Optical microscope with resonator

Abstract

Disclosed is an optical microscope (200) comprising: a light source (201) that emits illumination light (202); an optical device comprising a microscope objective (205); a resonator (212) disposed between the optical device and a sample and comprising, in order in a direction of an optical axis of the microscope objective, a first layer having a first optical refractive index, a spacer layer having a second optical refractive index, and a waveguide layer having a third optical refractive index, a resonator having a second optical refractive index smaller than the first and third optical refractive indexes, the resonator having a retaining surface opposite the optical device, the retaining surface being intended to receive a sample; an optical detector (206); the optical device is configured to collect light emitted from the resonator; the emergent light includes light (204) scattered by the sample and a reflective portion (215) of the illumination light that is not scattered.

Description

Field of the Invention

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

[0002] More specifically, the present invention relates to the field of detecting and characterizing nanoparticles by an optical microscope. The present invention can be used to detect objects with characteristic sizes in the range of 1 nm to 100 nm. Such objects include metal nanoparticles, nanoscale contaminants, and other objects of biological interest, such as proteins or peptides. Background Art

[0003] Methods for detecting nanoparticles involve labeling the particles, for example by fluorescence labeling. However, this method quickly reaches its limitations because the effect of fluorescence labeling is limited in time, thus limiting the duration of observation. The detection quality is also affected. In fact, the temporal resolution of the image is limited. In addition, fluorophores degrade rapidly over time. Finally, this technique is difficult to implement because fluorescence labeling requires a large amount of upstream preparation.

[0004] Various optical microscopy techniques allow the detection of nanoparticles by elastic scattering without prior labeling, including dark field techniques or interference techniques. Among the interference techniques, the most common are iSCAT (interference scattering) technique, COBRi (coherent bright field) technique, and IRIS (interference reflection imaging sensor) technique. They enable the visualization of particles of 10 nm or smaller at present.

[0005] The core problem of interference techniques and dark field techniques is the signal-to-noise ratio. In fact, their purpose is to detect the signal of interest, i.e., the light elastically scattered by the nanoparticles. However, different types of noise affect the detection of the signal of interest.

[0006] In fact, there are many technical noises associated with fluctuations in the measurement system. In addition, the incident light source used in the microscope is affected by intensity fluctuations (referred to as photon noise), which is inherent in the physical process. In addition, the scattering of the illumination beam causes random interference patterns (referred to as speckle noise).

[0007] Patent application WO2018 / 011591 discloses an iSCAT-type interference microscope, in which a spatial filter enables the attenuation of the illumination beam, thereby improving the contrast of the image. Summary of the Invention

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

[0009] According to one embodiment, the present invention provides an optical microscope, which comprises:

[0010] - A light source that emits illumination light suitable for illuminating a sample to be imaged;

[0011] - An optical device that includes a microscope objective;

[0012] - A resonator that sequentially includes, along the optical axis direction 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, where the second optical refractive index is less than the first optical refractive index and the third optical refractive index, and the resonator has a support surface facing away from the optical device, and the support surface is intended to receive the sample;

[0013] - An optical detector;

[0014] The optical device is configured to collect the light exiting from the resonator and guide the exiting light from the resonator to the optical detector to form an image of the sample on the optical detector;

[0015] The exiting light includes the light scattered by the sample and the unscattered portion of the illumination light.

[0016] With these features, several technical advantages can be achieved:

[0017] - Enhancing the effective scattering cross-section of particles, which is equivalent to enhancing the signal of interest collected by the optical detector,

[0018] - Concentrating the light scattered by the sample into a very small solid angle, which allows for effective selective spatial filtering of the scattered light.

[0019] These effects enable an improvement in the signal-to-noise ratio. The enhancement of the effective scattering cross-section increases the signal of interest collected by the optical detector. Concentrating the scattered light into a strictly limited solid angle allows the use of an attenuation filter to eliminate the stray light that introduces noise in the measurement of the signal of interest without losing the signal of interest.

[0020] Specifically, according to one embodiment, the present invention provides an optical microscope that includes:

[0021] - A light source that emits illumination light,

[0022] - An optical detector,

[0023] - An optical device that includes a microscope objective, and the optical device receives the illumination light to guide the illumination light onto the sample,

[0024] - A resonator, the resonator being disposed between the optical device and the sample, the resonator sequentially including, 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 less than the first optical refractive index and the third optical refractive index, the resonator having a support surface facing away from the optical device, the support surface being intended to receive the sample, the microscope objective being configured to direct the illumination light to the resonator at an incident angle greater than the critical angle of the interface between the first layer and the spacer layer, so that the illumination light resonantly excites at least one mode in the waveguide layer and irradiates the sample through an enhanced evanescent wave,

[0025] The optical device is configured to collect the light exiting from 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.

[0026] The exiting light includes the light scattered by the sample and the unscattered portion of the illumination light.

[0027] With these features, several technical advantages are achieved:

[0028] i) Enhancing the illumination intensity of the sample through resonant excitation of the waveguide, thereby enhancing the amount of light scattered by the particles contained in the sample. In fact, the resonant excitation of one or more modes generates energy accumulation in the resonator, which leads to an enhancement of the field in the resonator and near the resonator.

[0029] ii) Enhancing the effective scattering cross-section of the particles.

[0030] iii) Generating an evanescent wave illumination intensity that is confined near the resonator-sample interface and that is uniform in the plane parallel to the interface.

[0031] iv) Concentrating the light scattered by the sample into a very small solid angle.

[0032] These effects make it possible to improve the signal-to-noise ratio.

[0033] Generally, the light scattered by the sample corresponds to the light emitted from the sample and the resonator plate that interacts with the particles contained in the sample, and the unscattered portion of the illumination light corresponds to the portion of the illumination light that exists in the exiting light beam and does not interact with the particles.

[0034] The vicinity of the resonator plate corresponds to a sample thickness positioned less than a few hundred nanometers from the support surface of the resonator plate, for example, positioned at a distance less than 200 nanometers from the support surface.

[0035] According to an embodiment, the optical microscope as described above may include one or more of the following features.

[0036] According to one embodiment, the optical device includes an amplitude filter that is arranged between the microscope objective and the optical detector, for example, in the Fourier plane of the microscope objective or in the image plane of this plane. The amplitude filter is configured to apply a selective first attenuation to the unscattered portion of the illumination light.

[0037] Therefore, the scattered light represents a larger proportion in the intensity of the outgoing light detected by the optical detector. In other words, this filtering increases the ratio between the amplitude of the scattered field and the amplitude of the field not scattered by the sample.

[0038] There are various techniques for manufacturing such an amplitude filter, such as thin film deposition, particularly metal thin film deposition. The attenuation applied by the amplitude filter can be characterized by an intensity transmission coefficient. According to one embodiment, the intensity transmission coefficient associated with the first attenuation is between 10 -1 and 10 -6 , preferably between 10 -2 and 3·10 -4 . For example, an intensity transmission coefficient close to 10 -3 is suitable for using a camera with a well capacity of 10k electrons as a detector, which is common.

[0039] According to one embodiment, the intensity transmission coefficient associated with the first attenuation is less than 10 -6 . By ensuring that the transmittance of the reflected field is substantially zero in this way, a dark field configuration is obtained.

[0040] According to one embodiment, the light scattered by the sample includes: a first portion of the scattered light from the resonant excitation mode; and a second portion of the scattered light. The amplitude filter is further configured to apply a selective second attenuation to the second portion of the scattered light. Such an amplitude filter enables the selection of light scattered at a specific angle corresponding to the radiation leakage of one or more guided modes by attenuating the remaining scattered light. Therefore, the field scattered by the guided mode is not attenuated and is transmitted to the optical detector. Such an amplitude filter can be used in a dark field configuration or a bright field interference configuration.

[0041] According to one embodiment, the intensity transmission coefficient associated with the second attenuation is between 10 -1 and 10 -6 .

[0042] According to one embodiment, the intensity transmission coefficient associated with the second attenuation is less than 10 -6 .

[0043] According to an embodiment of the dark field configuration, the intensity transmission coefficient associated with the first attenuation and the intensity transmission coefficient associated with the second attenuation are less than 10 -6 . Thus, the amplitude filter is configured to apply a total attenuation to the reflected field and the field scattered by the sample, except around a specific angle corresponding to the radiative leakage of the guided mode.

[0044] According to an embodiment in the interference configuration, the amplitude filter is configured to apply a selective first attenuation to the non-scattered portion of the illuminating light and a selective second attenuation to the second portion of the scattered light. Preferably, in this case, 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 between 10 -1 and 10 -6 , and the intensity transmission coefficient associated with the second attenuation is between 10 -1 and 10 -6 .

[0045] According to an embodiment, the optical device includes two converging lenses arranged to image the Fourier plane of the microscope objective on the amplitude filter.

[0046] Thus, the amplitude filter can attenuate the non-scattered portion of the illuminating light, the position of which in the Fourier plane is known. This attenuation can be precisely selective.

[0047] According to an embodiment, the illuminating light is a light beam, and in particular, the illuminating light is a laser beam.

[0048] Thus, the illuminating light is coherent, and the illuminating light can be monochromatic.

[0049] According to an embodiment, the illuminating light is emitted by a light-emitting diode (LED).

[0050] According to an embodiment, the illuminating light is monochromatic and has a wavelength between 400 nanometers and 1300 nanometers, and preferably, the wavelength is between 450 nanometers and 532 nanometers.

[0051] According to an embodiment, the resonator further includes at least one partial mirror.

[0052] According to an embodiment, the mirror is a Bragg mirror. Specifically, the mirror is a partial mirror.

[0053] According to an embodiment, the resonator includes a plurality of spacers and a plurality of waveguides, and each of the plurality of spacers is in contact with at least one of the plurality of waveguides.

[0054] According to one embodiment, at least two first spacers among the plurality of spacers have different thicknesses. Suitable thicknesses can generally be between 100 nm and 1 μm.

[0055] According to one embodiment, at least two second spacers among the plurality of spacers are made of different materials.

[0056] According to one embodiment, at least one spacer among the plurality of spacers is composed of magnesium fluoride.

[0057] According to one embodiment, at least two first waveguides among the plurality of waveguides have different thicknesses. Suitable thicknesses can generally be between 10 nm and 500 nm.

[0058] According to one embodiment, at least two second waveguides among the plurality of waveguides are composed of different materials.

[0059] According to one embodiment, at least one waveguide among the plurality of waveguides is made of titanium dioxide.

[0060] According to one embodiment, the resonant mode of the resonator is a surface wave.

[0061] According to one embodiment, the optical device includes at least one converging lens through which the outgoing light passes, and the converging lens is configured to image the object plane of the microscope objective on the optical detector.

[0062] According to one embodiment, the microscope includes an image processing system or is connected to an image processing system, and the image processing system is configured to:

[0063] - Record a plurality of images detected by the optical detector at consecutive times;

[0064] - Synthesize the plurality of images into a reference image. For example, each pixel in the reference image can be calculated as the average or median of the corresponding pixels in multiple images;

[0065] - Process at least one image detected by the optical detector using the reference image to suppress static signals.

[0066] For this purpose, the reference image can be subtracted from the detected or each detected image. This will generate one or more images in which only the dynamic signals that change over time are retained, while the static signals are not retained. In this way, all static noise can be filtered.

[0067] According to one embodiment, the image processing system is configured to apply a convolutional filter to at least one image detected by the optical detector.

[0068] According to one embodiment, the convolutional filter is a Gaussian filter. According to one embodiment, the optical microscope includes an image processing system or is connected to an image processing system, and the image processing system is configured to:

[0069] Determine the contrast in the image detected by the optical detector;

[0070] Determine at least one parameter of the particles contained in the sample according to the contrast, and the parameter is selected from the mass of the particles and the position of the particles along the optical axis direction.

[0071] According to one embodiment, the optical detector can be a digital camera.

[0072] According to one embodiment, the light source and the optical device are arranged to irradiate the sample received by the support surface of the resonator in a reflective manner.

[0073] According to one embodiment, the optical device receives the irradiated light to guide the irradiated light to the sample, and the microscope objective of the optical device is configured to guide the irradiated light to the resonator at an incident angle greater than the critical angle of the interface between the first layer and the spacer layer, so that the irradiated light resonantly excites at least one mode in the waveguide layer and irradiates the sample through the enhanced evanescent wave.

[0074] According to one embodiment, the part of the outgoing light not scattered by the sample is the reflected part of the irradiated light.

[0075] According to one embodiment, the optical device includes a polarization or non-polarization beam splitter, and the polarization or non-polarization beam splitter reflects the irradiated light in the direction of the microscope objective and allows the outgoing light to pass through.

[0076] 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-polarization beam splitter and a phase mask, the non-polarization beam splitter receives the outgoing light and divides the outgoing light into a first part of the outgoing light guided to the first detector and a second part of the outgoing light guided to the second detector, the first part of the outgoing light includes a first part of the reflected light and a first part of the scattered light, the phase mask is arranged to allow the first part of the outgoing light to pass through, and the phase mask is configured to apply a phase shift between the first part of the reflected light and the first part of the scattered light.

[0077] Therefore, the light intensity difference between the first optical detector and the second optical detector can be measured.

[0078] According to one embodiment, the phase mask is a first phase mask, and the optical device further includes a second phase mask which is arranged for the second part of the outgoing light to pass through. The second part of the outgoing light includes the second part of the reflected light and the second part of the scattered light. The second phase mask is configured to apply a phase shift between the second part of the reflected light and the second part of the scattered light, and the first phase mask and the second phase mask have different phase characteristics.

[0079] Therefore, the phase shift between the first phase mask and the second phase mask can be configured to minimize background noise and maximize contrast by subtracting two images.

[0080] According to one embodiment, the optical device includes an optical condenser which receives the illuminating light emitted from the light source, and the optical condenser is configured to focus the illuminating light onto a region away from the optical axis of the microscope objective in the Fourier plane of the microscope objective to generate the incident angle.

[0081] Therefore, the incident angle can be selected to exceed the critical angle on the resonator interface.

[0082] According to one embodiment, the resonator is arranged between the microscope objective and the light source along the optical axis of the microscope objective, such that the light source is adapted to illuminate a sample received by the support surface of the resonator in a transmissive manner.

[0083] According to one embodiment, the light source is arranged to emit an incident beam which illuminates the support surface of the resonator in a normal incidence manner. Description of the Drawings

[0084] The present invention can be better understood by the following description of several specific embodiments of the present invention with reference to the accompanying drawings, and other objects, details, features and advantages of the present invention will also become clearer. These specific embodiments are given by way of illustration only and not by way of limitation.

[0085] Figure 1 An example of a transmission optical microscope is shown.

[0086] Figure 2 A reflective optical microscope including a resonator plate according to a first embodiment is shown.

[0087] Figure 3 A reflective optical microscope including a resonator plate according to a second embodiment is shown.

[0088] Figure 4 Shows Figure 2The microscope objective lens and the resonant plate of an optical microscope.

[0089] Figure 5 is similar to Figure 4 a view, Figure 5 showing the scattering of light by nanoparticles and Figure 2 the scattering of light in the resonant plate of an optical microscope.

[0090] Figure 6 is a schematic diagram of light waves coupled to Figure 2 the resonant plate of an optical microscope.

[0091] Figure 7 is a graphical representation of the angular distribution of the energy scattered by nanoparticles.

[0092] Figure 8 is Figure 7 a graphical representation of the dispersion relation of the resonant plate used in

[0093] Figure 9 a diagram of a spatial filter that can be used in an optical microscope according to a first variant operating in dark field. The black part is opaque. The spatial filter allows most of the scattered light to pass through, which is concentrated near the angles defined by the resonant plate and intersects with the reflected beam.

[0094] Figure 10 a diagram of a spatial filter that can be used in an optical microscope with a resonant plate according to a second variant, which is capable of attenuating the reflected field for operation in an interference mode.

[0095] Figure 11 a diagram of a spatial filter that can be used in an optical microscope with a resonant plate according to a third variant, which is capable of strongly attenuating the reflected beam and separating the useful signal from the noise.

[0096] Figure 12 a diagram of a resonant plate using a set of dielectric layers according to a first alternative.

[0097] Figure 13 a diagram of a resonant plate using a mirror according to a second alternative.

[0098] Figure 14 a diagram of a reflective optical microscope according to a third embodiment.

[0099] Figure 15 a graphical representation of the results that can be obtained in a configuration according to the prior art.

[0100] Figure 16 is using Figure 9 a graphical representation of the results that can be obtained in the dark field configuration of an optical microscope using the filter in

[0101] Figure 17 is a graphical representation of the results that can be obtained using the filter in Figure 10 the interference configuration of an optical microscope.

[0102] Figure 18 is a graphical representation of a set of results that can be obtained using different distance values between nanoparticles and a resonator plate.

[0103] Figure 19 shows a transmission optical microscope that includes a resonator plate according to a fourth embodiment. Detailed Embodiments

[0104] Embodiments of an optical microscope equipped with a resonator will be described below, which can perform high-performance detection of light scattered by very small objects. To this end, reference Figure 1 is made, and first, the concept of the optical microscope that helps to understand the present invention will be discussed.

[0105] Figure 1 shows an example of a transmission microscope 1. The transmission microscope 1 includes a light source (not shown) that emits an incident beam 2 having an incident intensity I′ i = M 2 I i where the incident intensity I i is defined in the image plane of the camera 9 and differs from the intensity I′ of the incident beam 2 defined in the particle plane by a factor M i where M represents the magnification of the microscope. The nanoparticle 3 is irradiated by the incident beam 2. The irradiation of the nanoparticle 3 by the incident beam 2 is the source of the scattered light 4, and the scattered light 4 has a scattered intensity I 2 in the camera image plane. S .

[0106] The transmission microscope 1 includes a microscope objective 5, an optical device, and a camera 6. The optical device includes two converging lenses 7 and 8 that form a support 4-f, a spatial filter 9, and a tube lens 10. The support 4-f allows the Fourier plane of the microscope objective 5 to be projected onto the spatial filter 9.

[0107] The objective 5, the optical device, and the camera 6 have a common optical axis. The incident beam 2 is parallel to the common optical axis. The incident beam 2 and the scattered light 4 are imaged on the camera 6 after passing through the objective 5 and the optical device.

[0108] The camera 6 receives the final signal, which is the superposition of the incident beam 2 and the scattered light 4. The final signal may include an interference term due to the phase shift Δθ between the incident beam and the scattered light.

[0109] The intensity of the detected final signal is expressed as:

[0110]

[0111] The nanoparticle 3 has an effective scattering cross-section σ that is proportional to the square of its volume. In addition, the scattered energy f collected by the receiving part of the camera 6 col . The magnification of the transmission microscope 1 is M. Thus, the power scattered into a camera pixel is written as:

[0112] P s = f col σI′ i = f col σM 2 I i .

[0113] In addition, the scattered power is distributed at the level of the surface S of the Airy disk corresponding to the image of the nanoparticle 3 on the camera 6. Thus, the scattered power can also be expressed as:

[0114] P S = SI S .

[0115] This allows the expression for the detected intensity to be reformulated as a function of the effective scattering cross-section σ, the fraction f of the scattered energy collected col and the magnification M:

[0116]

[0117] According to the quantitative example, the physical quantities have the following values:

[0118] · The wavelength of the incident light beam 2 is 450 nm, and the diameter of the nanoparticle 3 is 3 nm. The optical refractive index of the nanoparticle 3 is 1.5, and the nanoparticle 3 is suspended in water, whose optical refractive index is 1.33. Thus, the effective scattering cross-section is 7.810 -14 μm 2 .

[0119] · The magnification M is 100.

[0120] · The radius of the Airy disk in the image plane of the objective lens 5 can be 400 nm, i.e., 40 μm in the image plane of the camera 9. The surface area of the Airy disk in the image plane of the camera 9 is 5000 μm 2 . The microscope objective lens 5 can be an oil microscope and has a numerical aperture of 1.45. Then the collection coefficient can be 42%. In this case, the following values are obtained:

[0121]

[0122] The transmission microscope 1 can be used in an interference (or bright field) configuration or a dark field configuration. When the transmission coefficient of the attenuator (spatial filter 9) placed at the center is zero, the microscope is in the dark field configuration. Schematically, when this transmission coefficient is non-zero, the microscope is in the interference configuration. In fact, as long as the interference term can be neglected compared to the direct scattering term of the nanoparticles, the dark field configuration can be obtained.

[0123] In the interference configuration, the camera 6 receives both the incident beam 2 and the scattered light 4, and the two interfere with each other.

[0124] As Figure 1 shown, the incident beam 2 can be attenuated more or less by the spatial filter 9. In the dark field configuration, the transmission coefficient of the spatial filter 9 is zero.

[0125] The tube lens 7 is configured to focus the scattered light 4 onto the object focal plane of the intermediate converging lens 8.

[0126] The spatial filter 9 includes a mask placed on the common optical axis. The tube lens 7 and the intermediate converging lens 8 focus the incident beam 2 onto the mask of the spatial filter, and the spatial filter 9 is located at a certain focal length from the intermediate converging lens 8.

[0127] The spatial filter 9 allows the filtered beam 11 to pass through. Thus, the camera receives the scattered light 4 and the filtered beam 11. The scattered light 4 passes through the final converging lens 10 and is focused on the camera 6 in the image focal plane of the final converging lens 10.

[0128] In the dark field configuration, the incident intensity reaching the camera is zero, and the detected intensity is expressed as:

[0129]

[0130] When the magnification M is 100, the detected intensity is 6.5×10 -14 I i .

[0131] The average number N of photons detected by a pixel with an area of S′ in a period of time τ is I det S′τ. When the photon noise (or shot noise) is the main noise source and the intensity is expressed as the number of photons per second per unit area, the signal-to-noise ratio is expressed as:

[0132]

[0133] For the case where the area S′ is 100 μm 2 and the incident intensity is 10 10 s -1 μm -2 ², the signal-to-noise ratio is given by: 100×[1012 6 10 -18 τ] 1 / 2 = 0.2τ.

[0134] If ten pixels are averaged over a one - second acquisition time, the signal - to - noise ratio will be greater than 1. However, the background noise is actually greater than the 3nm particle signal. Detecting particles smaller than 10 nanometers in diameter is difficult. Therefore, improving the signal - to - noise ratio is a key issue.

[0135] For bright - field interference microscopy, the detected intensity is expressed as:

[0136]

[0137] Then the signal - to - noise ratio can be expressed as:

[0138]

[0139] The signal - to - noise ratio is twice that of the dark - field configuration. On the other hand, the signal to be measured in the interference configuration is much larger. The difference can be characterized by introducing a contrast c, which is defined as: Then we can write the signal recorded by the detector:

[0140] I det = I i (1 + 2c cos(Δθ)).

[0141] In the interference configuration, the amplitude of the signal is 2cI i , while in the dark - field configuration it is c 2 I i .

[0142] The intensity detected in the interference configuration is higher, which enables the background noise that limits detection in the dark - field configuration to be overcome. In addition, this allows electrons to be collected faster on the pixels of the camera, so it can be acquired faster.

[0143] The spatial fluctuations (speckle fluctuations) of the incident intensity I i are much larger than the signal and must be subtracted. Therefore, a detector capable of sensing very small changes in the signal is required to distinguish between the background noise and the useful signal. Therefore, in the bright - field configuration, the issue of improving the signal - to - noise ratio is also important.

[0144] Figure 2 , Figure 3 , Figure 14 and Figure 19 show four embodiments of optical microscopes 100, 200, 700, 800, including:

[0145] - Light sources 101, 201, 701, 801 that emit illumination light suitable for illuminating samples 133, 233, 733, 833 to be imaged;

[0146] - An optical device that includes microscope objectives 105, 205, 705, 805;

[0147] - A resonator that sequentially includes, along the optical axis direction of the microscope objective: at least one first layer 241, 541, 641 having a first optical refractive index, at least one spacer layer 242, 542, 642 having a second optical refractive index, and at least one waveguide layer 243, 543, 643 having a third optical refractive index, where the second optical refractive index is less than the first optical refractive index and the third optical refractive index, and the resonator has a support surface facing away from the optical device, and the support surface is intended to receive the samples 133, 233, 733, 833;

[0149] - Optical detectors 106, 206, 706, 716, 806;

[0150] The optical device is configured to collect the light exiting from the resonator and direct the exiting light from the resonator to the optical detector to form an image of the samples 133, 233, 733, 833 on the optical detectors 106, 206, 706, 716, 806;

[0151] The exiting light includes the light 104, 204, 504, 604, 704, 804 scattered by the sample and the unscattered part 115, 215, 715, 815 of the illumination light.

[0152] The resonator actually takes the form of resonator plates 112, 212, 512, 612, 712, 812.

[0153] In the first, second, and third embodiments of the optical microscopes 100, 200, 700, the light sources 101, 201, 701 and the optical device are arranged to illuminate the samples 133, 233, 733 received by the support surface of the resonator 112, 212, 712 in a reflective manner. For this purpose, the sample receives the incident light beam emitted by the light source after passing through the resonator plates 112, 212, 712, which will be described in more detail later. In this configuration, the support surface of the resonator plate is away from the light source.

[0154] To this end, the optical microscopes 100, 200, 700 include polarization beam splitters 114, 214, 714, which are arranged on the optical axes of the microscope objectives 105, 205, 705 and are arranged to reflect at least a part of the illumination light emitted by the light sources 101, 201, 701 in the direction of the resonant plates 112, 212, 712. In a variant, the beam splitter can also be non-polarizing.

[0155] In this reflection configuration, the incident beam penetrates the resonant plate via the lower surface of the resonant plate, which is opposite to the support surface for receiving the samples 133, 233, 733. This support surface will hereinafter be referred to as the upper surface of the resonant plates 112, 212, 712.

[0156] Generally, in the reflection configurations of the first, second, and third embodiments, the incident beam emitted by the light source propagates along an optical path that first passes through the converging incident lenses 107, 207, 761. After passing through the converging incident lenses 107, 207, 761, the incident beam is partially reflected by the polarization beam splitters 114, 214, 714 and passes through the microscope objectives 105, 205, 705, and then through the resonant plates 112, 212, 712 to reach the samples 133, 233, 733 received by the support surfaces of the resonant plates.

[0157] After the interaction between the samples 133, 233, 733 and the incident beam, the outgoing beam is emitted and exits from the resonant plates 112, 212, 712. The outgoing beam passes through the microscope objectives 105, 205, 705 and is transmitted by the polarization beam splitters 114, 214, 714. It propagates through the converging outgoing lenses 108, 208, 762. It includes the light 104, 204, 504, 604, 704 scattered by the samples 133, 233, 733 and the non-scattered part 115, 215, 715 of the illumination light. When the optical microscope is used with reflection illumination, the non-scattered part 115, 215, 715 corresponds to the part of the incident beam reflected at the interface between the resonant plates 112, 212, 712 and the samples without interacting with the samples 133, 233, 733. In this configuration, the non-scattered part 115, 215, 715 is thus the reflected part of the incident beam.

[0158] The fact that the incident beam passes through the resonant plates 112, 212, 712 before penetrating the sample makes it possible to enhance the illumination intensity of the sample by means of resonant excitation of the waveguide, thereby enhancing the amount of light scattered by the particles placed near the resonant plate.

[0159] In addition, the evanescent wave illumination intensity is confined near the interface between the resonator and the sample, and the evanescent wave illumination intensity is uniform in a plane parallel to the interface. Therefore, the illumination is confined within a very small thickness. This avoids noise caused by light scattered by particles far from the interface in the case of non-confined illumination.

[0160] The resonator plates 112, 212, 712 enhance the effective scattering cross-section of particles in their vicinity.

[0161] In addition, the resonator plates 112, 212, 712 change the radiation pattern of particles in their vicinity and concentrate their scattered light within a very small solid angle. Therefore, it is beneficial to selectively collect the light scattered by the sample, and the signal-to-noise ratio can be easily improved by spatial filtering, as described in more detail below.

[0162] In Figure 19 In the fourth embodiment of the optical microscope 800 shown, the light source 801 and the optical device are arranged to illuminate the sample 833 received on the support surface of the resonator 812 in a transmission manner. Here, the sample directly receives the incident light beam emitted by the light source 801, and the light beam does not interact with any other optical elements. The incident light beam 802 propagates in a normal incidence manner with respect to the support surface of the resonator plate. Therefore, the sample 833 is preferably illuminated at normal incidence. In this case, no optical element is 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 faces the light source 801.

[0163] In this transmission configuration, the incident light beam penetrates the sample placed on the support surface of the resonator plate 812 without passing through 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.

[0164] The outgoing light beam is emitted and exits from the resonator plate 812. It passes through the microscope objective 805 and propagates through the converging outgoing lens 808, just like in an optical microscope operating in a reflection manner.

[0165] The outgoing light beam includes the light 804 scattered by the sample 833 and the non-scattered part 815 of the illumination light. When the optical microscope is used with transmission illumination, the non-scattered part 815 corresponds to the part of the incident light beam that passes through the sample without interacting with the particles contained in the sample 833.

[0166] The resonator plate 812 enhances the effective scattering cross-section of particles located in its vicinity.

[0167] In addition, the coupling of the light scattered by the sample with the resonator plate 812 concentrates the light scattered by the sample into a very small solid angle. Therefore, it is beneficial to selectively collect the light scattered by the sample, and the signal-to-noise ratio can be improved by filtering.

[0168] Each embodiment shown in the drawings will now be described in more detail.

[0169] Figure 2 A first embodiment of an optical microscope 100 is shown, which operates in a reflection mode and includes a resonant plate 112, which is intended to be in contact with a sample 133, which includes one or more nanoparticles suspended in a solution. The diameter of the nanoparticles can be between 1 nanometer and 100 nanometers.

[0170] In practice, the sample 133 can rest on the upper surface of the resonant plate 112 under the action of its own weight. The resonant plate 112 is arranged horizontally. It can be understood that there can be other configurations in which the sample is placed in contact with the resonant plate 112 by other means.

[0171] The optical microscope 100 further includes a light source 101, a microscope objective 105, and a detector 106.

[0172] Here, the light source 101 is a laser source, and the incident light beam is an incident laser beam 102. The light source 101 is arranged to emit the incident laser beam 102, which has a wavelength, for example, between 400 nanometers and 1300 nanometers. The incident laser beam 102 can be a wide beam that irradiates a wide area of the sample 133, or a focused narrow beam that scans the region of interest of the sample 133.

[0173] The optical microscope 100 further includes an optical device, which includes a converging incident lens (hereinafter referred to as a first converging lens 107), a converging outgoing lens (hereinafter referred to as a second converging lens 108), and a polarization beam splitter 114.

[0174] The incident laser beam 102 passes through the first converging lens 107 and is reflected by the polarization beam splitter 114. The first converging lens 107 can be an optical condenser. The polarization beam splitter 114 has a semi-reflective surface, which is positioned such that the incident angle of the incident laser beam 102 on the polarization beam splitter 114 is 45°. According to one embodiment, the incident angle of the incident laser beam 102 on the polarization beam splitter 114 can have different values. The polarization beam splitter 114 reflects the polarization component of the incident laser beam 102, but allows the other polarization component of the incident laser beam 102 to pass through in a transmissive manner.

[0175] The incident laser beam 102 is reflected on the polarization beam splitter 114 and then directed towards the microscope objective 105. The microscope objective 105 can be an immersion objective, including immersion oil having the same refractive index as the refractive index of glass. The microscope objective 105 has a numerical aperture suitable for generating the excitation of the resonant mode described below.

[0176] The optical microscope 100 is also configured such that the incident laser beam 102 is focused with an intermediate eccentricity with respect to the optical axis of the microscope objective 105. Thus, the distance from the optical axis in the Fourier plane 113 enables the tilt of the incident laser beam 102 on the sample 133 to be controlled.

[0177] Thus, at the output of the microscope objective 105, the incident laser beam 102 irradiates the surface of the sample 133 at a predefined incident angle controlled by the parameters of the optical microscope 100.

[0178] The incident laser beam 102 passes through the retarder plate 199 and then through the objective 105 to irradiate the resonant plate 112. The retarder plate 199 introduces a phase shift between the two polarization components of the beam transmitted by the retarder plate. A reflected laser beam 115 and a scattered beam 104 are emitted from the resonant plate 112. The reflected laser beam 115 and the scattered beam 104 constitute the outgoing beam. The reflected laser beam 115 is the reflection of the incident laser beam 102 on the region of interest. The scattered beam 104 propagates over a wider angular range than the reflected laser beam 115.

[0179] The scattered beam 104 and the reflected laser beam 115 pass through the objective 105 and then through the polarization beam splitter 114. The scattered beam 104 and the reflected laser beam 115 pass through the second converging lens 108 and are imaged on the detector 106.

[0180] The second converging lens 108 can be a tube lens. The sensitive surface of the detector 106 can be located at the focal length of the second converging lens 108.

[0181] The detector 106 can be an image sensor, such as of the CMOS or CCD type. In particular, a sensor limited to 10,000 electrons per pixel can be used. The detector 106 can also include a memory for recording a plurality of consecutive images.

[0182] Reference Figure 3 , shows a second embodiment. The incident beam is here, for example, the incident laser beam 202. The incident laser beam 202 emitted by the light source 201 passes through a converging incident lens (here called the initial converging lens 207) and is then reflected on the polarization beam splitter 214. The incident laser beam 202 is then reflected in the direction of the microscope objective 205.

[0183] According to the second embodiment, the incident laser beam 202 is focused at a position offset from the center of the polarization beam splitter 214. The incident laser beam 202 is then reflected in the direction of the microscope objective.

[0184] The incident laser beam 202 passes through the retarder plate 299 which, according to one embodiment, is a quarter-wave plate.

[0185] The incident laser beam 202 is focused on the Fourier plane 213 of the microscope objective 205.

[0186] The optical microscope 200 is also configured such that the incident laser beam 202 is focused with a central eccentricity with respect to the optical axis of the microscope objective 205. Thus, the distance from the optical axis in the Fourier plane 213 enables the tilt of the incident laser beam 202 on the sample 233 to be controlled.

[0187] Thus, at the output of the microscope objective 205, the incident laser beam 202 irradiates the surface of the sample 233 at a predefined incident angle controlled by the parameters of the optical microscope 200.

[0188] The resonant plate 212 sends back the reflected laser beam 215 and the scattered light 204 emitted by the sample. Thus, the output beam consists 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 also focused on the Fourier plane 213 of the microscope objective 205, at an additional eccentric position.

[0189] The scattered light 204 and the reflected laser beam 215 pass through the polarization beam splitter 214, and then pass through a converging output lens, which here consists of a first converging output lens 208, a second converging output lens 217, a spatial filter 209, and a third converging output lens 218, and then are imaged on the camera 206.

[0190] The image focal plane of the first converging output lens 208 corresponds to the object focal plane of the second converging output lens 217. The spatial filter 209 is placed in the image focal plane of the second converging output lens 217. Thus, the reflected laser beam 215 is focused on the spatial filter 209.

[0191] The third converging output lens 218 focuses the reflected laser beam 215 and the scattered light 204 onto the sensitive surface of the camera 206, and the sensitive surface of the camera 206 is located within the image focal plane of the third converging output lens 218.

[0192] Reference Figures 4 to 6 , shows three details of Figure 3 . Figure 4 Shows the microscope objective 205 and the resonant plate 212, and the propagation of light in the resonant plate 212.

[0193] Of course, Figures 4 to 6 the arrows drawn in

[0194] The microscope objective lens 205 is an oil-based objective lens and includes an immersion oil 228 having an optical refractive index identical to that of glass. The resonant plate 212 is placed against the microscope objective lens 205 and is in contact with the immersion oil 228.

[0195] The resonant plate 212 is composed of a plurality of parallel layers arranged in sequence along the optical axis of the microscope. The resonant plate 212 includes a glass plate 241, and the glass plate 241 is in contact with the immersion oil 228.

[0196] The spacer 242 is placed adjacent to the glass plate 241. The optical refractive index of the spacer 242 is less than that of the glass plate 241. According to one embodiment, the spacer 242 is made of magnesium fluoride. According to one embodiment, the thickness of the spacer is 485 nanometers.

[0197] The spacer 242 is placed between the glass plate 241 and the waveguide 243. The optical refractive index of the spacer 242 is less than that of 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.

[0198] The waveguide 243 is in contact with the sample 233 in which one or more nanoparticles 203 are suspended.

[0199] Reference Figure 6 , the optical refractive index n of the glass plate 241 1 is greater than the optical refractive index n of the spacer 242 2 . Therefore, a critical angle exists at the interface between the glass plate 241 and the spacer 242, resulting in total internal reflection.

[0200] Reference Figure 4 , the incident laser beam 202 is focused on the Fourier plane at a critical distance from the optical axis of the microscope objective lens, and this critical distance causes the incident laser beam 202 to be projected onto the resonant plate 212 at an incident angle greater than the aforementioned critical angle.

[0201] Therefore, the incident laser beam 202 undergoes frustrated 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 incident angle and passes through the objective lens of the microscope 205.

[0202] The evanescent wave 252 penetrates the spacer 242. Since the thickness of the spacer 242 is selected to be of the same order of magnitude as the attenuation length of the evanescent wave 252, 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 field enhancement in the waveguide 243. The thicker the spacer 242, the greater the enhancement.

[0203] Reference Figure 6 , the optical refractive index n of the waveguide 2433 Greater than the optical refractive index of the spacer 242, the evanescent wave 252 from the spacer 242 is refracted in the waveguide 243 in the form of a guided wave 253.

[0204] The frequency and the incident angle of the incident laser beam 202 are configured such that the evanescent wave 252 resonantly excites the mode of the waveguide 243. For this purpose, a "phase matching" configuration is achieved.

[0205] Due to the resonant excitation of the mode of the waveguide 243, the enhanced wave 254 propagates from the waveguide 243 in the sample 233, and its amplitude can be enhanced by a high enhancement factor, for example, the enhancement factor can be as high as 1000. The enhanced wave 254 propagates in the sample 233 and irradiates the nanoparticles 203.

[0206] Reference Figure 5 , the nanoparticles 203 irradiated by the enhanced wave 254 emit scattered light 204, and the scattered light 204 propagates in the direction of the microscope objective (not shown) in the resonant plate 212. The dashed line corresponds to the scattered light 204, while the solid line corresponds to the incident laser beam 202, the refracted beam in the form of the guided wave 253, the reflected laser beam 215, and the radiative loss of the guided wave 253 through the spacer 242.

[0207] Reference Figure 7 , the energy density δ of the scattered light 204 is represented on the ordinate as a function of the scattering angle α on the abscissa. The nanoparticles are located on the resonant plate 212. The wavelength used is 515 nm. The refractive indices of the layers 241 to 243 are n1 = 1.518, n2 = 1.38, and n3 = 2.8 respectively, and the thicknesses of the layers 242 and 243 are 484 nm and 45 nm respectively.

[0208] The scattering angle α is measured with respect to the optical axis in the immersion oil 228. Due to the coupling of the scattered light 204 with the resonant mode of the waveguide 243, the radiation power of the scattered light 204 in the direction of the microscope objective is via the waveguide 243 according to the predetermined scattering peak angle α 0 escapes mostly (about 50%) at the output end of the resonant plate, here about 66° with respect to the optical axis. Therefore, the scattered light 204 is a light beam containing the maximum energy cone corresponding to the scattering peak angle α 0 .

[0209] Reference Figure 8 , the dispersion relation of the resonant plate 212 is shown, relating the spatial wave vector in the resonant plate 212 to the wavelength of the incident laser beam 202. Therefore, the resonant mode of the guided wave 253 is not the same according to the wavelength of the incident laser beam 202. Therefore, the scattering peak angle α of the scattered light 0 is also a function of the wavelength of the incident laser beam 202.

[0210] We now describe how the spatial filter 209 utilizes this angular distribution of the energy scattered by the nanoparticles 203.

[0211] Since the Fourier plane of the objective lens 205 is imaged on the spatial filter 209 at the output of the first converging exit lens 208 and the second converging exit lens 217, the angular distribution of the scattered light 204 is retained on the spatial filter 209. In terms of the transverse component of the propagation wave vector, the maximum energy cone substantially corresponds to a circle with a certain thickness, in other words, corresponds to a ring.

[0212] Thus, according to Figure 9 the first variant shown, the spatial filter 209 can be used to transmit only a portion of the scattered light present in the maximum energy circle.

[0213] Figure 9 The first filter 20 that can be used is shown. The first filter 20 includes a first mask 98 composed of two concentric parts, and the first mask 98 is configured to define a transmission ring 97, and the transmission ring 97 is configured to transmit only the scattered light corresponding to the maximum energy cone. Outside the transmission ring 97, the first mask 98 has a transmission coefficient that is preferably zero (total attenuation).

[0214] To form a dark field filter, the first filter 20 may further include a second mask 96 located in the transmission ring 97 and configured to attenuate the reflected laser beam 215. Similarly, the second mask 96 has a transmission coefficient that is preferably zero in intensity (total attenuation), that is, for example, less than 10 -6 .

[0215] Referring to Figure 10 , the second filter 21 can be used. The second filter 21 only includes the second mask 96, and the second mask 96 is configured to attenuate the reflected light beam 215, and the attenuation can be total attenuation or partial attenuation. All the scattered light 204 is transmitted. If the intensity transmission coefficient of the second mask 96 is substantially zero, for example, less than 10 -6 , then the second filter 21 is a dark field filter. If the transmission coefficient of the second mask 96 is not zero, for example, greater than 10 -3 , then the second filter 21 is an interference bright field filter. Then the optical microscope 200 is used in an interference configuration.

[0216] Referring to Figure 11 , the second interference bright field filter 22 is shown. The second interference bright field filter 22 has a structure similar to that of the dark field filter 20. However, here the second mask 96 has a non-zero transmission coefficient (partial attenuation), and preferably greater than the transmission coefficient of the first mask 98.

[0217] Referring to Figure 12 andFigure 13 , showing other embodiments of the resonant plate.

[0218] Reference Figure 12 , elements similar or identical to those of the second embodiment have the same reference numerals, but increased by 300. Figure 12 Shows that some layers can be added to the resonant plate 512. Specifically, a plurality of spacers 542 and a plurality of waveguides 543 can be alternately placed. The evanescent wave propagates in the spacer 542.

[0219] The spacers 542 can have different thicknesses and / or different materials. The waveguides 543 can also have different thicknesses and / or different materials. Suitable thicknesses can generally be between 100 nm and 1 μm.

[0220] The plurality of waveguides 543 enables coupling of the resonant mode to more values of the wavelength and the angle of incidence of the incident laser beam 202. Therefore, the optical microscope becomes more robust to changes in the wavelength and the angle of incidence of the incident laser beam 502.

[0221] In addition, the plurality of waveguides 543 enables an increase in the amplitude value of the resonant guided wave 553. Finally, the plurality of waveguides 543 allows the incident light beam 502 to simultaneously excite multiple resonant modes, which results in multiple angular peaks in the energy of the scattered light 504 of the nanoparticles 503, where the angular peaks of the energy are in multiple directions.

[0222] Reference Figure 13 , elements similar or identical to those of the second embodiment have the same reference numerals, but increased by 400. Figure 13 Shows that the resonant plate 612 can include a partial mirror 645. The mirror 645 can be a metal layer or a Bragg mirror, and can enhance the resonant phenomenon in the resonant plate 612. The evanescent wave propagates in the spacer 642.

[0223] Figure 12 and Figure 13 Schematically shows total internal reflection at the interface between the first layer 541 or 641 and the spacer 542 or 642. However, the position of the interface where frustrated total internal reflection occurs is not limited. Other material layers can be inserted below the interface where frustrated total internal reflection occurs.

[0224] Reference Figure 14 , a third embodiment of the optical microscope 700 including a resonant plate 712 can be used in an interference configuration employing balanced homodyne detection technology.

[0225] The light source 701 of the optical microscope 700 emits an incident laser beam 702, which passes through a converging incident lens (herein referred to as the initial converging lens 761), and then is reflected onto a polarization beam splitter 714, which is the first polarization beam splitter 714 herein. The incident laser beam 702 is directed towards the microscope objective 705, passes through the resonant plate 712 and irradiates the sample 733 disposed on the support surface of the resonant plate.

[0226] The outgoing beam includes scattered light 704 and a reflected beam 715, which pass through the microscope objective 705 and the first polarization beam splitter 714.

[0227] The scattered light 704 and the reflected beam 715 pass through a converging outgoing lens, which here consists 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 all converging lenses.

[0228] A second beam splitter 766 is placed after the third common lens 765. The second beam splitter 766 first separates the reflected beam 715 into a first reflected beam 778 and a second reflected beam 779, and secondly separates the scattered light 704 into a first scattered light 780 and a second scattered light 781.

[0229] The first reflected beam 778 and the first scattered light 780 pass through the initial lens of the first arm 767 and then pass through the first phase mask 768. The final lens of the first arm 769 focuses the first reflected beam 778 and the first scattered light 780 onto the first camera 706.

[0230] The second reflected beam 779 and the second scattered light 781 pass through the initial lens of the second arm 770 and the second phase mask 771. The final lens of the second arm 772 focuses the second reflected beam 779 and the second scattered light 781 onto the second camera 716.

[0231] The first phase mask 768 and the second phase mask 771 are respectively configured to cause a phase shift of the first reflected beam 778 relative to the first scattered light 780 and a phase shift of the second reflected beam 779 relative to the second scattered light 781, and the phase shift angles are respectively According to one embodiment, the phase shift angle For the first phase mask 768 can be π / 2, and for the second phase mask 771 can be -π / 2.

[0232] 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, and the difference between the first intensity and the second intensity is expressed as:

[0233]

[0234] Such an expression makes it possible to retain only one interference term and filter the reference signal.

[0235] Figures 15 to 18 Graphical results obtained by numerical simulation are shown. Figure 15 、 Figure 17 and Figure 18 show microscope results in an interference configuration, where in Figure 15 、 Figure 17 and Figure 18 the color scale represents the contrast c defined as above.

[0236] In fact, the contrast c can be calculated or measured by the following formula: c = I / I_ref, where I represents the intensity of the camera image and I_ref represents the intensity of the average image (or reference image). Figure 15 、 Figure 17 and Figure 18 The contrast images shown in

[0237] Figure 15 are obtained by pixel-by-pixel division.

[0238] Figures 16 to 17 Graphical results obtained by simulation in a scenario with a resonant plate are shown.

[0239] Figure 16 corresponds to the case of using the dark field filter 20. In this figure, the gray scale represents the relative intensity, and black (0) corresponds to a signal effectively zero. The central spot corresponds to the nanoparticle. Compared with Figure 15 the central spot is clearer, more circular and smaller in diameter, indicating an improvement in resolution.

[0240] Figure 17 corresponds to the case of using the interference filter 21. The reflected laser beam 215 is then filtered and attenuated. Figure 17 shows the central spot at the center of the interference pattern, which has more arcs than the comparison image. However, the central spot is more distinguishable than in the comparison image, which also indicates an improvement in sensitivity.

[0241] The spatial filtering technique used is similar to rejecting all wave vectors except one, which means that the image of a point is imaged in a series of circles. However, this does not prevent the detection of a single particle, even if the sample contains multiple particles, as long as these particles are spatially separated from each other in the sample.

[0242] Therefore, regardless of the filter used, the resonant plate can improve the sensitivity of the optical microscope.

[0243] Figure 18 A set of comparison images is shown, where the distance Z measured along the optical axis between the resonant plate 212 and the nanoparticle 203 varies between the individual images, as shown above each image. This example shows that the change in contrast and the shape of the interference pattern can be used to evaluate the distance from the nanoparticle 203 to the resonant plate 212.

[0244] To achieve better detection, image processing techniques can be implemented. The camera can record several consecutive images, and then combine the several consecutive images into a reference image. The reference image can be the average of the consecutive images.

[0245] Then the reference image can be subtracted from the received image to filter out the reference signal and stationary noise.

[0246] For the case of moving nanoparticles (e.g., suspended in a solution), two consecutive images can be subtracted to eliminate background noise.

[0247] In addition, a filter, such as a convolution filter, can be applied to the detected signal. The convolution filter can be a Gaussian filter.

[0248] The contrast of the particle image detected in bright-field mode is proportional to the mass of the particles present in the sample. Therefore, through image processing, this contrast can be quantitatively measured and the mass of the particles can be inferred therefrom by comparing with a calibration signal previously measured with particles of known mass. This technique is particularly effective for particles that do not absorb at the wavelength used (e.g., proteins in the visible spectrum).

[0249] Particles of known mass are selected, where the optical refractive index is very close to that of the particles to be characterized. For example, when the aim is to characterize organic materials, polymer materials are selected.

[0250] In a fourth embodiment of the optical microscope 800, as Figure 19 shown, the incident beam 802 passes through the sample 833: the nanoparticle scatters the light of the incident beam 802.

[0251] If the particle is located near the resonant plate 812, a portion of the scattered light is coupled to the guided mode.

[0252] Accordingly, a portion of the scattered light 804 is coupled to the guided mode of the resonant plate 812, while another portion is not coupled to the resonant plate 812. Then, the outgoing scattered light 804 includes a first portion of the scattered light that is coupled to the guided mode of the resonant plate 812 by particles placed near the plate; and a second portion of the scattered light that is not coupled. The outgoing beam is collected by the microscope objective 805 and propagated through a converging lens system.

[0253] The scattered light 804 and the non-scattered portion 815 of the incident beam pass through a converging outgoing lens, where the converging outgoing lens is composed of a first converging outgoing lens 808, a second converging outgoing lens 817, a spatial filter 809, and a third converging outgoing lens 818, and then are collected by an optical detector, here a camera 806.

[0254] The image focal plane of the first converging outgoing lens 808 corresponds to the object focal plane of the second converging outgoing lens 817. The spatial filter 809 is placed in the image focal plane of the second converging outgoing lens 817. The spatial filter 809 is placed in the image plane of the Fourier plane 813 of the microscope objective 805. Accordingly, the non-scattered portion 815 of the incident beam is focused on the spatial filter 809 and can be blocked by the filter.

[0255] The third converging outgoing lens 818 focuses the scattered light 804 onto the sensitive surface of the camera 806, and the camera 806 is located within the image focal plane of the third converging outgoing lens 218.

[0256] The filter 809 attenuates the non-scattered portion 815 of the outgoing beam and the second portion of the scattered light. The filter 809 is, for example, similar to Figure 11 filter 22. For example, the filter 809 can be an interference bright-field filter, and except for a second mask 96 located at the center of the filter, the structure of the filter 809 is similar to Figure 11 filter 22 in, and the second mask 96 attenuates the incident light. For example, the second mask and the first mask 98 have different transmittance coefficients. For example, the transmittance coefficient of the second mask is greater than that of the first mask.

[0257] As described in the first, second, and third embodiments, in the optical microscopes 100, 200, 700 that irradiate a sample in a reflective manner, approximately half of the energy scattered by each particle of the sample 833 is included in the resonant mode of the resonant plate 812 and is thus included in the first portion of the scattered light 804. The filter 809 largely filters the outgoing beam so as to block, as much as possible, the non-scattered portion 815 of the incident beam 802 without blocking the scattered light, and in particular, without blocking the first portion of the scattered light that has been coupled to the resonant mode of the resonant plate 812. This is achieved by directionally emitting the first portion of the scattered light that has been coupled to the resonant mode of the resonant plate 812, as described in the reflective embodiment of the reference optical microscope.

[0258] Advantageously, reducing the intensity of the incident beam increases the contrast of the sample image formed on the camera sensor. The same is true for the case of the above-described reflective optical microscope.

[0259] Unlike the embodiments of the optical microscope that use reflective irradiation of the sample, the incident beam 802 irradiates the entire thickness of the sample 833, rather than just the first few hundred nanometers of the sample in contact with the resonant plate (as is the case in the reflective configuration). Thus, in addition to the above functions, an optical microscope without the filter 809 can also be used. In this configuration, a larger volume of the sample is irradiated in order to detect slightly defocused particles. This is particularly useful for tracking the movement of scattered particles in the sample.

[0260] Due to the transmission configuration of the optical microscope 800, the outgoing beam is not affected by the back reflection of the incident beam on the multiple lenses of the microscope objective 805. These back reflections exist in the reflective configuration and constitute parasitic signals, in other words, noise. Their elimination improves the signal-to-noise ratio of the detected image.

[0261] A transmission embodiment of the optical microscope with a resonant plate is described here. Obviously, other transmission embodiments can be envisioned, particularly a simplified transmission embodiment similar to the first reflective embodiment, in which the second converging outgoing lens, the third converging outgoing lens, and the filter are eliminated. A transmission embodiment of the optical microscope used in an interference configuration can also be envisioned, using balanced in-phase detection technology, in which the outgoing beam travels along the optical path at the exit of the resonant plate, similar to the path that the outgoing beam travels after passing through the first polarization beam splitter in the above-described third reflective embodiment.

[0262] Although the present invention has been described in connection with several specific embodiments, it is obvious that the present invention is in no way limited thereto, and the present invention includes all technical equivalents of the described devices and their combinations, provided that these fall within the scope of the present invention.

[0263] The use of the verb “comprise” or “include” and its conjugations does not exclude the presence of elements or steps other than those listed in the claims.

[0264] In the claims, any reference signs in parentheses shall not be construed as limiting the claim.

Claims

1. An optical microscope (100, 200, 700, 800), the optical microscope comprises: - a light source (101, 201, 701, 801) that emits illumination light (102, 202, 502, 602, 702, 802) suitable for irradiating a sample to be imaged (133, 233, 733, 833); - an optical device that includes a microscope objective (105, 205, 705, 805); - a resonator (112, 212, 512, 612, 712, 812) that sequentially includes, along the direction of the optical axis of the microscope objective: at least one first layer (241, 541, 641) having a first optical refractive index, at least one spacer layer (242, 542, 642) having a second optical refractive index, and at least one waveguide layer (243, 543, 643) having a third optical refractive index, the second optical refractive index being less than the first optical refractive index and the third optical refractive index, the resonator having a support surface facing away from the optical device, the support surface being intended to receive the sample (133, 233, 733, 833); - an optical detector (106, 206, 706, 716, 806); the optical device is configured to collect light exiting from the resonator (112, 212, 512, 612, 712, 812) and direct the exiting light from the resonator (112, 212, 512, 612, 712, 812) to the optical detector to form an image of the sample (133, 233, 733, 833) on the optical detector (106, 206, 706, 716, 806); the exiting light includes light scattered by the sample (104, 204, 504, 604, 704, 804) and an unscattered portion (115, 215, 715, 815) of the illumination light (102, 202, 502, 602, 702, 802).

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

3. The optical microscope (100, 200, 700, 800) according to claim 2, wherein, The intensity transmission coefficient associated with the first attenuation is less than 10 -6 .

4. The optical microscope (100, 200, 700, 800) according to claim 2 or 3, wherein, The light scattered by the sample includes: a first part of the scattered light from the resonant excitation mode; and a second part of the scattered light, and the amplitude filters (209, 764, 20, 22, 809) are further configured to apply a selective second attenuation to the second part of the scattered light.

5. The optical microscope (100, 200, 700, 800) according to claim 4, wherein, The intensity transmission coefficient associated with the selective second attenuation is less than 10 -6 .

6. The optical microscope (100, 200, 700, 800) according to the combination of claim 2 and claim 4, wherein, The intensity transmission coefficient associated with the first attenuation is greater than the intensity transmission coefficient associated with the second attenuation.

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

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

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

10. The optical microscope (100, 200, 700, 800) according to any one of the foregoing claims, wherein, The optical microscope further includes an image processing system, and the image processing system is configured to: - Record a plurality of images detected by the optical detector at consecutive times; - Synthesize the plurality of images into a reference image; - Process at least one image detected by the optical detector using the reference image to suppress static signals.

11. The optical microscope (100, 200, 700, 800) according to any one of claims 1 to 10, wherein, The optical microscope further includes an image processing system, and the image processing system is configured to: Determine the contrast in the image detected by the optical detector; Determine at least one parameter of the particles contained in the sample according to the contrast, and the parameter is selected from the mass of the particles and the position of the particles in the direction along the optical axis.

12. The optical microscope (100, 200, 700) according to any one of claims 1 to 11, wherein, The light sources (101, 201, 701) and the optical device are arranged to irradiate the sample (133, 233, 733) placed on the support surface of the resonator (112, 212, 512, 612, 712) in a reflective manner.

13. The optical microscope (100, 200, 700) according to claim 12, wherein, the optical device receives the irradiated light (102, 202, 502, 602, 702) to direct the irradiated light to the sample (133, 233, 733), and the microscope objective (105, 205, 705) of the optical device is configured to direct the irradiated light (102, 202, 502, 602, 702) to the resonator (112, 212, 712) at an incident angle greater than the critical angle of the interface between the first layer (241, 541, 641) and the spacer layer (242, 542, 642), so that the irradiated light (102, 202, 502, 602, 702) resonantly excites at least one mode in the waveguide layer (243, 543, 643) and irradiates the sample (133, 233, 733) through the enhanced evanescent wave.

14. The optical microscope (100, 200, 700) according to any one of claims 12 to 14, wherein, the optical device includes a polarization beam splitter (114, 214, 714), and the polarization beam splitter (114, 214, 714) reflects the irradiated light (102, 202, 502, 602, 702) to the microscope objective (105, 205, 705) and allows the outgoing light to pass through.

15. The optical microscope (700) according to claim 15, wherein, the optical detector is a first optical detector (706), the optical microscope includes a second optical detector (716), the optical device includes an unpolarized beam splitter (766) and a phase mask (768), the unpolarized beam splitter receives the outgoing light (704, 715) and divides the outgoing light into a first part of the outgoing light directed to the first detector (706) and a second part of the outgoing light directed to the second detector (716), the first part of the outgoing light includes a first part of the reflected light (778) and a first part of the scattered light (780), the phase mask (768) is arranged to allow the first part of the outgoing light to pass through, and the phase mask (768) is configured to apply a phase shift between the first part of the reflected light and the first part of the scattered light.

16. The optical microscope (700) according to claim 16, wherein, The phase mask is a first phase mask (768), and the optical device further includes a second phase mask (771). The second phase mask is arranged for the second part of the outgoing light to pass through. The second part of the outgoing light includes a second part (779) of the reflected light and a second part (781) of the scattered light. The second phase mask (771) is configured to apply a phase shift between the second part of the reflected light and the second part of the scattered light, and the first phase mask and the second phase mask have different phase characteristics.

17. The optical microscope (100, 200, 700) according to one of the preceding claims, wherein, the optical device includes an optical condenser (107, 207, 761). The optical condenser receives the illuminating light emitted from the light source, and the optical condenser (107, 207, 761) is configured to focus the illuminating light into a region away from the optical axis of the microscope objective (105, 205, 705) in the Fourier plane (113, 213, 713) of the microscope objective to generate the incident angle.

18. The optical microscope (800) according to one of claims 1 to 11, wherein, the resonator (812) is arranged 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 a sample (833) received by the support surface of the resonator (812) in a transmissive manner.

Images