Optical microscope with resonator

Abstract

The optical microscope (200) includes a light source (201) that emits illumination light (202), an optical device including a microscope objective (205), a resonator (212) arranged between the optical device and the sample, the resonator having, in sequence in the direction of the optical axis of the microscope objective, at least one first layer having a first optical refractive index, at least one spacer layer having a second optical refractive index, and at least one waveguide layer having a third optical refractive index, the second optical refractive index being smaller than the first optical refractive index and the third optical refractive index, the resonator being oriented in a direction away from the optical device and having a support surface intended to receive the sample, and an optical detector (206), the optical device being arranged to collect light exiting the resonator, the exiting light including light scattered by the sample (204) and a non-scattered portion (215) of the illumination light.

Description

[Technical Field]

[0001] The present invention relates to the field of optical microscopy.

[0002] In particular, the present invention relates to the field of nanoparticle detection and characterization by optical microscopy. The present invention can be used to detect objects having characteristic dimensions in the range of 1 nanometer to 100 nanometers. Such objects include metal nanoparticles, nanoscale contaminants, and other objects of biological interest such as proteins and peptides. [Background technology]

[0003] Means for detecting nanoparticles include labeling said particles, for example by fluorescent labeling. However, such methods quickly reached their limits, as the effect of fluorescent labeling is time-limited, thus limiting the observation period. The quality of detection is also affected. In fact, the temporal resolution of images is limited. Furthermore, fluorophores degrade rapidly over time. Finally, this technique is difficult to implement, as fluorescent labeling requires extensive upstream preparation.

[0004] Various optical microscopy techniques, including dark-field or interference techniques, allow for the detection of nanoparticles by elastic scattering without the need for pre-labeling. The most common of these techniques are interferential scattering (iSCAT), coherent bright field (COBRi), and interferometric reflectance imaging sensor (IRIS). These currently allow for the observation of particles smaller than 10 nanometers.

[0005] A central issue in interferometry and dark field imaging is the signal-to-noise ratio (S / N ratio). Indeed, the goal of these techniques is to detect the signal of interest, which is the elastically scattered light by nanoparticles. However, various types of noise affect the detection of the signal of interest.

[0006] In fact, there is a lot of technical noise associated with fluctuations in measurement systems. In addition, the incident light source used in microscopes is subject to intensity fluctuations called photon noise, which are inherent in physical processes. Furthermore, scattering of the illuminating light beam causes random interference patterns called speckle noise.

[0007] WO 2018 / 011591 discloses an iSCAT type interference microscope in which a spatial filter can attenuate the illuminating light beam in order to obtain an enhanced contrast of the image. Summary of the Invention

[0008] The basic object of the present invention is to provide an optical microscope that allows for an improved signal-to-noise ratio for the individual detection of nanoparticles without labeling.

[0009] According to one embodiment, the present invention provides an optical microscope comprising: a light source emitting illumination light adapted to illuminate a sample to be imaged; an optical device including a microscope objective; a resonator having at least one first layer having a first optical refractive index, at least one spacer layer having a second optical refractive index, and at least one waveguide layer having a third optical refractive index, arranged sequentially in the direction of the optical axis of the microscope objective, wherein the second optical refractive index is smaller than the first optical refractive index and the third optical refractive index, the resonator having a support surface facing away from the optical device and intended to receive the sample; and an optical detector, wherein the optical device is arranged to collect light exiting the resonator and to guide the exit light from the resonator to the optical detector in order to form an image of the sample on the optical detector, the exit light including light scattered by the sample and a non-scattered portion of the illumination light.

[0010] These features enable several technical advantages, including increasing the effective scattering cross section of the particle, thereby increasing the signal of interest collected by the photodetector, and concentrating the light scattered by the sample into a very small solid angle, thereby enabling efficient, selective spatial filtering of the scattered light.

[0011] These effects allow for an improved signal-to-noise ratio: increasing the effective scattering cross section increases the signal of interest collected by the photodetector; concentrating the scattered light into a very limited solid angle without loss of the signal of interest allows the use of attenuating filters for stray light that introduce noise into the measurement of the signal of interest.

[0012] In particular, according to one embodiment, the present invention provides an optical microscope, comprising: a light source for emitting illumination light; an optical detector; an optical device including a microscope objective lens for receiving the illumination light in order to direct the illumination light onto a sample; and a resonator interposed between the optical device and the sample, the resonator comprising at least one first layer having a first optical refractive index, at least one spacer layer having a second optical refractive index, and at least one waveguide layer having a third optical refractive index, which are arranged sequentially in a direction of an optical axis of the microscope objective lens, the second optical refractive index being smaller than the first and third optical refractive indices, the resonator having a support surface oriented in a direction away from the optical device and intended to receive the sample, the objective lens directing the illumination light onto the resonator at an angle of incidence greater than a critical angle at an interface between the first layer and the spacer layer, such that the illumination light resonates and excites at least one mode in the waveguiding layer and generates enhanced evanescent waves. the optical device is arranged to collect light exiting the resonator and direct the exiting light from the resonator to the optical detector to form an image of the sample on the optical detector, the exiting light including light scattered by the sample and reflected portions of the illumination light.

[0013] These features enable several technical advantages, including i) resonant excitation of the waveguide increases the illumination intensity at the sample and also increases the amount of light scattered by particles contained within the sample. In effect, resonant excitation of one or more modes deposits energy within the resonator, increasing the electric field in and near the resonator; ii) increasing the effective scattering cross section of the particles; iii) producing an evanescent wave illumination intensity that is confined near the resonator-sample interface and uniform in a plane parallel to the interface; and iv) concentrating the light scattered by the sample into a very small solid angle.

[0014] These effects make it possible to improve the signal-to-noise ratio.

[0015] In general, the light scattered by the sample corresponds to the light emitted from the sample and the light emitted from the resonator plate that has interacted with particles contained in the sample, and the unscattered portion of the illumination light corresponds to the portion of the illumination light that is present in the emitted light beam without interacting with particles.

[0016] The vicinity of the resonator plate corresponds to a thickness of the sample located at a distance of less than a few hundred nanometers from the support surface of the resonator plate, for example less than 200 nanometers from this support surface.

[0017] According to some embodiments, the optical microscope described above may have one or more of the following features.

[0018] According to one embodiment, the optical device includes an amplitude filter arranged between the microscope objective and the optical detector, for example at the Fourier plane of the microscope objective or at an image plane of this plane, and configured to apply a first selective attenuation to the unscattered portion of the illumination light.

[0019] Therefore, scattered light makes up a larger portion of the intensity of the emitted light detected by the photodetector, in other words, such filtering increases the ratio between the amplitude of the scattered field and the amplitude of the field unscattered by the sample.

[0020] To manufacture such amplitude filters, various techniques are available, such as thin film deposition, in particular metal thin film deposition. The attenuation applied by the amplitude filter can be specified by an intensity transmission coefficient. According to one embodiment, the intensity transmission coefficient associated with the first attenuation is 10 -1 From 10 -6 Between 10 and 20, preferably 10 -2 From 3.10 -4 For example, 10 -3 An intensity transmission coefficient close to 0.01 is usually suitable when using a detector with a well capacity of 10,000.

[0021] According to one embodiment, the intensity transmission coefficient associated with the first attenuation is 10 -6 This ensures that the transmittance of the reflected field is essentially zero, resulting in a dark field.

[0022] According to one embodiment, the light scattered by the sample is composed of a first portion of scattered light from a resonant excitation mode and a second portion of scattered light, and the amplitude filter is further configured to apply a second selective attenuation to the second portion of scattered light. Such an amplitude filter allows for the selection of scattered light around a specific angle corresponding to radiative leaks of one or more waveguide modes by attenuating the remaining portion of scattered light. Thus, the scattered field via the waveguide modes is transmitted unattenuated to the photodetector. Such an amplitude filter can be used in a dark-field or bright-field interferometric configuration.

[0023] According to one embodiment, the intensity transmission coefficient associated with the second attenuation is 10 -1 From 10 -6 is included between.

[0024] According to one embodiment, the intensity transmission coefficient associated with the second attenuation is 10 -6 is less than.

[0025] According to one embodiment in a dark field configuration, the intensity transmission coefficient associated with the first attenuation and the intensity transmission coefficient associated with the second attenuation are 10 -6 Thus, the amplitude filter is configured to apply an overall attenuation to the reflected and scattered fields by the sample, except around a specific angle corresponding to the radiative leakage of a guided mode or modes.

[0026] According to one embodiment of the interference arrangement, the amplitude filter is configured to apply a first selective attenuation to the unscattered portion of the illumination light and a second selective attenuation to the second portion of the scattered light. Preferably, in this example, the intensity transmission coefficient associated with the first attenuation is greater than the intensity transmission coefficient associated with the second attenuation. For example, the intensity transmission coefficient associated with the first attenuation is greater than 10 -1 From 10 -6 and an intensity transmission coefficient associated with said second attenuation is between 10 -1 From 10 -6 is included between.

[0027] According to one embodiment, the optical device comprises two converging lenses arranged to image the Fourier plane of the microscope objective onto the amplitude filter.

[0028] Thus, the amplitude filter can attenuate the unscattered part of the illumination light, the position of which is already known on the Fourier plane, and this attenuation can be done precisely and selectively.

[0029] According to one embodiment, the illumination light is a light beam, preferably a laser beam.

[0030] Thus, the illumination light may be coherent and monochromatic.

[0031] According to one embodiment, the illumination light is emitted by a light emitting diode (LED).

[0032] According to one embodiment, the illumination light is monochromatic and has a wavelength comprised between 400 and 1300 nanometers, preferably between 450 and 532 nanometers.

[0033] According to one embodiment, the resonator further comprises at least one partially reflecting mirror.

[0034] According to one embodiment, said mirror is a Bragg mirror, in particular it is a partially reflecting mirror.

[0035] According to one embodiment, the resonator includes a plurality of spacers and a plurality of waveguides, each spacer of the plurality of spacers being arranged to contact at least one of the plurality of waveguides.

[0036] According to one embodiment, at least two first spacers of said plurality of spacers have different thicknesses, with suitable thicknesses generally comprised between 100 nanometers and 1 micrometer.

[0037] According to one embodiment, at least two second spacers of said plurality of spacers are made of different materials.

[0038] According to one embodiment, at least one spacer of said plurality of spacers consists of magnesium fluoride.

[0039] According to one embodiment, at least two first waveguides of said plurality of waveguides have different thicknesses, suitable thicknesses being generally comprised between 10 nanometers and 500 nanometers.

[0040] According to one embodiment, at least two second waveguides of said plurality of waveguides are made of different materials.

[0041] According to one embodiment, at least one waveguide of said plurality of waveguides is made of titanium dioxide.

[0042] According to one embodiment, the resonant mode of the resonator is a surface wave.

[0043] According to one embodiment, the optical device comprises at least one converging lens through which the emitted light passes, the converging lens being configured to image an object plane of the microscope objective onto the optical detector.

[0044] According to one embodiment, the microscope includes or is connected to an image processing system, which is configured to record a plurality of images successively detected by the optical detector, combine the plurality of images into a reference image, and process at least one image detected by the optical detector with the reference image to suppress static signals.

[0045] To do this, it is possible to subtract a reference image from the detected image or from each detected image, which results in an image or images in which only dynamic signals remain in time, and not static signals, thus filtering out all static noise.

[0046] According to one embodiment, the image processing system is configured to apply a convolution filter to at least one image detected by the photodetector.

[0047] According to one embodiment, the convolution filter is a Gaussian filter. According to one embodiment, the optical microscope includes or is connected to an image processing system, the image processing system being configured to determine a contrast of an image detected by the optical detector and to determine at least one parameter of particles contained in the sample as a function of the contrast, the parameter being selected from the group consisting of a mass of a particle and a position of the particle in the optical axis direction.

[0048] According to one embodiment, the optical detector may be a digital camera.

[0049] According to one embodiment, the light source and the optical device are arranged to illuminate a sample received by the reflective surface of the resonator in the reflective arrangement.

[0050] According to one embodiment, the optical device receives the illumination light to direct it to the sample, and the microscope objective lens of the optical device directs the illumination light onto the resonator at an angle of incidence greater than the critical angle at the interface between the first layer and the spacer layer, so that the illumination light resonantly excites at least one mode in the waveguiding layer and illuminates the sample with an enhanced evanescent wave.

[0051] According to one embodiment, the part of the exiting light that is not scattered by the sample is a reflected part of the illumination light.

[0052] According to one embodiment, the optical device comprises a polarizing or non-polarizing beam splitter plate that reflects the illumination light towards the microscope objective and is traversed by the exiting light.

[0053] According to one embodiment, the optical detector is a first optical detector, the optical microscope includes a second optical detector, the optical device includes a non-polarizing beam splitter plate that receives the emitted light and splits the emitted light into a first portion of emitted light toward the first detector and a second portion of emitted light toward the second detector, the first portion of emitted light including a first portion of reflected light and a first portion of scattered light, and a phase mask is positioned to be traversed by the first portion of emitted light, and the phase mask is configured to apply a phase shift between the first portion of reflected light and the first portion of scattered light.

[0054] Thus, a difference in light intensity can be measured between the first optical detector and the second optical detector.

[0055] According to one embodiment, the phase mask is a first phase mask, and the optical device further includes a second phase mask arranged to be traversed by the second output light portion, the second output light portion including a second reflected light portion and a second scattered light portion, the second phase mask configured to apply a phase shift between the second reflected light portion and the second scattered light portion, and the first phase mask and the second phase mask having different phase characteristics.

[0056] Thus, the phase shift between the first and second phase masks is configured to minimize background noise and maximize contrast by subtracting the two images.

[0057] According to one embodiment, the optical device includes an optical condenser that receives the illumination light emitted from the light source, and the optical condenser is configured to focus the illumination light to a region in the Fourier plane of the microscope objective lens away from the optical axis of the microscope objective lens to generate the incident angle.

[0058] Therefore, the angle of incidence can be selected to exceed the critical angle at the interface of the resonator.

[0059] According to one embodiment, the resonator is positioned along the optical axis of the microscope objective between the microscope objective and the light source, such that the light source is adapted to illuminate the sample received by the support surface of the resonator when transmitted through it.

[0060] According to one embodiment, the light source is arranged to emit an incident light beam that illuminates the support surface of the resonator at normal incidence.

[0061] The invention will be better understood and other objects, details, features and advantages will become more apparent from the description of some particular embodiments of the invention, given by way of non-limiting illustration and with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0062] [Figure 1] FIG. 1 illustrates an example of an optical microscope in a transmission configuration. [Figure 2] 1 shows an optical microscope in a reflective configuration according to a first embodiment, including a resonator plate; [Figure 3] FIG. 10 shows an optical microscope in a reflective configuration according to a second embodiment, including the resonator plate. [Figure 4] 3 is a diagram showing a microscope objective lens of the optical microscope in FIG. 2 and the resonator plate. FIG. [Figure 5] 4 shows the scattering of light within the cavity of the optical microscope in FIG. 2 and by nanoparticles. [Figure 6] 3 is a schematic diagram showing the coupling of light waves within the resonator plate of the optical microscope in FIG. 2. [Figure 7] 1 is a graphical representation of the angular distribution of energy scattered by a nanoparticle. [Figure 8] 8 is a graphical representation of the dispersion relation within the resonator plate in FIG. 7. [Figure 9] 1 shows a spatial filter that can be used in a first variant optical microscope operating in dark field. The black parts are opaque. The spatial filter passes most of the scattered light that is concentrated around the angle defined by the resonator and intersects with the reflected light beam. [Figure 10] FIG. 10 shows a spatial filter that can be used in an optical microscope with a resonator plate according to a second variant that attenuates the reflected field and allows it to operate in interference mode. [Figure 11] FIG. 10 shows a spatial filter that can be used in an optical microscope with a resonator plate according to a third variant, which significantly attenuates the reflected light and makes it possible to separate the useful signal from the noise. [Figure 12] FIG. 10 shows a representation of a resonator plate according to a first alternative using a pair of dielectric layers. [Figure 13] FIG. 10 shows a representation of a resonator plate according to a second alternative using a mirror. [Figure 14]FIG. 10 shows an optical microscope in a reflective configuration according to a third embodiment. [Figure 15] 1 is a graphical representation of the results obtainable from a prior art arrangement. [Figure 16] 10 is a graphical representation of the results that can be obtained in the dark field configuration of an optical microscope using the filter of FIG. 9. [Figure 17] 11 is a graphical representation of the results that can be obtained in an interference configuration of an optical microscope using the filter of FIG. 10. [Figure 18] 1 is a graphical representation of the results that can be obtained with different distances between the nanoparticles and the resonator plate. [Figure 19] FIG. 10 shows an optical microscope in a transmission configuration including a resonator plate according to a fourth embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0063] An embodiment of an optical microscope having a resonator capable of detecting light scattered by a minute object with high performance will be described below. To this end, the concept of an optical microscope that is useful for understanding the present invention will first be described with reference to FIG.

[0064] FIG. 1 shows an example of a transmission electron microscope 1. The transmission electron microscope 1 detects an incident intensity I′ i =M 2 I i For convenience, the incident intensity I i is defined in the image plane of the camera 9, and the intensity I′ of the incident ray 2 is defined in the plane of the particle i is the coefficient M 2 where M is the magnification of the microscope. Nanoparticle 3 is illuminated by incident light ray 2. The illumination of nanoparticle 3 by incident light ray 2 results in a scattered intensity I s This becomes a source of scattered light 4 with

[0065] The transmission microscope 1 comprises a microscope objective 5, an optical device, and a camera 6. The optical device comprises two converging lenses 7, 8, a spatial filter 9, and a tube lens 10, which form a mount 4-f. The mount 4-f allows the Fourier plane of the microscope objective 5 to be projected onto the spatial filter 9.

[0066] The objective lens 5, the optical device, and the camera 6 share a common optical axis. The incident light ray 2 is parallel to the common optical axis. The incident light ray 2 and the scattered light 4 pass through the objective lens 5 and the optical device before being imaged onto the camera 6.

[0067] The camera 6 receives a final signal that is a superposition of the incident light beam 2 and the scattered light beam 4. The final signal may include an interference term due to the phase shift Δθ between the incident light beam and the scattered light.

[0068] The detected strength of the final signal is given as:

number

[0069] The nanoparticle 3 has an effective scattering cross section σ, which is proportional to the square of its volume. Furthermore, the camera 6 measures the collected scattered energy f col The magnification of the transmission microscope 1 is M. The power scattered to the camera pixel is therefore:

number

[0070] Furthermore, said scattering power is distributed at the level of the surface S of the Airy spot corresponding to the image of the nanoparticle 3 on the camera 6. The scattering power can therefore also be expressed as:

number

[0071] This allows us to calculate the effective scattering cross section σ, the fraction of scattered energy collected f, and the equation for the detected intensity as follows: col , and can be rearranged as a function of the magnification M.

number

[0072] By way of quantitative example, these physical quantities have the following values: The wavelength of incident light ray 2 is 450 nanometers, and the diameter of nanoparticle 3 is 3 nanometers. The optical refractive index of nanoparticle 3 is 1.5, and nanoparticle 3 is suspended in water, which has an optical refractive index of 1.33. Therefore, the effective scattering cross section is 7.8 × 10 -14 μm 2 This becomes: The magnification M is 100. The radius of the Airy spot can be 400 nm on the image plane of the objective lens 5, i.e., 40 μm on the image plane of the camera 9. In this case, the surface area of ​​the Airy spot can be 5000 μm on the image plane of the camera 9. 2 The microscope objective lens 5 is an oil microscope, and the numerical aperture is 1.45. Therefore, the collection factor is 42%. In this case, the following numerical relationship is obtained:

number

[0073] The transmission microscope 1 can be used in an interference (or bright-field) or dark-field configuration. If the transmittance of the centrally located attenuator (spatial filter 9) is zero, the microscope is in a dark-field configuration. Schematically, if this transmittance is non-zero, the microscope is in an interference configuration. In fact, the dark-field configuration is obtained when the interference term becomes negligibly small compared to the direct scattering term of the nanoparticles.

[0074] In the interference configuration, the camera 6 receives both the incident light beam 2 and the scattered light 4, which interfere with each other.

[0075] As shown in Figure 1, the incident light beam 2 is more or less attenuated by the spatial filter 9. In the dark field configuration, the transmission coefficient of the spatial filter 9 is zero.

[0076] The tube lens 7 is configured to focus the scattered light 4 onto the object focal plane of an intermediate focusing lens 8 .

[0077] The spatial filter 9 includes a mask disposed on a common optical axis. The tube lens 7 and intermediate convergence lens 8 focus the incident light beam 2 onto the mask of the spatial filter, and the spatial filter 9 is disposed a focal distance away from the intermediate convergence lens 8.

[0078] The spatial filter 9 passes the filtered light beam 11. Thus, the camera receives the scattered light 4 and the filtered light beam 11, and the scattered light 4 passes through the final converging lens 10 and is focused onto the camera 6 at the image focal plane of the final converging lens 10.

[0079] In the dark field configuration, the incident intensity reaching the camera is zero and the detected intensity is given by:

number

[0080] If the magnification M value is 100, the detected intensity is 6.5 x 10 -14 I i is.

[0081] The average number N of photons detected by a pixel in area S' over a period τ is I det Considering photon noise (or shot noise) as the dominant noise source, and expressing the intensity in photons per second and per unit area, the signal-to-noise ratio is given by

number

[0082] 100 μm 2 The regions S′ and 10 10 s -1 μm -2 For an intensity of , the signal-to-noise ratio is therefore given by:

number

[0083] Averaging 10 pixels with a 1-second acquisition time results in a signal-to-noise ratio greater than 1. However, in practice, there is background noise that is greater than the 3-nm particle signal. It is difficult to detect particles smaller than 10 nanometers in diameter. Therefore, improving the signal-to-noise ratio is an important issue.

[0084] For bright-field interference microscopy, the detected intensity can be expressed as:

number

[0085] Therefore, the signal to noise ratio can be expressed as:

number

[0086] The signal-to-noise ratio is then twice as large as in the dark-field configuration, while the measured signal is significantly larger in the interferometric configuration. The difference can be defined by introducing the contrast c as follows:

number

number

[0087] In the interference configuration, the amplitude of the signal is therefore 2cI i which means that in the dark-field configuration, c2 I i becomes.

[0088] The intensity detected in the interferometric configuration is higher, allowing it to rise above the background noise that limits detection in the dark-field configuration. Furthermore, this speeds up the collection of electrons on the camera pixels and therefore image acquisition.

[0089] However, the incident intensity I i The spatial variation of the signal (speckle variation) is much larger than the signal and therefore needs to be subtracted. To distinguish between background noise and useful signals, a detector is needed that can sense very small variations in the signal. This concerns an improvement in the signal-to-noise ratio and is therefore also very important for bright-field configurations.

[0090] 2, 3, 14 and 19 show four embodiments of optical microscopes 100, 200, 700, 800, comprising a light source 101, 201, 701, 801 emitting an illumination light 102, 202, 502, 602, 702, 802 adapted to illuminate a sample 133, 233, 733, 833 to be imaged, an optical device comprising a microscope objective 105, 205, 705, 805, and a resonator having, arranged sequentially in the direction of the optical axis of the microscope objective, at least one first layer 241, 541, 641 with a first optical refractive index, at least one spacer layer 242, 542, 642 with a second optical refractive index and at least one waveguide layer 243, 543, 643 with a third optical refractive index, wherein the second optical refractive index is greater than the first optical refractive index. and an optical detector (106, 206, 706, 716, 806), the optical device being arranged to collect light exiting the resonator and direct exit light from the resonator to the optical detector (106, 206, 706, 716, 806) to form an image of the sample (133, 233, 733, 833) on the optical detector (106, 206, 706, 716, 806), the exit light including light scattered by the sample (104, 204, 504, 604, 704, 804) and a non-scattered portion (115, 215, 715, 815) of the illumination light.

[0091] The resonators are actually in the form of resonator plates 112, 212, 512, 612, 712, 812.

[0092] In the first, second and third embodiments of the optical microscope 100, 200, 700, the light source 101, 201, 701 and the optical device are arranged to illuminate the sample 133, 233, 733 in reflection, which is received by the support surface of the resonator 112, 212, 712. To this end, the sample receives the incident light emitted by the light source after passing through the resonator plate 112, 212, 712, as will be described in more detail below. In this configuration, the support surface of the resonator plate faces away from the light source.

[0093] For this purpose, the optical device comprises a polarizing beam splitter plate 114, 214, 714 arranged on the optical axis of the microscope objective 105, 205, 705 and arranged to reflect at least a part of the illumination light emitted by the light source 101, 201, 701 in the direction of the resonator plate 112, 212, 712. In one variant, the beam splitter plate can be non-polarizing.

[0094] In this reflective configuration, the incident light beam is transmitted through the resonator plate via the lower surface of the resonator plate opposite the support surface adapted to receive the sample 133, 233, 733, which will henceforth be referred to as the upper surface of said resonator plate 112, 212, 712.

[0095] Generally, in the reflective configurations of the first, second and third embodiments, an incident light beam emitted by the light source propagates along an optical path that first passes through a converging entrance lens 107, 207, 761. After passing through the converging entrance lens 107, 207, 761, the incident light beam is partially reflected by the polarizing beam splitter plate 114, 214, 714, passes through a microscope objective lens 105, 205, 705, and then through a resonator plate 112, 212, 712 to reach the sample 133, 233, 733 that is received by the support surface of the resonator plate.

[0096] After interaction of the incident light beam with the sample 133, 233, 733, an output light beam is emitted and leaves the resonator plate 112, 212, 712. The output light beam passes through the microscope objective lens 105, 205, 705 and is directed by the polarizing beam splitter plate 114, 214, 714. It propagates through the converging output lens 108, 208, 762. It includes scattered light 104, 204, 504, 604, 704 by the sample 133, 233, 733 and an unscattered portion 115, 215, 715 of the illumination light. When the optical microscope is used with reflected illumination, the unscattered portion 115, 215, 715 corresponds to the portion of the incident light beam that is reflected at the interface between the resonator plate 112, 212, 712 and the sample and does not interact with the sample 133, 233, 733. In this configuration, the non-scattering portions 115, 215, 715 are therefore reflective of the incident light ray.

[0097] The fact that the incident light beam passes through the resonator plate 112, 212, 712 before penetrating the sample allows for resonant excitation of the waveguide to enhance the illumination intensity of the sample and therefore enhance the amount of light scattered by particles placed in the vicinity of the resonator plate.

[0098] Furthermore, the evanescent wave illumination intensity is confined near the resonator-sample interface and is uniform in the plane parallel to this interface. The illumination is therefore confined to a small thickness, which avoids the noise introduced by scattered light from particles far from the interface in the case of unconfined illumination.

[0099] The resonator plate 112, 212, 712 enhances the effective scattering cross section of particles in its vicinity.

[0100] Additionally, the resonator plate 112, 212, 712 modifies the radiation pattern of particles in its vicinity, concentrating their scattered light into a very small solid angle, facilitating selective collection of light scattered by the sample and facilitating enhanced signal-to-noise ratios through spatial filtering, as described in more detail below.

[0101] In a fourth embodiment of an optical microscope 800 shown in FIG. 19, a light source 801 and an optical device are arranged to illuminate a sample 813 received by the support surface of a resonator plate 812 in transmission. Here, the sample receives the incident light beam emitted by the light source 801 directly without interacting with any other optical elements. The incident light beam 802 propagates at normal incidence relative to the support surface of the resonator plate. This sample 833 is therefore preferably illuminated at normal incidence. In this case, there are no optical elements arranged on the optical path of the incident light beam between the light source 801 and the resonator plate 812. The support surface of the resonator plate 812 is directed towards the light source 801.

[0102] In this transmission configuration, the incident light beam does not pass through the resonator plate 812 but penetrates the sample placed on the support surface of the resonator plate 812. The incident light beam 802 passes through the entire thickness of the sample 833 and then passes through the resonator plate 812.

[0103] The outgoing light beam is emitted away from the resonator plate 812. It passes through the microscope objective lens 105 and propagates through the converging exit lens 808, like an optical microscope operating in reflection.

[0104] The exiting light beam includes light 804 scattered by the sample 833 and an unscattered portion of the illuminating light 815. When an optical microscope is used with transmitted illumination, the unscattered portion 815 corresponds to the portion of the incident light beam that is transmitted through the sample and does not interact with particles contained within the sample 833.

[0105] The resonator plate 812 enhances the effective scattering cross section of particles placed in its vicinity.

[0106] The coupling of the light scattered by the sample to the resonator plate 112 concentrates the light scattered by the sample into a very small solid angle, thus facilitating selective collection of the light scattered by the sample and allowing for filtering to improve the signal-to-noise ratio.

[0107] The embodiments illustrated in the accompanying drawings will now be described in more detail.

[0108] 2 shows a first embodiment of an optical microscope 100 operating in reflection, comprising a resonator plate 112 intended to be in contact with a sample 133 containing one or more nanoparticles suspended in a solution. These nanoparticles can have a diameter comprised between 1 nanometer and 100 nanometers.

[0109] In fact, the sample 133 can rest on the upper surface of the resonator plate 112 by the action of its own weight. The resonator plate 112 is positioned horizontally. It is understood that there may be other configurations in which the placement of the sample in contact with the resonator plate 112 is ensured by other means.

[0110] The optical microscope 100 further includes a light source 101, a microscope objective lens 105, and a detector .

[0111] Here, the light source 101 is a laser light source and the incident light beam is an incident laser beam 102. The light source 101 is arranged to emit this incident laser beam 102 having a wavelength comprised between 400 nanometers and 1300 nanometers, for example. The incident laser beam 102 can be a broad beam that illuminates an extended area on the sample 133, or a focused narrow beam that scans an area of ​​interest on the sample 133.

[0112] The optical microscope 100 further includes optical devices including a converging entrance lens, herein referred to as the first converging lens 107, a converging exit lens, herein referred to as the second converging lens 108, and a polarizing beam splitter plate 114.

[0113] The incident laser beam 102 passes through the first converging lens 107 and is reflected by the polarizing beam splitter plate 114. The first converging lens 107 may be an optical condenser. The polarizing beam splitter plate 114 has a semi-reflective surface arranged such that the incident laser beam 102 has an incident angle of 45 degrees on the polarizing beam splitter plate 114. According to one embodiment, the incident angles of the incident laser beam 102 on the polarizing beam splitter plate 114 may have different values. The polarizing beam splitter plate 114 reflects one polarization component of the incident laser beam 102 and allows the other polarization component of the incident laser beam 102 to pass through in a transmitted form.

[0114] The incident laser beam 102 is reflected off a polarizing beam splitter plate 114 and directed towards a microscope objective 105. The microscope objective 105 may be an immersion objective containing immersion oil with a refractive index similar to that of glass. The microscope objective 105 has a numerical aperture adapted to generate excitation of a resonant mode as described below.

[0115] The optical microscope 100 is further configured such that the incident laser beam 102 is focused off-center relative to the center of the optical axis of the microscope objective 105. Thus, the tilt of the incident laser beam 102 on the sample 133 can be controlled by the distance from the optical axis at the Fourier plane 113.

[0116] Thus, at the output of the microscope objective 105 , the incident laser beam 102 illuminates the surface of the sample 133 at a predetermined angle of incidence controlled by the parameters of the optical microscope 100 .

[0117] The incident laser beam 102 passes through a delay plate 199 and then through an objective lens 105 to illuminate a resonator plate 112. The delay plate 199 introduces a phase difference between the two polarization components of the beam passing through the plate. The resonator plate 112 emits a reflected laser beam 115 and scattered light 104. The reflected laser beam 115 and scattered light 104 constitute the output beam. The reflected laser beam 115 is a reflection of the incident laser beam 102 at the region of interest. The scattered light 104 propagates over a wider angular range than the reflected laser beam 115.

[0118] The scattered light 104 and the reflected laser beam 115 pass through the objective lens 105 and through a polarizing beam splitter plate 114. The scattered light 104 and the reflected laser beam 115 pass through a second focusing lens 108 and are imaged onto a detector 106.

[0119] The second converging lens 108 may be a tube lens. The sensitive surface of the detector 106 may be positioned at the focal length of the second converging lens 108.

[0120] The detector 106 can be, for example, a CMOS or CCD type photo sensor. In particular, a sensor limited to 10,000 electrons per pixel can be used. The detector 106 also includes a memory for recording multiple successive images.

[0121] 3, a second embodiment is shown, in which the incident light beam is, for example, an incident laser beam 202. The incident laser beam 202 emitted from a light source 201 passes through a converging incident lens, referred to herein as a first converging lens 207, and is reflected by a polarizing beam splitter plate 214. The incident laser beam 202 is then reflected towards a microscope objective lens 205.

[0122] In the second embodiment, the incident laser beam 202 is focused off-center onto the polarizing beam splitter plate 214. The incident laser beam 202 is then reflected towards the microscope objective.

[0123] The incident laser beam 202 passes through a retardation plate 299, which, according to one embodiment, is a quarter wave plate.

[0124] An incident laser beam 202 is focused into the Fourier plane 213 of the microscope objective 205 .

[0125] The optical microscope 200 is further configured such that the incident laser beam 202 is focused off-center relative to the center of the optical axis of the microscope objective 205. Thus, the tilt of the incident laser beam 202 on the sample 233 can be controlled by the distance from the optical axis at the Fourier plane 213.

[0126] Thus, at the output of the objective microscope 205 , the incident laser beam 202 illuminates the surface of the sample 233 at a predetermined angle of incidence controlled by the parameters of the optical microscope 200 .

[0127] The resonator plate 212 sends back the reflected laser beam 215 and the scattered light 204 emitted from the sample, where the output beam is composed of the reflected laser beam 215 and the scattered light 204. The reflected laser beam 215 and the scattered light 204 pass through the microscope objective 205. The reflected laser beam 215 is focused at another off-center position on the Fourier plane 213 of the microscope objective 205.

[0128] The scattered light 204 and the reflected laser beam 215 pass through a polarizing beam splitter plate 214 and then through a convergent exit lens, here consisting of a first convergent exit lens 208, a second convergent exit lens 217, a spatial filter 209 and a third convergent exit lens 218, before being imaged onto a camera 206.

[0129] The image focal plane of the first convergent output lens 208 corresponds to the object focal plane of the second convergent output lens 217. The spatial filter 209 is disposed at the image focal plane of the second convergent output lens 217. Thus, the reflected laser beam 215 is focused on the spatial filter 209.

[0130] A third converging exit lens 218 focuses the reflected laser beam 215 and the scattered light 204 onto the sensitive surface of a camera 206 located at the image focal plane of the third converging exit lens 218 .

[0131] 4 to 6, three details of FIG. 3 are shown: FIG. 4 shows the microscope objective lens 205 and the resonator plate 212, and shows the propagation of light in the resonator plate 212;

[0132] Of course, the arrows depicted in Figures 4 to 6 are very schematic and partial representations of electromagnetic field propagation. They do not indicate the dimensions of the rays, but are intended only to indicate some important directions of propagation.

[0133] The microscope objective 205 is an oil-based objective that includes an immersion oil 228 having an optical refractive index identical to that of glass. The resonator plate 212 is positioned relative to the microscope objective 205 and is in contact with the immersion oil 228.

[0134] The resonator plate 212 is made up of a number of parallel layers arranged successively along the optical axis of the microscope. The resonator plate 212 comprises a glass plate 241, which is in contact with the immersion oil 228.

[0135] A spacer 242 is disposed adjacent to the glass plate 241. The spacer 242 has a smaller optical refractive index than the glass plate 241. According to one embodiment, the spacer 242 is composed of magnesium fluoride. According to one embodiment, the thickness of the spacer is 485 nanometers.

[0136] The spacer 242 is disposed between the glass plate 241 and the waveguide 243. The spacer 242 has a smaller optical refractive index than the waveguide 243. According to one embodiment, the waveguide 243 is made of titanium dioxide. According to one embodiment, the thickness of the waveguide 243 is 45 nanometers.

[0137] The waveguide 243 is in contact with the sample 233, in which one or more nanoparticles 203 are suspended.

[0138] 6, the optical refractive index n1 of the glass plate 241 is greater than the optical refractive index n2 of the spacer 242. Therefore, there is a critical angle at the interface between the glass plate 241 and the spacer 242, and total reflection occurs.

[0139] Referring to FIG. 4, an incident laser beam 202 is focused onto the Fourier plane at a critical distance from the optical axis of the microscope objective, the critical distance being such that the incident laser beam 202 is projected onto the resonator plate 212 at an angle of incidence greater than the critical angle mentioned above.

[0140] Thus, the incident laser beam 202 undergoes total internal reflection at the interface between the glass plate 241 and the spacer 242. The reflected laser beam 215 is projected towards the objective lens of the microscope 205 at the same angle of incidence and passes through the objective lens of the microscope 205.

[0141] The evanescent wave 252 penetrates the spacer 242. The thickness of the spacer 242 is selected to be on the same order of magnitude as the decay length of the evanescent wave 252 so that the evanescent wave 252 is not completely attenuated at the interface between the spacer 242 and the waveguide 243. The thickness of the spacer 242 can be selected to control the electric field enhancement within the waveguide 243. The thicker the spacer 242, the greater the enhancement.

[0142] Referring to FIG. 6, when the optical refractive index n3 of the waveguide 243 is greater than the optical refractive index of the spacer 242, the evanescent wave 252 caused by the spacer 242 is refracted within the waveguide 243 and becomes a guided wave 253.

[0143] The frequency and angle of incidence of the incident laser beam 202 are configured so that the evanescent wave 252 resonantly excites a mode of the waveguide 243. To this end, a "phase-matching" configuration is realized.

[0144] Due to the resonant excitation of the modes of the waveguide 243, an augmented wave 254 propagates from the waveguide 243 into the sample 233, and its amplitude is amplified by a large amplification factor, for example, by a factor of 1000. The augmented wave 254 propagates through the sample 233 and illuminates the nanoparticles 203.

[0145] 5, nanoparticles 203 illuminated by augmented wave 254 emit scattered light 204 that propagates within resonator plate 212 toward a microscope objective (not shown). The dotted lines correspond to scattered light 204, and the solid lines correspond to the incident laser beam 202, the refracted light in the form of guided wave 253, reflected laser beam 215, and the radiation losses of guided wave 253 passing through spacer 242.

[0146] Referring to Figure 7, the energy density δ of scattered light 204 is plotted on the vertical axis as a function of the scattering angle α on the horizontal axis. Nanoparticles are disposed on the resonator plate 212. The wavelength used is 515 nanometers. The refractive indices of layers 241 to 243 are n1 = 1.518, n2 = 1.38, and n3 = 2.8, respectively, and the thicknesses of layers 242 and 243 are 484 nanometers and 45 nanometers, respectively.

[0147] The scattering angle α is measured relative to the optical axis within the immersion oil 228. Due to the coupling of the scattered light 204 with the resonant mode of the waveguide 243, based on a given scattering peak angle α at the output of the resonator plate, here about 66 degrees relative to the optical axis, the radiated power of the scattered light 204 in the direction of the microscope objective via the waveguide 243 is the largest portion (about 50%). The scattered light 204 is therefore a beam containing a cone of maximum energy corresponding to the scattering peak angle α.

[0148] 8, the dispersion relation of the resonator plate 212 is shown, which links the spatial wave vector within the resonator plate 212 with the wavelength of the incident laser beam 202. Therefore, the resonant modes of the guided wave 253 are not the same depending on the wavelength of the incident laser beam 202. Therefore, the scattering peak angle α of the scattered light is also a function of the wavelength of the incident laser beam 202.

[0149] We next explain how a spatial filter 209 can take advantage of such an angular distribution of energy scattered by the nanoparticles 203 .

[0150] The Fourier plane of the objective lens 205 is imaged onto the spatial filter 209 at the output of the first converging exit lens 208 and the second converging exit lens 217, so that the angular distribution of the scattered light 204 is preserved on the spatial filter 209. With respect to the transverse component of the propagating wave vector, the cone of maximum energy corresponds substantially to a circle, i.e., a ring, having a given thickness.

[0151] Thus, according to a first variant shown in FIG. 9, a spatial filter 209 can be used to transmit only the part of the scattered light that is present within the circle of maximum energy.

[0152] A usable first filter 20 is shown in Figure 9. The first filter 20 includes a first mask 98 configured to define a transmission ring 97 made up of two concentric sections and configured to transmit only scattered light corresponding to the maximum energy cone. Outside the transmission ring 97, the transmission coefficient of the first mask 98 is preferably zero (total attenuation).

[0153] To form a dark field filter, the first filter 20 may also include a second mask 96 located within the transmission ring 97 and configured to attenuate the reflected laser beam 215. Similarly, the second mask 96 preferably has a transmission coefficient of zero intensity (total attenuation), i.e., e.g., 10 -6 has a permeability coefficient that is less than

[0154] 10, a second filter 21 can be used. The second filter 21 includes only a second mask 96 configured to attenuate the reflected light 215, which can be totally or partially attenuated. All scattered light 204 is transmitted. The intensity transmission coefficient of the second mask 96 is substantially zero, e.g., 10 -6 If the transmission coefficient of the second mask 96 is not zero, e.g., 10 -3 If λ is greater than λ, then the second filter 21 is an interferometric brightfield filter. The optical microscope 200 is used in an interferometric configuration.

[0155] 11, there is shown a second interference brightfield filter 22. The second interference brightfield filter 22 has the same structure as the darkfield filter 20. However, here the second mask 96 has a non-zero transmission coefficient (partial attenuation) and is preferably greater than the transmission coefficient of the first mask 98.

[0156] 12 and 13, another embodiment of the resonator plate is shown.

[0157] Referring to Figure 12, elements similar or identical to those of the second embodiment are given the same reference numerals, increased by 300. Figure 12 shows that the number of layers in the resonator plate 512 can be increased. In particular, multiple spacers 542 and multiple waveguides 543 can be alternated. The evanescent wave propagates within the spacers 542.

[0158] The spacers 542 can have different thicknesses and / or different materials. The waveguides 543 can similarly have different thicknesses and / or different materials. Suitable thicknesses are typically between 100 nanometers and 1 micrometer.

[0159] The multiple waveguides 543 allow for coupling of resonant modes to more values ​​of wavelength and angle of incidence of the incident laser beam 202. Thus, the optical microscope becomes more robust to changes in wavelength and angle of incidence of the incident laser beam 502.

[0160] Additionally, multiple waveguides 543 allow for increased amplitude values ​​of resonant guided waves 553. Finally, multiple waveguides 543 allow incident light beam 502 to simultaneously excite multiple resonant modes, resulting in multiple angular peaks of energy in scattered light 504 relative to nanoparticle 503, with angular peaks of energy occurring in multiple directions.

[0161] Referring to Figure 13, elements similar or identical to those of the second embodiment are given the same reference numerals, increased by 400. Figure 13 shows that the resonator plate 612 can include a partially reflecting mirror 645. The mirror 645 can be a metal layer or a Bragg mirror, and can enhance the resonance phenomenon in the resonator plate 612. A decaying wave propagates within the spacer 642.

[0162] 12 and 13 show a schematic representation of total internal reflection at the interface between the first layer 541 or 641 and the spacer 542 or 642. However, this location of the interface where frustrated total internal reflection occurs is not limited. Layers of other materials can be inserted below the interface where frustrated total internal reflection occurs.

[0163] Referring to FIG. 14, a third embodiment of an optical microscope 700 including a resonator plate 712 can be used in an interferometric configuration using a balanced homodyne detection technique.

[0164] A light source 701 of the optical microscope 700 emits an incident laser beam 702 that passes through a converging incident lens, referred to herein as an initial converging lens 761, and is reflected on a polarizing beam splitter plate 714, referred to herein as a first polarizing beam splitter plate 714. The incident laser beam 702 is directed towards a microscope objective lens 705, passes through a resonator plate 712, and illuminates a sample 733 that is placed on the support surface of the resonator plate.

[0165] The exiting beam includes scattered light 704 and reflected light 715 that passes through the microscope objective lens 705 and the first polarizing beam splitter plate 714 .

[0166] The scattered light 704 and the reflected light ray 715 now pass through a converging exit lens made up of a first common lens 762, a second common lens 763, a spatial filter 764, and a third common lens 765. The first common lens 762, the second common lens 763, and the third common lens 765 are converging lenses.

[0167] The second beam splitter plate 766 is positioned behind the third general lens 765. The second beam splitter plate 766 first splits the reflected light beam 715 into a first reflected light beam 778 and a second reflected light beam 779, and then splits the scattered light beam 704 into a first scattered light beam 780 and a second scattered light beam 781.

[0168] The first reflected light ray 778 and the second reflected light ray 779 pass through the initial lens in the first arm 767 and then through the first phase mask 768. The final lens in the first arm 769 focuses the first reflected light ray 778 and the first scattered light ray 780 onto the first camera 706.

[0169] The second reflected light ray 779 and the second scattered light ray 781 pass through the initial lens in the second arm 770 and the second phase mask 771. The final lens in the second arm 772 focuses the second reflected light ray 779 and the second scattered light ray 781 onto the second camera 716.

[0170] The first phase mask 768 or the second phase mask 771 is configured to phase shift the first reflected light ray 778 with respect to the first scattered light ray 780 and the second reflected light ray 779 with respect to the second scattered light ray 781 by a phase shift angle φ, respectively. According to one embodiment, the phase shift angle φ can be π / 2 for the first phase mask 768 and −π / 2 for the second phase mask 771.

[0171] The data processing system 90 can subtract the first intensity received by the first camera 706 and the second intensity received by the second camera 716, where the difference between the first intensity and the second intensity is expressed as:

number

[0172] Such a representation allows us to keep only one interference term and filter the reference signal.

[0173] Figures 15 to 18 show the graphical results obtained by numerical simulations. In Figures 15, 17 and 18, which show the microscopy results for the interferometric configuration, the colour scale represents the contrast c as defined above.

[0174] In practice, the contrast c can be calculated or measured as c = I / I_ref, where I is the intensity of the camera image and I_ref is the intensity of the average image (or reference image). The contrast images shown in Figures 15, 17, and 18 are obtained by performing division on a pixel-by-pixel basis.

[0175] Figure 15 shows a comparison image obtained with a conventional microscope without a resonator plate. The central spot in the center of the interference pattern corresponds to the detection of a nanoparticle.

[0176] Figures 16 to 17 show the graphical results obtained by simulation in a scenario using a resonator plate.

[0177] Figure 16 corresponds to the case where a dark field filter 20 is used. In this figure, the gray scale represents relative intensity, with black (0) corresponding to essentially zero signal. The central spot corresponds to a nanoparticle. The central spot is sharper, more circular, and has a smaller diameter than in Figure 15, indicating improved resolution.

[0178] Figure 17 corresponds to the case where an interference filter 21 is used. The reflected laser beam 215 is filtered and attenuated. Figure 17 shows a central spot in the center of the interference pattern with more arcs than the comparison image. Nevertheless, the central spot is easier to distinguish than in the comparison image, indicating an increase in sensitivity.

[0179] The spatial filtering technique used means that the image of a point is taken in a series of circles, as well as rejecting all wave vectors except one, but this does not prevent the detection of individual particles, even if the sample contains multiple particles, provided that the particles are spatially separated from each other within the sample.

[0180] In this way, the resonator plate makes it possible to increase the sensitivity of the optical microscope, independent of the filters used.

[0181] 18 shows a set of contrast images in which the distance Z measured along the optical axis between the resonator plate 212 and the nanoparticle 203 varies from image to image, as indicated above each image. This example shows that the changes in contrast and shape of the interference pattern can be used to assess the distance from the nanoparticle 203 to the resonator plate 212.

[0182] To achieve better detection, image processing techniques can be implemented: a camera can record several consecutive images and combine them to create a reference image, which can also be the average of the consecutive images.

[0183] The reference image can then be subtracted from the received image to remove the reference signal and stationary noise.

[0184] In the case of moving nanoparticles, such as those suspended in solution, two successive images can be subtracted to remove background noise.

[0185] Additionally, a filter may be applied to the detected signal, for example a convolution filter, which may be a Gaussian filter.

[0186] The contrast of an image of a particle detected in bright-field mode is proportional to the mass of the particle present in the sample. Image processing therefore makes it possible to perform a quantitative measurement of this contrast and, by comparing it with a calibration signal previously measured with particles of known mass, to infer the particle's mass. This technique is particularly effective for particles that do not absorb at the wavelengths used, such as proteins in the visible spectrum.

[0187] Particles of known mass are chosen that closely match the optical refractive index of the particles to be characterized, for example polymeric materials if the aim is to characterize organic materials.

[0188] In a fourth embodiment of an optical microscope 800 shown in FIG. 19, an incident light beam 802 passes through a sample 833 and nanoparticles scatter the light of the incident light beam 802 .

[0189] When the particle is near the resonator plate 812, some of the scattered light is coupled into the guided mode.

[0190] Thus, a portion of the scattered light 804 is coupled into the guided mode of the resonator plate 812, and another portion is not coupled. The outgoing scattered light 804 includes a first portion of scattered light that is coupled into the guided mode of the resonator plate 812 by particles located near the resonator plate 812, and a second portion of scattered light that is not coupled. The outgoing light is collected by the microscope objective lens 805 and propagates through a converging lens system.

[0191] The scattered light 804 and unscattered portion 815 of the incident light beam are collected by a photodetector, here a camera 806, after passing through a convergent exit lens, here consisting of a first convergent exit lens 808, a second convergent exit lens 817, a spatial filter 809, and a third convergent exit lens 818.

[0192] The image focal plane of the first convergent exit lens 808 corresponds to the object focal plane of the second convergent exit lens 817. The spatial filter 809 is located at the image focal plane of the second convergent exit lens 817, which is located at the image plane of the Fourier plane 813 of the microscope objective lens 805. Thus, the unscattered portion 815 of the incident light beam can be focused onto the spatial filter 809 and blocked by the filter.

[0193] A third converging exit lens 818 focuses the scattered light 804 onto the sensitive surface of a camera 806 positioned at the image focal plane of the third converging exit lens 218 .

[0194] Filter 809 attenuates the unscattered portion 815 of the exiting light beam and a second portion of the scattered light. This filter 809 may be, for example, similar to filter 22 of FIG. 11. For example, it may be an interference bright-field filter having a structure similar to filter 22 of FIG. 11, except that a second mask 96 that attenuates the illumination light is located in the center of the filter. For example, the second mask and the first mask 98 have different transmission coefficients. For example, the second mask has a transmission coefficient that is greater than the transmission coefficient of the first mask.

[0195] As with the optical microscopes 100, 200, 700 that illuminate the sample in reflection, as described in the first, second, and third embodiments, approximately half of the energy scattered by each particle in the sample 833 is contained in the resonant modes of the resonator plate 812, i.e., the first portion of scattered light 804. The filter 809 filters most of the outgoing beam so as to block as much of the unscattered portion 815 of the incoming beam 802 as possible, without blocking the scattered light, and in particular the first portion of scattered light that is coupled to the resonant modes of the resonator plate 812. This is possible due to the directional emission of the first portion of scattered light that is coupled to the resonant modes of the resonator plate 812, as described with reference to the reflection embodiments of the optical microscope.

[0196] Advantageously, reducing the intensity of the incident light increases the contrast of the image of the sample formed on the camera sensor, as is the case with the reflected light microscope described above.

[0197] Unlike optical microscope embodiments that use reflected illumination of the sample, the incident light beam 802 illuminates the entire thickness of the sample 833, rather than just the first few hundred nanometers of the sample in contact with the resonator plate, as in the reflected configuration. Thus, in addition to the above capabilities, the optical microscope without filter 809 can also be used in a configuration that illuminates a larger volume of the sample to detect particles that are slightly out of focus. This is particularly useful for tracking the movement of scattered particles within the sample.

[0198] The transmission configuration of the optical microscope 800 prevents the exiting light beam from being subjected to back-reflections of the incident light beam at the multiple lenses of the microscope objective 805. These back-reflections exist in the reflection configuration and constitute parasitic signals, i.e., noise. Their removal improves the signal-to-noise ratio of the detected image.

[0199] Here, a transmission-type embodiment of an optical microscope with a resonator plate has been described. Obviously, other transmission embodiments are conceivable, in particular a simplified transmission embodiment similar to the first reflection embodiment, in which the second and third converging exit lenses and filters are eliminated. It is also conceivable to conceive a transmission embodiment of an optical microscope used in an interference configuration using the technique of balanced homodyne detection, in which the exiting light beam at the exit of the resonator plate travels an optical path similar to the optical path traveled by the exiting light beam after the first polarizing beam splitter plate in the third reflection embodiment described above.

[0200] Although the present invention has been described with reference to some particular embodiments, it is clear that the invention is not limited to these in any way, but encompasses all technical equivalents of the described means as well as combinations thereof, provided they fall within the scope of the invention.

[0201] Use of the verbs "comprise" and "include" and their conjugations does not exclude the presence of elements or steps other than those stated in a claim.

[0202] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Claims

1. a light source (101, 201, 701, 801) that emits illumination light (102, 202, 502, 602, 702, 802) adapted to illuminate a sample (133, 233, 733, 833) to be imaged; an optical device including a microscope objective (105, 205, 705, 805); a resonator (112, 212, 512, 612, 712, 812) having at least one first layer (241, 541, 641) with a first optical refractive index, at least one spacer layer (242, 542, 642) with a second optical refractive index, and at least one waveguide layer (243, 543, 643) with a third optical refractive index, which are arranged in this order in the direction of the optical axis of the microscope objective, wherein the second optical refractive index is smaller than the first optical refractive index and the third optical refractive index, and the resonator (112, 212, 512, 612, 712, 812) has a support surface facing away from the optical device and intended to receive the sample (133, 233, 733, 833); an optical detector (106, 206, 706, 716, 806); the optical device is positioned to collect light exiting the resonator (112, 212, 512, 612, 712, 812) and direct the exit light from the resonator (112, 212, 512, 612, 712, 812) to the optical detector (106, 206, 706, 716, 806) to form an image of the sample (133, 233, 733, 833) on the optical detector; the emitted light includes light scattered by the sample (104, 204, 504, 604, 704, 804) and a non-scattered portion (115, 215, 715, 815) of the illumination light (102, 202, 502, 602, 702, 802); Optical microscope (100, 200, 700, 800).

2. 2. The optical microscope (100, 200, 700, 800) of claim 1, wherein the optical device includes an amplitude filter (209, 764, 20, 21, 22, 809) disposed between the microscope objective lens (105, 205, 705, 805) and the optical detector (106, 206, 706, 716, 806) and configured to apply a first selective attenuation to the unscattered portion (115, 215, 715, 815) of the illumination light.

3. The intensity transmission coefficient associated with the first attenuation is 10 －６ 3. The optical microscope (100, 200, 700, 800) of claim 2, wherein the optical microscope (100, 200, 700, 800) has a resolution of less than 1000 s.p.m.

4. 4. The optical microscope (100, 200, 700, 800) of claim 2 or claim 3, wherein the scattered light by the sample is composed of a first portion of scattered light from a resonant excitation mode and a second portion of scattered light, and the amplitude filter (209, 764, 20, 22, 809) is further configured to apply a second selective attenuation to the second portion of scattered light.

5. The intensity transmission coefficient associated with the second selective attenuation is 10 －６ 5. The optical microscope (100, 200, 700, 800) of claim 4, wherein the optical microscope (100, 200, 700, 800) has a resolution of less than 1000 s.p.m.

6. 5. The optical microscope (100, 200, 700, 800) of claim 4, which relies on claim 2, wherein the intensity transmission coefficient associated with the first attenuation is greater than the intensity transmission coefficient associated with the second attenuation.

7. An optical microscope (100, 200, 700, 800) according to any one of claims 2 to 6, wherein the optical device comprises two converging lenses (208, 217, 808, 817) arranged to image the Fourier plane (213, 713, 813) of the microscope objective lens (205, 705, 805) onto the amplitude filter (209, 764, 809).

8. 8. The optical microscope (100, 200, 700, 800) of any one of claims 1 to 7, wherein the illumination light (102, 202, 502, 602, 702, 802) is a laser beam.

9. 9. The optical microscope (100, 200, 700, 800) of any one of claims 1 to 8, wherein the optical device includes at least one converging lens (108, 218, 769, 772, 808) through which the emitted light passes, the converging lens (108, 218, 769, 772, 808) being configured to image an object plane of the microscope objective lens (105, 205, 705, 805) onto the optical detector (106, 206, 706, 716, 806).

10. The optical microscope (100, 200, 700, 800) further comprises an image processing system; The image processing system includes: recording a plurality of images successively detected by said optical detector; combining the plurality of images into a reference image; configured to process at least one image detected by the optical detector with the reference image to suppress static signals; An optical microscope (100, 200, 700, 800) according to any one of claims 1 to 9.

11. The optical microscope (100, 200, 700, 800) further comprises an image processing system; The image processing system includes: determining the contrast of the image detected by the optical detector; determining at least one parameter of particles contained in the sample as a function of the contrast, the parameter being configured to be selected from the group consisting of a mass of a particle and a position of the particle in the optical axis direction; An optical microscope (100, 200, 700, 800) according to any one of claims 1 to 10.

12. 12. An optical microscope (100, 200, 700) according to any one of claims 1 to 11, wherein the light source (101, 201, 701) and the optical device are arranged to illuminate the sample (133, 233, 733) placed on the support surface of the resonator (112, 212, 512, 612, 712) by reflection.

13. the optical device receives the illumination light (102, 202, 502, 602, 702) for directing the illumination light onto the sample (133, 233, 733); 13. The optical microscope (100, 200, 700) of claim 12, wherein the microscope objective lens (105, 205, 705) of the optical device directs the illumination light (102, 202, 502, 602, 702) onto the resonator (112, 212, 712) at an angle of incidence greater than a critical angle at the interface between the first layer (241, 541, 641) and the spacer layer (242, 542, 642), such that the illumination light (102, 202, 502, 602, 702) resonantly excites at least one mode in the waveguiding layer (243, 543, 643) and illuminates the sample (133, 233, 733) with an enhanced evanescent wave.

14. the optical device includes a polarizing beam splitter plate (114, 214, 714); An optical microscope (100, 200, 700) according to any one of claims 12 to 14, wherein the polarizing beam splitter plate (114, 214, 714) reflects the illumination light (102, 202, 502, 602, 702) towards the microscope objective lens (105, 205, 705) and is traversed by the emitted light.

15. the optical detector is a first optical detector (706); the optical microscope includes a second optical detector (716); the optical device includes a non-polarizing beam splitter plate (766) that receives the output light (704, 715) and splits the output light into a first output light portion toward the first detector (706) and a second output light portion toward the second detector (716), the first output light portion including a first reflected light portion (778) and a first scattered light portion (780); 16. The optical microscope (700) of claim 15, wherein a phase mask (768) is positioned to be traversed by the first portion of the emitted light, and the phase mask (768) is configured to apply a phase shift between the first portion of the reflected light and the first portion of the scattered light.

16. the phase mask is a first phase mask (768); the optical device further includes a second phase mask (771) positioned to be traversed by the second portion of the emitted light; the emitted light second portion includes a reflected light second portion (779) and a scattered light second portion (781); the second phase mask (771) is configured to apply a phase shift between the second portion of the reflected light and the second portion of the scattered light; 17. The optical microscope (700) of claim 16, wherein the first phase mask and the second phase mask have different phase characteristics.

17. the optical device includes an optical condenser (107, 207, 761) that receives the illumination light emitted from the light source; An optical microscope (100, 200, 700) according to any one of claims 1 to 16, wherein the optical condenser (107, 207, 761) is configured to focus the illumination light in a region away from the optical axis of the microscope objective lens (105, 205, 705) at the Fourier plane (113, 213, 713) of the microscope objective lens to generate the angle of incidence.

18. 12. An optical microscope (800) according to any one of claims 1 to 11, wherein the resonator (812) is positioned along the optical axis of the microscope objective (805) between the microscope objective (805) and the light source (801), such that the light source (802) is adapted to illuminate the sample (833) received by the support surface of the resonator (812) when transmitted through it.

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