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

Abstract

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

Description

Title of the invention: Optical blade for total internal reflection microscopy, total internal reflection microscopy device comprising such a blade, and method for manufacturing such a blade technical field

[0001] The invention falls within the field of optical microscopy. More particularly, the invention relates to a new concept of optical plate based on a multilayer stacking as a support for enhancing the electromagnetic field, adapted to total internal reflection microscopy.

[0002] The invention applies particularly, but not exclusively, to the field of imaging 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. Previous technique

[0003] Evanescent wave fluorescence microscopy is a fluorescence microscopy technique in which the excitation of fluorescent molecules in the observed sample is confined to a nanometer-thick region located in the immediate vicinity of the sample holder. In particular, it allows for the selective observation of structures and processes located on a cell membrane, with spatial resolution in an axial direction far exceeding the diffraction limit. Furthermore, compared to more conventional epifluorescence techniques, it provides better contrast in the fluorescence image and reduces the effects of photobleaching and irradiation damage to cells.

[0004] The principle underlying evanescent wave fluorescence microscopy is illustrated in Figure IA. Consider the case of a substrate SB with refractive index n2, having a surface Se in contact with an ambient medium MA with refractive index ni <n2. Par exemple, le substrat SB peut être constitué par une lamelle porte-échantillon, ou par un élément en verre sur lequel est posée une telle lamelle, tandis que le milieu ambiant MA peut être une solution aqueuse contenant, en suspension, des cellules marquées par des fluorophores. Un faisceau lumineux FLI, provenant du substrat SB, est incident sur la surface Se ; sa direction de propagation forme avec la normale zs à la surface un angle 0 greater than a critical value 0C (limit angle) — arcsin-^- (1)-

[0005] Consequently, the FLI beam undergoes total internal reflection (TIR), forming a reflected beam FLR and producing an evanescent wave OE in the surrounding medium MA. This evanescent wave has an intensity that decreases exponentially with the distance z from the surface S'■ I( z) — -, where the penetration length ô is given by / 5 —at------(Ibis), X being the wavelength of the radiation luminous. The evanescent wave excites the fluorophores contained in the surrounding medium, but only over a thickness on the order of λ, because beyond this its intensity quickly becomes negligible. As an example, for X=488 nm, n2=1.514 (BK7 glass), n1 = 1.33 (water) and λ = 67 λ > 3C ~ 61.45°, we find 5 ≈ 93 nm, which means that only the fluorophores located in a layer approximately 100 nm thick are excited and contribute to the formation of a fluorescence image.

[0006] [Fig. IB] illustrates the most commonly used configuration in TIRF microscopy. In the case of [Fig. IB], the same microscope objective OBJ, located on the substrate side opposite the MA medium, is used both to generate the evanescent waves by RTI and to collect the fluorescence radiation.

[0007] Thus, by this known technique, the images obtained have multiple qualities: they benefit first of all 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.

[0008] 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 rely on the principle of surface plasmon resonance, 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 restricts usable illumination conditions and biocompatibility, which is not optimal.

[0009] Another known technique, described in US patent document 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 performed with this technique remain limited, particularly in terms of sensitivity and resolution.

[0010] Furthermore, it is known from application FR2108879 to produce microscopy slides coated with stacks of nanometric layers of dielectric materials designed to enhance the evanescent electromagnetic field at the interface between the coverslip and the biological objects placed on it. This type of structure improves the sensitivity of total internal reflection microscopes that use a microscope objective to reach the critical angle of total reflection as illustrated in [Fig.1B].

[0011] The use of these dielectric stacks makes it possible to generate this enhancement at all wavelengths of the visible / near-infrared spectrum and at all illumination angles beyond the critical angle of RTL. However, this method is optimized for illuminations with plane waves, which is not the case in a microscope illuminating the object through the objective.

[0012] Indeed, one of the main limitations of resonant structures in RTI is their low angular tolerance, which is all the lower the more resonant the structure. However, unlike sensor applications which seek the most intense and finest spectrally and angularly evanescent field enhancement, in microscopy, the desired resonance is associated with a lower enhancement (from tens to a few hundred) with an angular tolerance at least on the order of magnitude of the divergence of the illumination by the microscope objective.

[0013] Consequently, there is a need for an optical slide for a total internal reflection microscope that enhances the evanescent field at the interface between the slide and the biological objects deposited on it with increased angular tolerance compared to prior art slides. Summary of the invention

[0014] To this end, an object of the invention is a method for manufacturing an optical slide intended to receive a biological sample in a total internal reflection microscopy device, said method comprising a design step of said optical slide and a material manufacturing step 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 that > ​​7¾ - the selection of a so-called illumination wavelength and an so-called optimal angle of incidence 6in according to the said total internal reflection microscopy device for which the said optical slide is intended - the design of a first structure comprising a first stack of dielectric thin films arranged above a transparent substrate layer (SB) in a predetermined spectral region including said illumination wavelength, said first stack comprising

[0015]

[0016]

[0017]

[0018] N > 7 of successive and alternating thin layers of the first dielectric material and the second dielectric material, the respective first thickness of each dielectric thin layer and said number N of dielectric thin layers being adapted so that the first structure exhibits resonant absorption at the optimal illumination wavelength and angle of incidence θ1 in the total internal reflection regime - the determination of a first function F1 representing a transmission value of the first structure in the predetermined spectral region as a function of the number of dielectric thin layers and the thickness of each dielectric thin layer, and the determination of a second function F2 representing the intensity of an evanescent electric field generated by the optical plate in total internal reflection at the illumination wavelength and the optimal angle of incidence θ1 as a function of the number of dielectric thin layers and the said first thickness of each dielectric thin layer - the design of said optical plate which includes a second stack of dielectric thin films called the optimized stack arranged above the substrate (SB), the optimized stack comprising a number M < N, M>7 of successive and alternating dielectric thin films of the first dielectric material and the second dielectric material, each of the layers having a respective optimized thickness, the optimized thicknesses and the number M of dielectric thin films being determined by an optimization of a merit function F based on the first function F y and the second function F2 so that said optical plate has a first resonant absorption at the illumination wavelength and at a first angle of incidence in the total internal reflection regime and has a second resonant absorption at the illumination wavelength and at a second angle of incidence &2 in the total internal reflection regime, with _ m _ ni G Fq 4 o.3 o 1 and #2 < Gin < 0i or < 0^ < #2- . he 21 [ ' ~ J Preferably, the merit function F is such that F = aF^ + ( 1 - a ) F2, with 0 < a < 1 a parameter allowing to weight a respective weight of the first function F} and the second function F2 in said optimization. Preferably, a is between 0.3 and 0.7. Preferably, the optimization is such that _ p - e [0 4 0 ' 1 5 0 J According to one embodiment, the optimization consists of finding the optimized thicknesses and the number M of dielectric thin films 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.

[0019] According to one embodiment, in the first stacking, an upper layer of the first stacking is in the second dielectric material and has a first thickness between 1. ^in! 4 and 1 -82^ / 4 and wherein the N -1 lower layers of the first stacking each have a first thickness between 0.94;„ / 4 and 1-14^ / 4 and wherein in the optimized stacking an upper layer of the first stacking is in the second dielectric material.

[0020] According to one embodiment, the first function Fl is representative of a transmission of fluorescence radiation indirectly induced by an absorption of said illumination wavelength in total internal reflection in said first illuminated stack with said optimal angle of incidence 6fn, 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°.

[0021] Another object of the invention is an optical slide for receiving a biological sample in a total internal reflection microscopy device, the optical slide comprising: - a substrate layer transparent in a predetermined spectral region - an optimized stack arranged above the substrate and comprising M > 7 successive and alternating dielectric thin layers of a first dielectric material and a second dielectric material, each layer having a respective optimized thickness, the optimized thicknesses and the number M of dielectric thin layers being adapted so that said optical plate has at least: • a first resonant absorption at a wavelength called illumination Δn and at a first angle of incidence in the regime of total internal reflection, and • a second resonant absorption at the illumination wavelength and at a second angle of incidence #2 in the regime of total internal reflection, with Ae = |01-02|e[0.40;3°]-

[0022] According to one embodiment: - the first dielectric material has a refractive index ni between 1.8 and 3.5; - the second dielectric material has a refractive index n2 between 1.2 and 1.7.

[0023] According to one embodiment, the first material is based on Nb2O5, the second The material is SiO2 based.

[0024] According to one embodiment, the number M of dielectric thin films is less than 30, preferably less than 20.

[0025] According to one embodiment, the optimized thicknesses and the number M of dielectric thin films are such that said optical plate has a thickness less than 5 and preferably between 0.5 ltm and 2 lim.

[0026] According to one embodiment, the optimized thicknesses are greater than or equal to 10 nm.

[0027] Another object of the invention is a total internal reflection microscopy device comprising: - an optical blade according to the invention - a light source suitable for emitting a light beam with 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 9in - a detector adapted to detect fluorescence radiation emitted when a sample is deposited in correspondence to 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. Brief description of the drawings

[0028] Other features, details and advantages of the invention will become apparent from the description given with reference to the accompanying drawings provided by way of example, which represent, respectively:

[0029] [Fig. 1 A] and

[0030] [Fig.1B] a schematic view of a total internal reflection microscope of the prior art;

[0031] [Fig.2], a schematic view of a total reflection microscopy device internal according to the invention;

[0032] [Fig. 3], a schematic view of an optical blade according to the invention, intended to receive a biological sample in a total internal reflection microscopy device;

[0033] [Fig.4A], the absorption of an optical plate according to a preferred embodiment of the invention in total internal reflection regime, as a function of the angle of incidence and the wavelength of the incident beam;

[0034] [Fig.4B], the enhancement factor of the evanescent electric field generated at the surface of an optical blade according to a preferred embodiment of the invention in total internal reflection regime, as a function of the angle of incidence of the incident beam;

[0035] [Fig.5A], and

[0036] [Fig.5B], the enhancement factor of the evanescent electric field generated at the surface of an optical blade according to a preferred embodiment of the invention in total internal reflection regime, as a function of the angle of incidence of the incident beam, for two different divergences of the incident beam

[0037] [Fig.6], the normalized enhancement factor with respect 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 regime, as a function of the angle of incidence of the incident beam,

[0038] [Fig.7], a schematic representation of the steps of the design phase of the manufacturing process according to the invention.

[0039] In the figures, unless otherwise indicated, the elements are not to scale and identical reference numerals designate identical elements or identical steps. Description of embodiments

[0040] Figure 2 illustrates in a simplified manner a total internal reflection microscopy device D according to the invention. The device D comprises an optical plate LO according to the invention, a light source SL, an optical device Obj and a fluorescence detector Det.

[0041] We will first describe the operation of device D before detailing the structure of the optical blade LO. Finally, we will describe a manufacturing process according to the invention for obtaining the optical blade LO.

[0042] The optical LO blade is a blade, preferably biocompatible, intended to receive a biological sample E for microscopy imaging purposes according to an RTL configuration. An example of an optical blade structure according to the invention is described later in relation to Figure 3, which schematically illustrates a section along the plane of the optical LO blade according to the invention.

[0043] The light source SL is adapted to emit a light beam Lin having a predetermined illumination wavelength. The source SL 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, which is at least partially transmitted by the optical plate LO.

[0044] The optical device Obj is adapted to shape the light beam Lin so that it illuminates the optical plate with an optimized angle of incidence 0in greater than the critical angle.

[0045] Preferably, as illustrated in Figure 2, the optical device Obj is a microscope objective. The microscope objective may have a variable focal length and a variable numerical aperture (greater than 1.45). It comprises optics or a more or less complex assembly of optical lenses capable of forming the light beam Lin by focusing it towards the optical slide. It is also capable of collecting the reflected and / or backscattered beam from the optical slide within 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 may have a large numerical aperture (NA), for example, on the order of 1.45, which makes it possible to obtain a high lateral spatial resolution (perpendicular to the z-direction) since the lateral spatial resolution d is given by j_. Furthermore, an objective lens with a high numerical aperture (ON) is advantageous because it allows the incident light beam (Lin) to be displaced relative to the optical axis (OA), thus achieving a strong deviation of the incident beam (Lin) by the lens. The beam (Lin) can then propagate at a high angle of incidence, sufficient to achieve a RTL (Reverse Light Throat).

[0046] Alternatively, the optical device Obj comprises one or more lenses and / or mirrors for focusing the light beam Lin and optionally a prism or a metasurface so that the light beam Lin illuminates the optical plate with the optimized angle of incidence 8irr

[0047] This optimized angle of incidence 8jn is greater than the critical angle ôc at the interface between a surface SE of the slide intended to receive the sample E and the surrounding medium (typically liquid) containing the sample. The optimized angle of incidence 8in 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 Lin on said interface produces evanescent waves OE. Since the sample E is placed corresponding to a region of the surface SE where the evanescent waves are produced, the fluorophores of the sample will generate fluorescence radiation FL excited by the evanescent waves OE.

[0048] The emitted fluorescence radiation FL is then collected by the objective lens Obj and directed to the detector Det for detection. This detector Det is typically a CCD or CMOS camera whose spectral band is adapted for detecting the re-emitted fluorescence radiation FL from the sample E. It converts the received light intensity into an electrical signal 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 lens. Obj so as to be able to control these elements for the purpose of acquiring images of sample E in RTI regime.

[0049] By way of non-limiting example, the device D illustrated in Figure 2 is based on the principle of epifluorescence, the observation of which is carried out in a reflection configuration using, for example, a dichroic plate or mirror 50. This particular configuration makes it possible to separate the optical path taken by the excitation light Lin from the optical path taken by the fluorescence radiation FL.

[0050] Preferably, the optimized angle of incidence is between 62 and 80 degrees. The lower limit of the aforementioned range (62 degrees) is given by the refractive index value of the sample under study. As for the upper limit of the aforementioned range (80 degrees), it is defined according to the numerical aperture value used for microscopy observation. The typical numerical aperture of the microscope objective Obj of device D is greater than or equal to 1.45. The microscope objective Obj preferably has a variable numerical aperture and focal length.

[0051] We will now describe in more detail the structure of the optical blade according to the invention, as represented in [Fig.3].

[0052] The optical plate LO has a 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 Lin. The first face SE constitutes the interface where an enhancement of the evanescent electromagnetic field OE is produced by the optical plate 10.

[0053] According to the embodiment illustrated in Figure 2, the optical slide LO and the microscope objective Obj are arranged so 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, along the x-direction.

[0054] According to the invention, the optical LO slide comprises a substrate SB that is optically transparent in a spectral region that includes the illumination wavelength and the spectral range of the fluorescence FL radiation. For example, the substrate SB is a 1.5 index soda-lime glass microscope slide (or any other optically transparent support calibrated in thickness).

[0055] Furthermore, the optical blade LO comprises a stack of dielectric layers (hereafter referred to as the optimized EO stack) arranged directly above the substrate SB. This optimized EO stack is particularly well-suited to enable enhancement of the evanescent electric field OE produced at the surface SE in a confi RTI configuration of the Lin beam. Thus, when the device in Figure 2 is operating, the biological sample E, which is placed on the top layer, is illuminated by the Lin beam at the illumination wavelength after passing through the optical plate. More precisely, the Lin beam passes through the substrate SB, then the dielectric stack EO, until it reaches the interface between the top layer and the sample E at the optimized angle of incidence 0in. The evanescent wave OE created by the RTI has its intensity amplified by the resonant structure of the EO stack, which significantly increases the imaging performance of TIRE microscopy, particularly in terms of sensitivity and axial resolution.

[0056] As illustrated in Figure 3, this EO stacking is formed of a succession of M > 7 alternating thin films of a first dielectric material with refractive index (thin films referenced Cj) and a second dielectric material with refractive index n2 (thin films referenced C2), with «2 < nl ■

[0057] The thin layer intended to be in contact with sample E (called the top layer) is made of one of two dielectric materials. Preferably, this layer is made of a biocompatible dielectric material, for example SiO2, TiO2, Nb2O5... The upper face of this top layer corresponds to the SE face previously introduced. As for the SI face illuminated by the Lin beam, it corresponds to the lower face of the substrate SB.

[0058] In the LO optical plate of the invention, each of the dielectric layers has a respective optimized thickness chosen in particular as a function of the illumination wavelength Δθ„, the optimized angle of incidence of the incident polarization, and the refractive indices λ and ν. The manufacturing process and the design face of the LO optical plate of the invention will be detailed later in the description of Figure 7. It will be limited to briefly stating that the optimized thicknesses and the number M of dielectric thin layers are adapted so that the LO optical plate exhibits at least one double resonance at the illumination wavelength Δθ„ in RTI for two distinct angles of incidence. More precisely, the optimized thicknesses and the number M of dielectric thin layers are such that the optical plate exhibits: - 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 2;„ and at a second angle of incidence ^2 in RTI.

[0059] The angles of incidence and 72 are chosen so that Ai = |0 - 0^ s°it is between 0.4° and 3°, preferably between 0.4° and 1.5°. More precisely, we wish A0 to be on the order of magnitude of the microscope divergence therefore according to the systems

[0060] It is understood that the optical blade LO of the invention can include more than two resonances as long as the aforementioned conditions remain verified.

[0061] The EO stacking of dielectric multilayers coupled to the SB substrate ensures significant enhancement of the evanescent electromagnetic field OE at the interface between the optical plate and the sample, with an angular tolerance far superior to that of prior art optical plates. This enhancement increases the intensity of the fluorescence radiation FL, thereby improving the sensitivity of microscopic imaging and the axial resolution of the device D.

[0062] Indeed, prior art resonant optical plates are optimized to exhibit a narrow spectral and angular resonance for a predetermined optimal angle of incidence and for a theoretically planar illumination wave. They thus exhibit a very high "theoretical" enhancement factor for the evanescent electromagnetic field. However, according to the laws of geometric optics, the Lin beam exhibits a non-negligible natural angular divergence after passing through the lens (typically between 0.5° and 1°). Also, the beam incident on the LO optical plate actually presents a plurality of angles of incidence centered around the optimal angle of incidence. This implies that the enhancement factor of the evanescent waves OE induced by the optical plate for rays at angles of incidence other than the optimal angle of incidence is in practice significantly lower than the "theoretical" enhancement factor.

[0063] In the invention, the presence of two (or more) closely spaced resonances allows for partial overlap between the resonance bands. This overlap makes it possible to widen the angular tolerance of the optical plate LO so that it exhibits a "real" enhancement factor six to ten times greater than prior art optical plates (see Figure 6) (see more in the case of multi-resonances). The angular tolerance of the plate of the invention is specifically chosen to be greater than or equal to the divergence of the beam Lin after passing through the microscope objective Obj, which is typically between 0.5° and 1°.

[0064] Through numerous simulations and experiments, the inventors observed that this multiple resonance was only possible with an EO stack comprising a number M > 7 of dielectric thin films.

[0065] As mentioned previously, the optimized thickness of each layer of the stack is chosen, in particular, according to the illumination wavelength, the optimized angle of incidence, the polarization, and the refractive indices ν and ω². The optimized angle of incidence θin corresponds here to the angle of incidence at which the Lin beam must illuminate the optical plate at the illumination wavelength to obtain an evanescent wave enhancement factor ε₀ with a tolerance Optimized angular. It is between and 63 (or between and 9] if > 02) to benefit from the overlap of the first resonant absorption band and the second resonant absorption band. Preferably, the optimized thicknesses and the number M are such that , + ^) / 2-

[0066] 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 fabrication of the LO optical layer. Indeed, the precise control of the thickness of layers less than 10 nm thick is technically complex to achieve.

[0067] Preferably, the first dielectric material has a refractive index ni 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 Nb2O5, the second material is based on SiO2.

[0068] Preferably, the number M of dielectric thin layers is less than 30, preferably less than 20. Indeed, the inventors have observed that, compared to an optical LO plate 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 allows only a minimal (or even zero) gain in the enhancement factor of the evanescent waves.

[0069] Preferably, in order to limit the optical aberrations induced by the use of the optical plate in the device D, the optimized thicknesses and the number M of dielectric thin films are such that said optical plate has a thickness less than 5 and preferably between 0.5 Lim and 2 Lim. Preferred example:

[0070] According to a preferred embodiment of the invention, the optimized stack EO of the optical plate LO comprises 14 alternating dielectric layers of SiO2 and Nb2O5. The top layer of the stack (the one intended to be in contact with the sample) is made of SiO2. The optimized thicknesses of the dielectric thin films are, from the top layer to the bottom 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.

[0071] This structure is optimized for an illumination wavelength of 561 nm and for an optimized incidence angle θin = 68°. These optimized thicknesses are determined by the method in [Fig. 7] described later. The refractive indices of the layers are n2(SiO2) = 1.486 and ni(Nb2O5) = 2.292 at 561 nm, respectively.

[0072] The average transmission of this structure, angularly integrated between 0 and 80 degrees, and spectrally integrated between 561 and 700 nm, is 53%.

[0073] 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 top 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 θ = 68.4° and the second resonant absorption is centered on the angle θ = 67.65°. It should be noted 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 by RTI on the SE surface.Indeed, the relationship 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 approximately 1°. As mentioned previously, this angular tolerance value corresponds to the upper limit of the typical divergence of the Lin beam after its passage through the objective lens of the microscope Obj.

[0074] Figure 4B is a curve representing the value of the evanescent field enhancement factor generated in RTI by the optical plate according to the preferred embodiment of the invention as a function of the beam incidence angle at the illumination wavelength λin = 561 nm. The curve in Figure 4B corresponds to a horizontal cross-section of the preceding figure for the illumination wavelength λ2 = 561 nm, but for the intensity of the evanescent field and not the absorption. As expected, the curve in Figure 4B includes the two enhancement peaks associated with the first and second resonant absorptions B2. It can be observed that these resonant absorptions allow for an respective enhancement factor of the evanescent field intensity of approximately 75. However, this value of the enhancement factor is a "theoretical" value obtained for illumination with a plane wave.In practice, the divergence of the Lin beam must be taken into account to estimate the true value of the excitation factor.

[0075] To this end, Figures 5A and 5B represent a curve of the value of the enhancement factor of the intensity of the evanescent field generated in RTI as a function of the angle of incidence of the Lin beam, at an illumination wavelength of 561 nm and for two different divergences of the Lin beam. The curve in [Fig. 5A] is obtained for a divergence of 0.6°, while the curve in [Fig. 5B] is obtained for a divergence of 0.9°.

[0076] It is observed that taking into account the divergence of the beam produces an angular integration of the first and second resonant absorptions B^ B2 which makes this double resonance BY, B2 disappear in favor of a single resonance "Averaged" centered on the optimized angle of incidence 0in = 68°. This "averaged" resonance has an exaltation factor of lower value than the values ​​associated with simple resonances B^ B2 (i.e. the "theoretical" value of [Fig.4B]) but higher than that which can be obtained with a single optimized resonance.

[0077] Thus, for a Litl beam with an illumination wavelength of 56Imn and an optimized angle of incidence of 68°, the LO optical plate according to the preferred embodiment of the invention makes it possible to obtain an enhancement factor of approximately 35 for a divergence of 0.6° and approximately 30 for a divergence of 0.9°. Logically, a greater divergence implies a greater angular averaging of the first and second resonant absorptions B^B2 and therefore a greater reduction in the enhancement factor.

[0078] Figure 6 is a curve showing the value of the enhancement factor of the fluorescence radiation produced by the optical LO plate according to the preferred embodiment of the invention as a function of the divergence of the incident beam, for the optimized angle of incidence θin = 68°. The value of the enhancement factor is calculated by integrating over an angular cone from 0° to 80° of the fluorescence radiation FL, taking into account the average transmission of the optical LO plate, and is then normalized with respect to the value of the enhancement factor obtained by a glass plate.

[0079] In addition, [Fig.6] includes a shaded area which corresponds to the divergence range of a laser beam focused by a commercial microscope.

[0080] Figure 6 shows that the LO optical plate according to the preferred embodiment of the invention makes it possible to obtain high enhancement factors over a wide divergence range that encompasses most existing commercial microscopes. More specifically, the LO optical plate makes it possible to obtain a normalized fluorescence enhancement factor of approximately 7.

[0081] Although the curves in Figures 4A to 6 have been described in relation to the optical plate LO according to the preferred embodiment of the invention, those skilled in the art will be able to reproduce equivalent results without going out of the scope of the invention with different dielectric materials, and / or different optimized thicknesses and / or a different number of layers M by adapting them to a predetermined wavelength and optimized angle of incidence 0in.

[0082] We will now describe the manufacturing process for the optical blade LO according to the invention. This manufacturing process comprises a design phase for the optical blade followed by a physical manufacturing phase for the designed optical blade. The various steps A to E of the design phase are illustrated in [Fig. 7].

[0083] In a first step A, the first dielectric material with index / 2iet the second dielectric material of index tl2 such that «j > ^2- The material of index n2 is preferably silica for biological compatibility and the material of index ni can be for example Nb2O5, TiO2, HfO2 or Ta2O5.

[0084] In a second step B, the illumination wavelength Δn and the optimal angle of incidence are selected according to the total internal reflection microscopy device for which the optical plate is intended. Typically, this involves choosing an illumination wavelength equal to the wavelength of the light source of the microscopy device. The optimal angle of incidence θ is chosen so as to be greater than the critical angle of TIR for an interface formed between a liquid medium and a layer in the second dielectric material, and according to the numerical aperture value used for microscopy observation.

[0085] After step B, the design phase includes a step C consisting of designing a first structure comprising a first stack of dielectric thin films arranged on top of a substrate SB. The substrate SB is transparent in a predetermined spectral region comprising the illumination wavelength and the fluorescence radiation FL generated indirectly by the illumination wavelength Δλ.

[0086] The first stack comprises N >7 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 N of dielectric thin layers are adapted so that the first structure exhibits a single resonant absorption at the illumination wavelength Δin and the optimal angle of incidence in the total internal reflection regime.

[0087] This step C is known in itself and is carried out by means of optical thin film simulation and optimization software such as Optilayer or by means of a direct calculation.

[0088] According to one embodiment, in the first stack, the top layer of the first stack is made of the second dielectric material and has a first thickness between 1.92^ / 4 and 1.84.zn / 4. The exact value of the thickness of the top layer depends on the refractive indices of the dielectric materials, the desired resonant wavelength, and the desired resonant angle. Furthermore, the N - 1 lower layers of the first stack (i.e., those below the top layer) each have a first thickness between 0.92^ / 4 and 1.92^ / 4. Preferably, these are layers of thickness 1.92^ / 4.

[0089] For example, in order to design the optical plate of the preferred embodiment of the invention described above, the first stacking in step B comprises N=15 alternating dielectric layers of SiO2 and Nb2O5. The top layer is made of SiO2 and has a first thickness equal to 1.442^ / 4. The other layers have a first thickness equal to ÀitJ 4.

[0090] After step C, the design phase includes a step D consisting of determining two functions F1 and F2, each representing the two critical parameters of the optical plate for the intended application: the transmission of fluorescence radiation and the enhancement of the evanescent field generated in RTI.

[0091] More precisely, the function represents the value of the transmission of the fluorescence (FL) radiation as a function of the number of dielectric thin layers and the thickness of each dielectric thin layer. To clarify, this refers to the fluorescence (FL) radiation indirectly induced by 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 the angle of incidence of the fluorescence radiation on the optical plate, for example, for an angle of incidence between 0° and 80°, as well as over the spectral range associated with the fluorescence emission.

[0092] The function F2 represents the intensity of the evanescent electric field generated by the first RTI stack at the illumination wavelength and the optimal angle of incidence Bin as a function of the number of dielectric thin layers and the thickness of each dielectric thin layer

[0093] Indeed, the actual enhancement factor of the optical plate, that is to say that of the fluorescence radiation collected by the microscope objective, is multifactorial and depends on: - the transmission of the structure to fluorescence radiation. This must be maximized in order to increase the amount of fluorescence radiation collected. This means, for example, 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 enhancement factor of the evanescent field generated in RTI, which must also be maximized - the angular tolerance of this exaltation factor, as explained previously.

[0094] Simultaneous optimization of these three parameters is only truly possible by increasing the number of degrees of freedom. Therefore, it is necessary to construct the functions F1 and F2, which will be used in the next step E.

[0095] For example, according to the preferred embodiment of the invention, the functions Fj and F2 have the following form for the design of the optical blade LO: rZ=8OO7im r@=80deg | T(À,3)dÂdO Â=400nm J0=0 with T the transmission of the structure for a plane wave illuminated at an angle of incidence at the wavelength ' F2= ]E(Atn,ff)p dff

[0096] with E the amplitude of the electric field at the upper interface of the structure, A0 the angular range chosen for field optimization, typically between 0.2 and 1.5 degrees.

[0097] Finally, after the determination of functions Fj and F2, the design phase includes a final step E of optimizing the first stacking so that Obtain the optimized EO stacking of the LO optical plate. The optimized thicknesses and the number M<N, M > The 7 dielectric thin films of the optimized EO stack are determined by optimizing a merit function F based on the first function F1 and the second function F2 such that the optical plate presents: - the first resonant absorption at the Fit illumination wavelength and the RTI incidence angle, and - the second resonant absorption at the illumination wavelength and the second angle of incidence 02 in RTL

[0098] As mentioned above, the optimization is such that e^0 4° ' 3° ] and (F < 6in < or < Bin < 02.

[0099] This optimization is carried out using optical thin film simulation and optimization software such as Optilayer.

[0100] Preferably, the merit function F to be maximized is such that F = aF ] + ( 1 - a ) F2> with ® < a < a parameter allowing the respective weighting of the first function F1 and the second function F2 in the optimization. The parameter alpha influences the respective weights of fluorescence transmission through the slide with respect to the enhancement of the evanescent field and is preferably between 0.3 and 0.7 because both parameters are important and should both be considered non-negligible.

[0101] 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.

[0102] The optimization typically consists of finding the optimized thicknesses and the number M of dielectric thin films for which the value of the transmission of the fluorescence radiation is maximal and for which the intensity of the evanescent electric field is maximal.

[0103] According to a preferred embodiment, the optimization is such that — p - e [fl 4 ° ■ 1 5 ° J 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 actual amplification factor of the optical plate LO of the invention.

[0104] Regarding 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.

[0105] The invention thus offers the possibility of producing electromagnetic field-enhanced optical plates whose characteristics can be easily adapted according to the imaging parameters required by the microscopy system.

[0106] As mentioned 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 for the study, with known and controlled dispersion values ​​of the refractive index and absorption coefficient, will be preferred. Such characteristics should allow, at an optimized angle of incidence and a predetermined illumination wavelength of the optical plate in total internal reflection, improved angular tolerance optical absorption in the top layer of the optimized stack, thus enhancing the evanescent electromagnetic field at the interface of said stack.

Claims

Claims

1. 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: A. selecting a first dielectric material having a refractive index ni and a second dielectric material having a refractive index such that Wj > H2 B. the selection of a so-called illumination wavelength and an optimal angle of incidence Gin as a function of said total internal reflection microscopy device for which said optical slide is intended C. the design of a first structure comprising a first stack of dielectric thin layers arranged above a so-called substrate layer (SB) transparent in a predetermined spectral region comprising said illumination wavelength ^in, said first stack comprising N > 7 of successive and alternating thin layers of the first dielectric material and of the second dielectric material, a first respective thickness of each dielectric thin layer and said number N of dielectric thin layers being adapted so that the first structure exhibits resonant absorption at the illumination wavelength and at the optimal angle of incidence in the total internal reflection regime D. determining a first function F] 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 F2 representative of an intensity of an evanescent electric field generated by the optical plate in the total internal reflection regime at the illumination wavelength and at the optimal angle of incidence Gin as a function of a number of thin dielectric layers and of said first thickness of each thin dielectric layer E.the design of said optical plate which comprises a second stack of dielectric thin layers called optimized stack arranged above the substrate (SB), the optimized stack comprising a number M £ N, M>1 of successive and alternating dielectric thin layers of the first dielectric material and of the second dielectric material, each of the layers having a respective so-called optimized thickness, the optimized thicknesses and the number M of dielectric thin layers being determined by an optimization of a merit function F based on the first function F} and the second function F2 so that said optical plate has a first resonant absorption at the illumination wavelength and at a first angle of incidence in the total internal reflection regime and has a second resonant absorption at the illumination wavelength and at a second angle of incidence in the total internal reflection regime, with A0 = j# - 07\ G [ 0.4 0 ; 3 ° ] and ^2 < < #1 or 6*] < 6in < É*2.

2. Manufacturing method according to the preceding claim, in which said merit function F is such that F — aF^ + ( 1 - a ) F2, with 0 < a < 1 a parameter making it possible to weight a respective weight of the first function F} and of the second function F2 in said optimization.

3. Manufacturing method according to the preceding claim, in which a is between 0.3 and 0.

7.

4. A manufacturing method according to any preceding claim, wherein said optimization is such that A3 = 1.5°]-

5. A manufacturing method according to any preceding claim, wherein said optimization consists of finding the optimized thicknesses and the number M of dielectric thin layers for which said transmission value of the first structure in the predetermined spectral region is maximum and for which said intensity of the evanescent electric field is maximum.

6. A manufacturing method according to any one of the preceding claims, 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 1 - 14; / 4 and 1.84,M and wherein the N- 1 lower layers of the first stack each have a first thickness of between 0.9^, / 4 and 1.14, / 4.

7. Manufacturing method according to any one of the preceding claims, in which the first function Fl is representative of a transmission of fluorescence radiation induced indirectly by an absorption of said illumination wavelength 4 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°.

8. Device (D) for total internal reflection microscopy comprising: - an 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 layer (SB) transparent in a predetermined spectral region • a so-called optimized stack arranged (EO) above the substrate (SB) and comprising a number M > 7 of successive and alternating dielectric thin layers (Cj, 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 M 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 the total internal reflection regime,and a second resonant absorption at the illumination wavelength and at a second angle,

9. of incidence 02 in total internal reflection regime, with = ^e[0.4o;3°] - a light source (SL) adapted to emit a light beam (Lin) having the illumination wavelength Àin - an optical device (Obj) adapted to shape the light beam (Lin) so that it illuminates said optical blade with said optimized angle of incidence 0ln - a detector (Det) adapted to detect fluorescence radiation (FL) emitted when a sample (E) is deposited in correspondence with 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 (Lin), 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.

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