces diélectriques étant adaptés de manière à ce que la première structure présente une absorption résonnante à la longueur d’onde d’illumination et à l’angle d’incidence optimal en régime de réflexion totale interne
• la détermination d’une première fonction représentative d’une valeur d’une transmission de la première structure dans la région spectrale prédéterminée en fonction d’un nombre de couches minces diélectriques et d’une épaisseur de chaque couche mince diélectrique, et la détermination d’une deuxième fonction représentative d’une intensité d’un champ électrique évanescent généré par la lame optique en régime de réflexion totale interne à la longueur d’onde d’illumination et à l’angle d’incidence optimal en fonction d’un nombre de couches minces diélectriques et de ladite première épaisseur de chaque couche mince diélectrique
• la conception de ladite lame optique qui comprend un deuxième empilement de couches minces diélectriques dit empilement optimisé disposé au dessus du substrat (SB), l’empilement optimisé comprenant un nombre de couches minces diélectriques successives et alternées du premier matériau diélectrique et du deuxième matériau diélectrique, chacune des couches présentant une épaisseur dite optimisée respective, les épaisseurs optimisées et le nombre de couches minces diélectriques étant déterminés par une optimisation d’une fonction de mérite basée sur la première fonction et la deuxième fonction de manière à ce que ladite lame optique présente une première absorption résonnante à la longueur d’onde d’illumination et à un premier angle d’incidence en régime de réflexion totale interne et présente une deuxième absorption résonnante à la longueur d’onde d’illumination et à un deuxième angle d’incidence en régime de réflexion totale interne, avec et ou .

For this purpose, an object of the invention is a method of manufacturing an optical slide intended to receive a biological sample in a total internal reflection microscopy device, said method comprising a step of designing said optical slide and a step of material manufacturing of said optical slide thus designed, said method being characterized in that the design phase comprises the following steps:

• the selection of a first dielectric material having a refractive index and a second dielectric material having a refractive index such as
• the selection of a so-called illumination wavelength and a so-called optimal angle of incidence depending on said total internal reflection microscopy device for which said optical slide is intended
• the design of a first structure comprising a first stack of thin dielectric layers arranged above a so-called substrate (SB) layer transparent in a predetermined spectral region comprising said illumination wavelength said first stack comprising of successive and alternating thin layers of the first dielectric material and the second dielectric material, a first respective thickness of each dielectric thin layer and said number of thin dielectric layers being adapted so that the first structure exhibits resonant absorption at the illumination wavelength and at the optimum angle of incidence in total internal reflection mode
• the determination of a first function representative of a value of a transmission of the first structure in the predetermined spectral region as a function of a number of dielectric thin layers and a thickness of each dielectric thin layer, and determining a second function representative of an intensity of an evanescent electric field generated by the optical blade in the total internal reflection regime at the illumination wavelength and at the optimum angle of incidence depending on a number of dielectric thin layers and said first thickness of each dielectric thin layer
• the design of said optical blade which comprises a second stack of thin dielectric layers called optimized stack arranged above the substrate (SB), the optimized stack comprising a number of successive and alternating thin dielectric layers of the first dielectric material and the second dielectric material, each of the layers having a respective so-called optimized thickness, the optimized thicknesses and the number of thin dielectric layers being determined by an optimization of a merit function based on the first function and the second function such that said optical blade exhibits a first resonant absorption at the illumination wavelength and at a first angle of incidence in the total internal reflection regime and exhibits a second resonant absorption at the illumination wavelength and at a second angle of incidence in total internal reflection mode, with And Or .

Preferably, the merit function is such that , with a parameter allowing to weight a respective weight of the first function and the second function in said optimization.

Preferably, is included in 0.3 and 0.7.

Preferably, the optimization is such that .

According to one embodiment, the optimization consists of finding the optimized thicknesses and the number of thin dielectric layers for which said value of the transmission of the first structure in the predetermined spectral region is maximum and for which said intensity of the evanescent electric field is maximum.

According to one embodiment, in the first stack, an upper layer of the first stack is in the second dielectric material and has a first thickness of between And and in which the lower layers of the first stack each have a first thickness of between And and wherein in the optimized stack a top layer of the first stack is in the second dielectric material.

According to one embodiment, the first function F1 is representative of a transmission of fluorescence radiation induced indirectly by an absorption of said illumination wavelength. in total internal reflection in said first stack illuminated with said optimal angle of incidence , the fluorescence radiation having a spectrum included in said spectral region, the transmission being integrated over an angular range of incidence of the fluorescence radiation on said optical plate between 0 and 80°.

• une couche dite substrat transparente dans une région spectrale prédéterminée
• un empilement dit optimisé disposé au dessus du substrat et comprenant un nombre de couches minces diélectriques successives et alternées d’un premier matériau diélectrique et d’un deuxième matériau diélectrique, chacune des couches présentant une épaisseur dite optimisée respective, les épaisseurs optimisées et le nombre de couches minces diélectriques étant adaptés de manière à ce que ladite lame optique présente au moins :

• une première absorption résonnante à une longueur d’onde dite d’illumination et à un premier angle d’incidence en régime de réflexion totale interne, et
• une deuxième absorption résonnante à la longueur d’onde d’illumination et à un deuxième angle d’incidence en régime de réflexion totale interne, avec .

Another object of the invention is an optical slide intended to receive a biological sample in a total internal reflection microscopy device, the optical slide comprising:

• a so-called substrate layer transparent in a predetermined spectral region
• a so-called optimized stack placed above the substrate and comprising a number of successive and alternating thin dielectric layers of a first dielectric material and a second dielectric material, each of the layers having a respective so-called optimized thickness, the optimized thicknesses and the number of thin dielectric layers being adapted so that said optical blade has at least:

• a first resonant absorption at a so-called illumination wavelength and at a first angle of incidence in total internal reflection mode, and
• a second resonant absorption at the illumination wavelength and at a second angle of incidence in total internal reflection mode, with .

• le premier matériau diélectrique présente un indice de réfraction compris entre 1,8 et 3,5 ;
• le deuxième matériau diélectrique présente un indice de réfraction compris entre 1,2 et 1,7.

According to one embodiment:

• the first dielectric material has a refractive index between 1.8 and 3.5;
• the second dielectric material has a refractive index between 1.2 and 1.7.

According to one embodiment, the first material is based on Nb ₂ O ₅ , the second material is based on SiO ₂ .

According to one embodiment, the number of thin dielectric layers is less than 30, preferably less than 20.

According to one embodiment, the optimized thicknesses and the number of thin dielectric layers are such that said optical blade has a thickness of less than 5 and preferably between 0.5 and 2 .

According to one embodiment, the optimized thicknesses are greater than or equal to 10 nm.

• une lame optique selon l’invention
• une source de lumière adaptée pour émettre un faisceau lumineux présentant la longueur d’onde d’illumination
• un dispositif optique adapté pour mettre en forme le faisceau lumineux de manière à ce qu’il illumine ladite lame optique avec ledit angle d’incidence optimisé
• un détecteur adapté pour détecter un rayonnement de fluorescence émis lorsqu’un échantillon est déposé en correspondance d’une région d’une surface de ladite lame optique où sont produites des ondes évanescentes par une réflexion totale interne dans ladite lame optique du faisceau lumineux mis en forme, ledit rayonnement de fluorescence étant excité par lesdites ondes évanescentes.

Another object of the invention is a total internal reflection microscopy device comprising:

• an optical blade according to the invention
• a light source adapted to emit a light beam having the illumination wavelength
• an optical device adapted to shape the light beam so that it illuminates said optical blade with said optimized angle of incidence
• a detector adapted to detect fluorescence radiation emitted when a sample is deposited in correspondence with a region of a surface of said optical plate where evanescent waves are produced by total internal reflection in said optical plate of the shaped light beam, said fluorescence radiation being excited by said evanescent waves.

Other characteristics, details and advantages of the invention will emerge from reading the description given with reference to the appended drawings given by way of example and which represent, respectively:

And

a schematic view of a prior art total internal reflection microscope;

, a schematic view of a total internal reflection microscopy device according to the invention;

, a schematic view of an optical slide according to the invention, intended to receive a biological sample in a total internal reflection microscopy device;

, the absorption of an optical plate according to a preferred embodiment of the invention in total internal reflection mode, as a function of the angle of incidence and the wavelength of the incident beam;

, the enhancement factor of the evanescent electric field generated on the surface of an optical plate according to a preferred embodiment of the invention in total internal reflection mode, as a function of the angle of incidence of the incident beam;

, And

, the enhancement factor of the evanescent electric field generated on the surface of an optical plate according to a preferred embodiment of the invention in total internal reflection mode, as a function of the angle of incidence of the incident beam, for two different divergences of the incident beam

, the normalized enhancement factor relative to a glass plate of the evanescent electric field generated on the surface of an optical plate according to a preferred embodiment of the invention in total internal reflection mode, as a function of the angle of incidence of the incident beam,

, a schematic representation of the steps of the design phase of the manufacturing method according to the invention.

In the figures, unless otherwise indicated, elements are not to scale and identical references designate identical elements or identical steps.

There illustrates in a simplified manner a device D for total internal reflection microscopy according to the invention. The device D comprises an optical slide LO according to the invention, a light source SL, an optical device Obj and a fluorescence detector Det.

We will first describe the operation of the device D before detailing the structure of the optical blade LO. Finally, we will describe a manufacturing method according to the invention for obtaining the optical blade LO.

The optical slide LO is a slide, preferably biocompatible, intended to receive a biological sample E for microscopy imaging purposes according to an RTI configuration. An example of an optical slide structure in accordance with the invention is described further on in relation to FIG. 3 which schematically illustrates a section along the plane of the optical blade LO according to the invention.

The light source SL is adapted to emit a light beam L _in having a so-called illumination wavelength predetermined. The SL source is a laser source configured to emit at a wavelength typically between 350 and 1300 nm so as to excite the fluorophores contained in the sample E and thus produce the emission of fluorescence radiation FL while being at least partially transmitted by the optical plate LO.

The optical device Obj is adapted to shape the light beam L _in so that it illuminates the optical blade with a so-called optimized angle of incidence greater than the critical angle.

Preferably, as illustrated in Figure 2, the optical device Obj is a microscope objective. The microscope objective can have a variable focal length and a variable numerical aperture (greater than 1.45). It comprises an optic or a more or less complex assembly of optical lenses capable of allowing the formation of the light beam L _in by focusing it in the direction of the optical plate. It is also capable of collecting the reflected and/or backscattered beam from the optical plate in an angular cone from 0 to a maximum angle given by the numerical aperture of the objective. The objective is generally of the oil immersion type and can have a large numerical aperture (NA), for example of the order of 1.45, which makes it possible to obtain a high lateral spatial resolution (perpendicular to the z direction) because the lateral spatial resolution is given by . Additionally, an Obj objective with a high numerical aperture ON is advantageous because it allows the incident light beam to be moved relative to the optical axis OA and thus obtain a strong deviation of the incident beam through the lens, the beam thus being able to propagate with an angle of incidence high and sufficient to obtain an RTI.

Alternatively, the optical device Obj comprises one or more lenses and/or mirrors to focus the light beam L _in and optionally a prism or a metasurface so that the light beam L _in illuminates the optical plate with the optimized angle of incidence.

This optimized angle of incidence is greater than the critical angle at the interface between a surface SE of the blade intended to receive the sample E and the ambient medium (typically liquid) comprising the sample. The optimized angle of incidence is typically between 62 and 80 degrees for a biological environment with a refractive index between 1.33 and 1.35. Thus, the RTI of the light beam L _in on said interface produces evanescent waves OE. The sample E being placed in correspondence with a region of the surface SE where the evanescent waves are produced, the fluorophores of the sample will generate a fluorescence radiation FL excited by the evanescent waves OE.

The emitted fluorescence radiation FL is then collected by the objective Obj and is directed towards the detector Det in order to be detected. This detector Det is typically a CCD or CMOS camera whose spectral band is adapted to the detection of the fluorescence radiation FL re-emitted from the sample E. It converts the light intensity received into an electrical signal intended for a processing unit (not shown in the figures). The processing unit is electrically connected to the light source SL, the detector Det and the microscope objective Obj so as to be able to control these elements for the purpose of acquiring images of the sample E in RTI mode.

As a non-limiting example, the device D illustrated in FIG. 2 is based on the principle of epifluorescence, the observation of the fluorescence of which is carried out in a configuration by reflection using a slide or a dichroic mirror 50 for example. This particular configuration makes it possible to dissociate the optical path taken by the excitation light. , of the optical path taken by the FL fluorescence radiation.

Preferably, the optimized angle of incidence is between 62 and 80 degrees. The lower limit of the above range (62 degrees) is given by the refractive index value of the sample studied. As for the upper limit of the above range (80 degrees), it is defined according to the value of the numerical aperture used for microscopy observation. The typical numerical aperture of the microscope objective Obj of the device D is greater than or equal to 1.45. The microscope objective Obj is preferably of numerical aperture and variable focal length.

The structure of the optical blade according to the invention, as shown in the figure, will be described in more detail below. .

The optical blade LO has the first face SE, and a second face SI, opposite the first, and defines a stacking axis extending between these two opposite faces in the direction The first face SE is intended to receive the biological sample E to be observed and the second face SI is the face illuminated by the light beam. . The first face SE constitutes the interface where an exaltation of the evanescent electromagnetic field OE is produced by the optical blade 10.

According to the embodiment illustrated in FIG. 2, the optical slide LO and the microscope objective Obj are arranged such that the stacking axis of the slide coincides with the optical axis OA of the objective. In other words, the optical slide LO and the microscope objective Obj are oriented relative to each other such that the optical interface formed between the optical slide LO and the sample E is perpendicular to the optical axis OA, according to the direction .

According to the invention, the optical blade LO comprises an optically transparent substrate SB in a spectral region which comprises the illumination wavelength. and which includes the spectral range of FL fluorescence radiation. For example, the SB substrate is a soda-lime glass microscope slide with an index of 1.5 (or any other optically transparent support calibrated in thickness).

In addition, the optical blade LO comprises a stack of dielectric layers (hereinafter called optimized stack EO) arranged directly above the substrate SB. This optimized stack EO is particularly suitable for enabling an enhancement of the evanescent electric field OE produced at the surface SE in an RTI configuration of the beam. . Thus, when the device of Figure 2 is in operation, the biological sample E which is placed on the upper layer is illuminated by the beam at the illumination wavelength after passing through the optical blade. More precisely, the beam passes through the SB substrate, then the EO dielectric stack until reaching the interface between the upper layer and the E sample at the optimized angle of incidence . The evanescent wave OE created by the RTI sees its intensity amplified thanks to the resonant structure of the EO stack, which makes it possible to significantly increase the imaging performance of TIRF microscopy, in particular in terms of sensitivity and axial resolution.

As illustrated in Figure 3, this EO stack is formed by a succession of alternating thin layers of a first dielectric material with a refractive index (thin layers referenced ) and a second dielectric material with a refractive index (thin layers referenced ), with .

The thin layer intended to be in contact with the sample E (called the upper layer) is made of one of the two dielectric materials. Preferably, this layer is made of a biocompatible dielectric material, for example SiO ₂ , TiO ₂ , Nb ₂ O ₅ … . The upper face of this upper layer corresponds to the face SE previously introduced. As for the face SI illuminated by the beam , it corresponds to the lower face of the SB substrate.

• une première absorption résonnante à la longueur d’onde d’illumination et à un premier angle d’incidence en RTI, et
• une deuxième absorption résonnante à la longueur d’onde d’illumination et à un deuxième angle d’incidence en RTI.

In the optical blade LO of the invention, each of the dielectric layers has a respective so-called optimized thickness chosen in particular as a function of the illumination wavelength. , of the optimized angle of incidence , incident polarization and refractive indices And . The manufacturing process and the design face of the optical blade LO of the invention will be detailed later in the description of figure 7. We will briefly limit ourselves to specifying that the optimized thicknesses and the number of thin dielectric layers are adapted so that the optical plate LO exhibits at least a double resonance at the illumination wavelength in RTI for two distinct angles of incidence. More precisely, the optimized thicknesses and the number of thin dielectric layers are such that the optical blade has:

• a first resonant absorption at the illumination wavelength and at a first angle of incidence in RTI, and
• a second resonant absorption at the illumination wavelength and at a second angle of incidence in RTI.

Angles of incidence And are chosen so that is between 0.4° and 3°, preferably between 0.4° and 1.5°. More precisely, we want to have of the order of magnitude of the divergence of the microscope therefore according to the systems

It is understood that the optical blade LO of the invention can comprise more than two resonances as long as the aforementioned conditions remain verified.

The EO stack of dielectric multilayers coupled to the SB substrate ensures a significant enhancement of the evanescent electromagnetic field OE at the interface between the optical blade and the sample with an angular tolerance much greater than the optical blades of the prior art. This enhancement increases the intensity of the FL fluorescence radiation, which improves the sensitivity of microscopic imaging as well as the axial resolution of the device D.

Indeed, the prior art resonant optical blades are optimized to exhibit a spectrally and angularly narrow resonance, for a predetermined optimal angle of incidence and for a theoretically planar illumination wave. They thus exhibit a very high "theoretical" enhancement factor of the evanescent electromagnetic field. However, according to the laws of geometric optics, the beam has a non-negligible natural angular divergence after passing through the objective (typically between 0.5° and 1°). Also, the beam incident on the optical plate LO actually has a plurality of angles of incidence centered around the optimum angle of incidence. This implies that the evanescent wave enhancement factor OE induced by the optical plate for rays at angles of incidence other than the optimum angle of incidence is in practice significantly lower than the “theoretical” enhancement factor.

In the invention, the presence of two (or more) angularly close resonances allows a partial overlap between the resonance bands. This overlap allows to widen the angular tolerance of the optical plate LO so that it has a "real" enhancement factor six to ten times greater than the optical plates of the prior art (see figure 6) (see more in the case of multi-resonances). The angular tolerance of the plate of the invention is specifically chosen in order to be greater than or equal to the divergence of the beam after passing through the microscope objective Obj which is typically between 0.5° and 1°.

Through numerous simulations and experiments, the inventors observed that this multiple resonance was only possible with an EO stack comprising a number of thin dielectric layers.

As mentioned above, the optimized thickness of each layer of the stack is chosen in particular according to the illumination wavelength. , of the optimized angle of incidence , polarization and refractive indices And . The optimized angle of incidence corresponds here to the angle of incidence at which the beam must illuminate the optical blade at illumination wavelength to obtain an evanescent wave exaltation factor OE with an optimized angular tolerance. It is between And (or between And if ) to benefit from the overlap of the band of the first resonant absorption and the band of the second resonant absorption. Preferably, the optimized thicknesses and the number are such that .

The optimized thicknesses are generally between 5 and 500 nanometers. Preferably, the optimized thicknesses are greater than or equal to 10 nm in order to facilitate the manufacture of the LO optical blade. Indeed, the precise control of the thickness of layers with a thickness of less than 10 nm is technically complex to achieve.

Preferably, the first dielectric material has a refractive index between 1.8 and 3.5 and the second dielectric material has a refractive index between 1.2 and 1.7. For example, the first material is based on Nb ₂ O ₅ , the second material is based on SiO ₂ .

Preferably, the number of thin dielectric layers is less than 30, preferably less than 20. Indeed, the inventors have observed that, compared to an optical blade LO with a number of layers less than 20, a number of dielectric layers greater than or equal to 20 increases the complexity of the manufacturing process but only allows a minimal gain (or even zero) in the evanescent wave exaltation factor.

Preferably, in order to limit the optical aberrations induced by the use of the optical blade in the device D, the optimized thicknesses and the number of thin dielectric layers are such that said optical blade has a thickness of less than 5 and preferably between 0.5 and 2 .

Favorite example:

According to a preferred embodiment of the invention, the optimized stack EO of the optical blade LO comprises 14 alternating dielectric layers of SiO ₂ and Nb ₂ O _{5 .} The upper layer of the stack (the one intended to be in contact with the sample) is made of SiO ₂ . The optimized thicknesses of the thin dielectric layers are, from the upper layer to the lower layer in contact with the substrate, respectively: 20 nm, 26.5 nm, 91.6 nm, 101.8 nm, 242.7 nm, 60.25 nm, 30.2 nm, 20 nm, 273.4 nm, 29.1 nm, 83.1 nm, 20 nm, 530.6 nm, 97.6 nm.

This structure is optimized for an illumination wavelength and for an optimized angle of incidence These optimized thicknesses are determined by the method of described further below. The layer indices are respectively n₂(SiO₂) = 1.486 and n₁(Nb₂O₅) = 2.292 at 561 nm.

The average transmission of this structure, angularly integrated between 0 and 80 degrees, and spectrally between 561 and 700 nm is 53%.

Figure 4A is a graph showing the optical absorption of the EO stack according to the preferred embodiment of the invention, with a liquid medium above the upper layer, as a function of the angle of incidence and the illumination wavelength. Four resonance bands are observed, including the band of the first resonant absorption (referenced B1 in Figure 4A) and the band of the second resonant absorption (referenced B2 in Figure 4A). At the illumination wavelength , the first resonant absorption is centered on the angle and the second resonant absorption is centered on the angle It is recalled here that the plot in Figure 4A represents an absorption curve and, consequently, is only an indirect plot of the enhancement of the evanescent field generated in RTI on the SE surface. Indeed, the dependence between enhancement and absorption is not linear, and the angular tolerance of the resonant enhancement cannot be directly determined from Figure 4A. The exact calculation shows that it is nevertheless about 1°. As mentioned previously, this angular tolerance value corresponds to the upper limit of the typical divergence of the beam after passing through the microscope objective Obj.

Figure 4B is a curve which represents the value of the enhancement factor of the evanescent field generated in RTI by the optical blade according to the preferred embodiment of the invention as a function of the angle of incidence of the beam. , at the illumination wavelength . The curve in Figure 4B corresponds to a horizontal section of the previous figure for the illumination wavelength , but for the evanescent field intensity and not the absorption. As expected, the curve in Figure 4B includes the two exaltation peaks associated with the first and second resonant absorption . It is observed that these resonant absorptions make it possible to obtain a respective exaltation factor of the intensity of the evanescent field of the order of 75. However, this value of the exaltation factor is a "theoretical" value obtained for illumination with a plane wave. In practice, the divergence of the beam must be taken into account. to estimate the real value of the exaltation factor.

For this, figures 5A and 5B represent a curve of the value of the factor of the exaltation of the intensity of the evanescent field generated in RTI as a function of the angle of incidence of the beam. , at the illumination wavelength and for two different beam divergences . The curve of the is obtained for a divergence of 0.6° while the curve of the is obtained for a divergence of 0.9°.

It is observed that taking into account the beam divergence produces an angular integration of the first and second resonant absorptions which makes this double resonance disappear in favor of a single “averaged” resonance centered on the optimized angle of incidence . This "averaged" resonance has an exaltation factor of lower value than the values associated with simple resonances. (i.e. the “theoretical” value of the ) but higher than what can be achieved with a single optimized resonance.

So, for a beam at the illumination wavelength and for the optimized angle of incidence the optical blade LO according to the preferred embodiment of the invention makes it possible to obtain an enhancement factor of about 35 for a divergence of 0.6° and of about 30 for a divergence of 0.9°. Logically, a greater divergence implies a greater angular averaging of the first and second resonant absorption and therefore a greater reduction in the excitement factor.

Figure 6 is a curve which shows the value of the enhancement factor of the fluorescence radiation produced by the optical blade LO according to the preferred embodiment of the invention as a function of the divergence of the incident beam, for the optimized angle of incidence. The value of the enhancement factor is calculated with an integration over an angular cone ranging from 0° to 80° of the FL fluorescence radiation, taking into account the average transmission of the LO optical slide, then is normalized with respect to the value of the enhancement factor obtained by a glass slide.

In addition, the includes a gray area that corresponds to the divergence range of a laser beam focused by a commercial microscope.

There allows to observe that the LO optical slide according to the preferred embodiment of the invention makes it possible to obtain high enhancement factors for a wide divergence range which includes most existing commercial microscopes. More precisely, the LO optical slide makes it possible to obtain a normalized fluorescence enhancement factor of the order of 7.

Although the curves of Figures 4A to 6 have been described in relation to the optical blade LO according to the preferred embodiment of the invention, those skilled in the art will be able to reproduce equivalent results without departing from the scope of the invention with different dielectric materials, and/or different optimized thicknesses and/or a number of layers different by adapting them to a wavelength and an optimized angle of incidence predetermined.

We now focus on describing the manufacturing process of the optical blade LO according to the invention. This manufacturing process comprises a phase of designing the optical blade and then a phase of material manufacturing of the designed optical blade. The different steps A to E of the design phase are illustrated in the .

In a first step A, we select the first dielectric material of index and the second dielectric material of index such as . The material of index n ₂ is preferably silica for biological compatibility and the material of index n ₁ can be for example Nb ₂ O ₅ , TiO ₂ , HfO ₂ or Ta ₂ O ₅ .

In a second step B, the illumination wavelength is selected and the optimal angle of incidence depending on the total internal reflection microscopy device for which the optical slide is intended. This typically involves choosing an illumination wavelength equal to the wavelength of the light source of the microscopy device. The optimal angle of incidence is chosen to be greater than the critical angle of RTI for an interface formed between a liquid medium and a layer in the second dielectric material and depending on the value of the numerical aperture used for microscopy observation.

After step B, the design phase comprises a step C consisting of designing a first structure comprising a first stack of dielectric thin layers arranged above a substrate SB. The substrate SB is transparent in a predetermined spectral region comprising the illumination wavelength. and the FL fluorescence radiation generated indirectly by the illumination wavelength .

The first stack includes successive and alternating thin layers of the first dielectric material and the second dielectric material, each dielectric thin layer having a respective first thickness. The first thicknesses and the number of thin dielectric layers are adapted so that the first structure exhibits a unique resonant absorption at the illumination wavelength and at the optimum angle of incidence in a regime of total internal reflection.

This step C is known in itself and is carried out using thin optical layer simulation and optimization software such as Optilayer or by direct calculation.

According to one embodiment, in the first stack, the upper layer of the first stack is in the second dielectric material and has a first thickness of between And . The exact value of the thickness of the top layer depends on the indices of the dielectric materials, the desired resonance wavelength and the desired resonance angle. In addition, the lower layers of the first stack (i.e. those below the upper layer) each have a first thickness of between And . Preferably, these are layers of thickness .

For example, in order to design the optical blade of the preferred embodiment of the invention previously described, the first stack of step B comprises N=15 alternating dielectric layers of SiO ₂ and Nb ₂ O ₅ . The upper layer is made of SiO ₂ and has a first thickness equal to . The other layers have a first thickness equal to .

After step C, the design phase includes a step D consisting of determining two functions And each representative of the two critical parameters of the optical blade for the intended application: the transmission of fluorescence radiation and the enhancement of the evanescent field generated in RTI.

More precisely, the function is representative of the value of the transmission of the FL fluorescence radiation as a function of the number of dielectric thin layers and the thickness of each dielectric thin layer. To clarify, this is the FL fluorescence radiation induced indirectly by the absorption in the first stack of the illumination wavelength in RTI with the optimal angle of incidence . In order to take into account the Lambertian emission of the fluorescence radiation, the transmission is integrated over a wide angular range of incidence angle of the fluorescence radiation on the optical plate, for example for an incidence angle between 0° and 80° as well as over the spectral range associated with the fluorescence emission.

The function is representative of the intensity of the evanescent electric field generated by the first RTI stack at the illumination wavelength and at the optimum angle of incidence depending on the number of dielectric thin layers and the thickness of each dielectric thin layer

• la transmission de la structure pour le rayonnement de fluorescence. Celui-ci doit être maximisée afin d’augmenter le rayonnement de fluorescence collecté. Cela revient par exemple à maximiser la transmission de la lame pour un angle d’incidence compris entre 0 et 80 degrés, pour une illumination avec une longueur d’onde comprise entre 400 nm et 800 nm,
• le facteur d’exaltation du champ évanescent généré en RTI, qui doit aussi être maximisé
• la tolérance angulaire de ce facteur d’exaltation, comme expliqué précédemment.

Indeed, the actual enhancement factor of the optical slide, that is to say that of the fluorescence radiation collected by the microscope objective, is multifactorial and depends on:

• the transmission of the structure for the fluorescence radiation. This must be maximized in order to increase the fluorescence radiation collected. This amounts, for example, to maximizing the transmission of the slide for an angle of incidence between 0 and 80 degrees, for illumination with a wavelength between 400 nm and 800 nm,
• the exaltation factor of the evanescent field generated in RTI, which must also be maximized
• the angular tolerance of this exaltation factor, as explained previously.

Simultaneous optimization of these three parameters is only really possible by increasing the number of degrees of freedom. This is why it is necessary to construct the functions And which will be used for the next step E.

For example, according to the preferred embodiment of the invention, the functions And have the following form for the design of the optical blade LO:

with T the transmission of the structure for a plane wave illuminated under incidence at the wavelength

with E the amplitude of the electric field at the upper interface of the structure,Δθ the angular interval chosen for field optimization, typically between 0.2 and 1.5 degrees.

• la première absorption résonnante à la longueur d’onde d’illumination et à l’angle d’incidence en RTI, et
• la deuxième absorption résonnante à la longueur d’onde d’illumination et au deuxième angle d’incidence en RTI.

Finally, after determining the functions And , the design phase includes a final step E of optimization of the first stack in order to obtain the optimized stack EO of the optical blade LO. The optimized thicknesses and the number of thin dielectric layers of the optimized EO stack are determined by the optimization of a merit function based on the first function and the second function so that the optical blade presents:

• the first resonant absorption at the illumination wavelength and at the angle of incidence in RTI, and
• the second resonant absorption at the illumination wavelength and at the second angle of incidence in RTI.

As mentioned above, the optimization is such that And Or .

This optimization is carried out using optical thin layer simulation and optimization software such as Optilayer.

Preferably, the merit function to maximize is such that , with a parameter allowing to weight a respective weight of the first function and the second function in optimization. The alpha parameter influences the respective weights of the fluorescence transmission by the slide with respect to the exaltation of the evanescent field and is preferably between 0.3 and 0.7 because both parameters being important, it is appropriate to consider them both in a non-negligible manner.

This form of merit function has a sufficient number of degrees of freedom to simultaneously optimize the three parameters influencing the actual exaltation factor of the optical plate.

Optimization typically involves finding the optimized thicknesses and the number of thin dielectric layers for which the value of the transmission of the fluorescence radiation is maximum and for which the intensity of the evanescent electric field is maximum.

According to a preferred embodiment, the optimization is such that . This makes it possible to obtain a double resonance separated angularly by a value corresponding to the typical divergence of a laser beam focused by a commercial microscope objective. This maximizes the value of the real enhancement factor of the optical blade LO of the invention.

Concerning the material manufacturing phase, the deposition of each thin layer is carried out using any technique known to those skilled in the art, for example by one of the following techniques: vacuum evaporation, vacuum spraying, sol-gel process, centrifugal coating, chemical vapor deposition, plasma deposition.

The invention thus offers the possibility of producing optical slides with electromagnetic field enhancement whose characteristics can be easily adapted according to the imaging parameters required by the microscopy system.

As indicated above, the thickness, number and type of material are characteristics of the stack according to the invention that can be adapted on a case-by-case basis, depending in particular on the imaging parameters of the system and the desired or imposed lighting conditions. Optically transparent materials in the spectral band used to conduct the study will be preferred, whose refractive index and absorption coefficient dispersion values are known and controlled. Such characteristics must allow, at a predetermined optimized angle of incidence and illumination wavelength of the optical plate in total reflection mode, optical absorption with improved angular tolerance in the upper layer of the optimized stack, thus enhancing the evanescent electromagnetic field at the interface of said stack.

Claims (15)

Method for manufacturing an optical slide (LO) intended to receive a biological sample (E) in a total internal reflection microscopy device, said method comprising a step of designing said optical slide (LO) and a step of materially manufacturing said optical slide (LO) thus designed, said method being characterized in that the design phase comprises the following steps:

1. the selection of a first dielectric material having a refractive index and a second dielectric material having a refractive index such as
2. the selection of a so-called illumination wavelength and a so-called optimal angle of incidence depending on said total internal reflection microscopy device for which said optical slide is intended
3. the design of a first structure comprising a first stack of thin dielectric layers arranged above a so-called substrate (SB) layer transparent in a predetermined spectral region comprising said illumination wavelength said first stack comprising of successive and alternating thin layers of the first dielectric material and the second dielectric material, a first respective thickness of each dielectric thin layer and said number of thin dielectric layers being adapted so that the first structure exhibits resonant absorption at the illumination wavelength and at the optimum angle of incidence in total internal reflection mode
4. the determination of a first function representative of a value of a transmission of the first structure in the predetermined spectral region as a function of a number of dielectric thin layers and a thickness of each dielectric thin layer, and determining a second function representative of an intensity of an evanescent electric field generated by the optical blade in the total internal reflection regime at the illumination wavelength and at the optimum angle of incidence depending on a number of dielectric thin layers and said first thickness of each dielectric thin layer
5. the design of said optical blade which comprises a second stack of thin dielectric layers called optimized stack arranged above the substrate (SB), the optimized stack comprising a number of successive and alternating thin dielectric layers of the first dielectric material and the second dielectric material, each of the layers having a respective so-called optimized thickness,

optimized thicknesses and number of thin dielectric layers being determined by an optimization of a merit function based on the first function and the second function such that said optical blade exhibits a first resonant absorption at the illumination wavelength and at a first angle of incidence in total internal reflection regime and exhibits a second resonant absorption at the illumination wavelength and at a second angle of incidence in total internal reflection mode, with And Or . Manufacturing method according to the preceding claim, wherein said merit function is such that , with a parameter allowing to weight a respective weight of the first function and the second function in said optimization. Manufacturing method according to the preceding claim, in which is included in 0.3 and 0.7. A manufacturing method according to any preceding claim, wherein said optimization is such that . A manufacturing method according to any preceding claim, wherein said optimization comprises finding the optimized thicknesses and the number of thin dielectric layers for which said value of the transmission of the first structure in the predetermined spectral region is maximum and for which said intensity of the evanescent electric field is maximum. A manufacturing method according to any preceding claim, wherein in the first stack an upper layer of the first stack is in the second dielectric material and has a first thickness of between And and in which the lower layers of the first stack each have a first thickness of between And . Manufacturing method according to any one of the preceding claims, in which the first function F1 is representative of a transmission of fluorescence radiation induced indirectly by an absorption of said illumination wavelength. in total internal reflection in said first stack illuminated with said optimal angle of incidence , the fluorescence radiation having a spectrum included in said spectral region, the transmission being integrated over an angular range of incidence of the fluorescence radiation on said optical plate between 0 and 80°. Optical slide (LO) intended to receive a biological sample (E) in a device (D) for total internal reflection microscopy, the optical slide comprising:

• a so-called substrate (SB) layer transparent in a predetermined spectral region
• a so-called optimized stack arranged (EO) above the substrate (SB) and comprising a number of successive and alternating thin dielectric layers ( of a first dielectric material and a second dielectric material, each of the layers having a respective so-called optimized thickness, the optimized thicknesses and the number of thin dielectric layers being adapted so that said optical blade has at least:
  • a first resonant absorption at a so-called illumination wavelength and at a first angle of incidence in total internal reflection mode, and
  • a second resonant absorption at the illumination wavelength and at a second angle of incidence in total internal reflection mode, with .

Optical blade according to the preceding claim, in which:
- the first dielectric material has a refractive index between 1.8 and 3.5;
- the second dielectric material has a refractive index between 1.2 and 1.7. Optical blade according to the preceding claim, in which the first material is based on Nb ₂ O ₅ , the second material is based on SiO ₂ . An optical blade according to any one of claims 8 to 10, wherein the number of thin dielectric layers is less than 30, preferably less than 20. An optical blade according to any one of claims 8 to 11, wherein the optimized thicknesses and the number of thin dielectric layers are such that said optical blade has a thickness of less than 5 and preferably between 0.5 and 2 . Optical blade according to any one of claims 8 to 12, in which the optimized thicknesses are greater than or equal to 10 nm. Device (D) for total internal reflection microscopy comprising:

• an optical blade (LO) according to any one of claims 8 to 13,
• a light source (SL) adapted to emit a light beam (L _in ) having the illumination wavelength
• an optical device (Obj) adapted to shape the light beam (L _in ) so that it illuminates said optical blade with said optimized angle of incidence
• a detector (Det) adapted to detect fluorescence radiation (FL) emitted when a sample (E) is deposited in correspondence of a region of a surface (SE) of said optical plate where evanescent waves (OE) are produced by total internal reflection in said optical plate of the shaped light beam (L _in ), said fluorescence radiation (Fl) being excited by said evanescent waves (OE).

Device according to the preceding claim, in which the optical device (Obj) is a microscope objective.