Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N2021/6417—Spectrofluorimetric devices
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N2021/6417—Spectrofluorimetric devices
  • G01N2021/6421—Measuring at two or more wavelengths

Definitions

• the present invention relates to a device and a method for exalted analysis of a sample of particles.
• the present invention relates to the field of devices for analyzing luminescent or optically diffusing particles.
• a device for analyzing a sample of particles in solution comprising a microscopy system, said microscopy system comprising means for supporting said sample, illumination means capable of emitting a beam excitation light, focusing means of said excitation light beam at a focusing point at the sample and spatial filtering means able to define a volume of analysis around the focusing point.
• Such a device is intended to be used in any type of optical analysis application on particles having a luminescence or diffusion marking, that is to say having a determined response in luminescence or diffusion to excitatory light.
• the particles to be analyzed are molecules or molecular assemblies, for example molecular complexes, nanocrystals or nanobeads, and have dimensions smaller than the wavelength of the light emitted by the illumination means. It finds particularly its application in the field of the detection of fluorescent molecules in solution, and more particularly in fluorescence correlation spectroscopy. It also finds an application in biological tests and more specifically the microarray DNA or protein test.
• the state of the art in this field comprises analysis devices comprising a confocal microscopy system.
• a system aims to analyze a sample, for example particles in solution, luminescent or optically diffusing.
• the system comprises a laser source emitting a laser beam at a given wavelength, a dichroic mirror, a microscope objective having a very high magnification and numerical aperture, optical imaging means, spatial filtering means and a detector.
• the laser beam is made convergent by the objective into a focusing point located at the level of the analysis volume that one wishes to analyze.
• the light of the focused laser beam is then absorbed by the particles and then reemitted at a wavelength greater than that of the laser beam and then directed to the detector level.
• the optical imaging means are arranged so as to combine the focusing point of the laser beam with the detector.
• the spatial filtering means make it possible to delimit an analysis volume and thus obtain a spatial resolution of less than one micrometer.
• They comprise an opening disposed upstream of the detector, in a plane conjugated to the focal plane of the microscope objective. In this way, only the photons located in a volume around the focusing point of the laser beam participate in the formation of the image at the detector.
• a correlator is connected to the detector in order to analyze the temporal fluctuations of the fluorescent light emitted by the analysis volume, so as to perform detection by spectroscopy.
• fluorescence correlation FCS
• the fluorescence as well as the temporal fluctuations thereof are thus analyzed.
• These fluctuations are directly related to the diffusion of fluophores through the volume of analysis.
• the autocorrelation function of the detected luminous intensity makes it possible to have access to the photophysical parameters of the emitters as well as to the average number of molecules detected. At long times, this function gives information on the average residence time - the diffusion time - of the molecules and their diffusion mode through the analysis volume.
• Such a device makes it possible to benefit from a large opening in order to collect the maximum of light and therefore of fluorescence, as well as to reduce the volume of analysis and consequently to reduce the diffusion noise in the solution. Nevertheless, for an application where it is desired to have an analysis resolution on a single molecule, such a device has several drawbacks. Indeed, the diffraction phenomena set a fundamental limit to the size of the focal spot of the laser, and therefore to the size of the analysis volume. These phenomena lead to limitations on the count rate per molecule at fixed power and on the maximum concentration for the detection of single molecules, since one seeks to analyze only one molecule per volume of analysis.
• a device for measuring a sample by correlation spectroscopy comprises a confocal microscope comprising an optical focusing system whose field defines a collection volume - or analysis volume - means capable of producing a beam of light. excitation and directing it on the sample through the microscope, means for detecting the intensity of the luminous flux produced by the interaction of the excitation beam on the sample and collected by the microscope, as well as means signal processing produced by the detection means.
• This device also comprises a photonic structure increasing the collected luminous flux, placed at the focus of the optical microscope focusing system and forming interference fringes in the collection volume.
• This photonic structure may be for example a dielectric mirror resistant to water and consisting of a stack of layers of very small optical thickness. It is placed at the focus of the optical focusing system of the microscope to form interference fringes in the collection volume.
• the implementation of this structure makes it possible, on the one hand, to significantly increase the signal collected per molecule and, on the other hand, to solve the problem of defining the collection volume.
• the disadvantage of this solution lies in the difficulty of obtaining a sufficient rejection of the noise induced by the reflection of the excitation beam on the mirror and the parasitic luminescence induced in the mirror.
• the maximum reduction The volume of analysis that can be obtained is about 3, which does not bring a substantial gain in practical applications.
• the object of the present invention is to remedy these technical problems by allowing the significant reduction of the analysis volume so that it contains only a quantity of molecules up to unit and by the implementation of means of exaltation of both the excitation light beam and the collection of emitted luminescence.
• These excitation means are arranged in such a way as to make the focal field defined by the excitation light beam at the level of the sample more intense and more condensed, this field being capable of reaching dimensions smaller than the diffraction limit, which allows the analysis on a volume lower than the femtolitre and thus likely to contain only one molecule in solutions of high concentrations (higher than 10 nanomolar).
• the approach of the solution consisted in implementing nanophotonic emission devices - called “photonic jet” - in order to reduce the focal field defined by the excitation light beam at the level of the sample and to increase the collection efficiency of the emitted light.
• the state of the art of this type of device comprises a microbead disposed in the path of a collimated beam in order to focus this beam. This confers on said beam a small divergence, as well as an invariance along its axis, with respect to a normal beam, capable of diffracting significantly over a small area.
• This type of device is therefore apparently not compatible with use on a highly focused light beam path, such as the excitation light beam of a microscopy analysis device, rendered converging at a focusing point.
• the study of the behavior of the excitation light beam crossing in part such a nanophotonic emission device, as well as its implementation have made it possible to demonstrate a significant reduction in the volume of analysis on the sample.
• the subject of the invention is an analysis device of the above-mentioned type in which, in addition to the features already mentioned, the microscopy system also comprises means for exalting said excitation light beam, said means exaltation having a strictly positive focus and a refractive index greater than the refractive index of said sample, at least a portion of said enhancement means being disposed in the path of said excitation light beam downstream of said support means and upstream of said focus point, at least a portion of said enhancement means not being integral with the support means.
• the exaltation means thus implemented make it possible to over-focus the excitation light beam into a zone of smaller dimensions than the initial analysis volume. Indeed, the portion of the incident light beam passing through the exaltation means converges in a zone adjacent to said enhancement means, this area being made narrower by the focal length and the refractive index of said exciting means. The part of the light beam that does not pass through the said means of exaltation will interfere with the part that went through them according to a phenomenon of destructive interference. Most of the light intensity of the incident beam is thus concentrated in a volume of longitudinal dimension of the order of half the wavelength and axial dimension of the order of the wavelength, therefore of dimensions less than the diffraction limit.
• the response light beam produced in response to the interaction of the excitation light beam on the sample and collected by the focusing means , is privilegedly directed in one or more specific directions of space.
• the particles will emit light in a privileged way towards the means of exaltation because its refractive index is the largest.
• an even higher light response flow will be collected and detected, which also allows to exalt also the collection of emitted luminescence.
• the analysis device thus constituted by the combination of the excitation means with a conventional microscopy system comprising spatial filtering means, thus makes it possible to significantly reduce the volume of analysis at the level of the sample to analyze single molecules, while concentrating the excitation light beam in this volume so as to benefit from a signal collection per molecule sufficient for analysis, for example for correlation spectroscopy applications.
• the microstructured interface defined by the means of exaltation can be simple to manufacture and extended on a large scale.
• at least part of the exaltation means is integral with the support means.
• the portion of the integral extinguishing means of the support means is constituted by a projection on said support means, said projection having a strictly positive curvature.
• the part of the non-integral exaltation means of the support means comprises at least one microlens, the part of the non-integral exaltation means of the support means comprises at least one microdrop, and
• the part of the non-integral exaltation means of the support means comprises at least one microbead.
• the (or) microbead (s) has a diameter substantially between 1 and 5 micrometers. This makes it possible to optimally focus the light intensity of the excitation light beam in a small analysis volume. In order to optimally optimize the dimensions of the analysis volume, it is expected that the (or) microbead (s) has a diameter substantially equal to 2 micrometers.
• the exaltation means are axially centered on the axis of the excitation light beam
• the exaltation means are centered longitudinally on the focusing point of the excitation light beam.
• the means of exaltation are covered at least in part by at least a thin metallic layer.
• the microscopy system also comprises means for detecting the intensity of the response light beam produced in response to the interaction of the excitation light beam on the sample and collected by the focusing means. This makes it possible to perform measurements only on the analysis volume, by measuring the luminous intensity received by the detection means in response to the excitation of the particles of the sample contained in the analysis volume.
• the microscopy system also comprises signal processing means provided by the detection means. This allows measurements not only of light response, but also temporal fluctuations of this light response, giving access to a significantly larger number of information on the particles contained in the sample.
• the support means consist of a glass substrate.
• the illumination means consist of a laser source. Upstream of the focusing means, there is thus a collimated, monochromatic excitation light beam of good optical wavefront quality.
• the focusing means it is expected that they consist of a high numerical aperture objective (typically greater than 0.7).
• a second embodiment of said focusing means it is expected that they consist of a low numerical aperture lens (typically less than 0.7).
• the focusing means it is expected that they consist of a high numerical aperture objective (typically greater than 0.7).
• a second embodiment of said focusing means it is expected that they consist of a low numerical aperture lens (typically less than 0.7).
• the microscopy system is confocal, the spatial filtering means comprising an opening of variable size. This makes it possible to adjust the size of the analysis volume, which in combination with the exaltation means, allows the significant reduction of the analysis volume.
• the excitation means of the excitation light beam are arranged in parallel. This makes it possible to have a volume of analysis covering a large but thin surface. Detection can then be done with a sensor comprising a plurality of pixels.
• the present invention also relates to a method for analyzing a sample of particles in solution by an analysis device comprising a microscopy system, said microscopy system comprising means for supporting said sample, illumination means capable of emitting an excitation light beam, focusing means of said excitation light beam at a focusing point at the sample and spatial filtering means able to define a volume of analysis around the focusing point, characterized in that that: at least a part of said excitation light beam is exalted downstream of said support means and upstream of said focusing point by a strictly convergent focusing and a refraction index greater than the refractive index of said sample, at least a part of which is not integral with said support means, and the intensity of the light beam of response is measured e as a function of time, said response light beam being derived from the interaction of the excitation light beam with the sample at the level of the analysis volume.
• FIG. 1 a diagram of a fluorescence correlation spectroscopy analysis device according to a first embodiment of the invention
• FIGS. 2A to 2D various embodiments of the excitation means according to FIG. invention
• FIG. 3 is a diagram of a fluorescence correlation spectroscopy analysis device according to a second embodiment of the invention.
• focusing point of the excitation light beam is meant the point of focus of the beam when no part of the beam passes through at least a portion of the excitation means.
• it may be said, for example, of some of the means of exaltation that it is arranged upstream of the focusing point of the excitation light beam, so as to understand that the center of this portion of the excitation means is disposed upstream of the beam focusing point if There was no means of exaltation.
• FIG. 1 represents a diagram of a fluorescence correlation spectroscopy analysis device according to a first embodiment of the invention.
• the analysis device aims to analyze a sample 2.
• This sample can be a liquid medium, gaseous, or a biological object containing particles to be analyzed.
• the particles to be analyzed are molecules or molecular assemblies, for example molecular complexes, nanocrystals or nanobeads, and have dimensions smaller than the wavelength of the light emitted by the illumination means.
• the analysis device comprises a confocal microscopy system 1.
• This system is an arrangement comprising support means 3, illumination 4, focusing 5, dichroic separation 6, spatial filtering 7, detection 8, treatment 9 and exaltation 14.
• the support means 3 consist of a glass substrate.
• This substrate may be a microscope slide having a thickness of between 100 and 200 microns.
• This substrate supports the sample 2, which is advantageously enclosed in a sealed box whose side walls are formed by self-adhesive wedges of thickness between 50 and 100 microns and resistant to water. The substrate is thus disposed at the top of said sealed box.
• the illumination means 4 emit an excitation light beam 10.
• This beam 10 is collimated at the output of the illumination means 4.
• These means advantageously consist of a laser.
• the laser may be a diode-pumped solid laser operating at a wavelength of 488 nanometers or a helium-neon laser operating at a wavelength of 633 nanometers.
• the excitation light beam 10 is directed towards the dichroic separation means 6.
• These means 6 are advantageously constituted by a dichroic filter of the notch type. This filter is chosen so that its cut-off wavelength and its bandwidth make it possible to reflect the excitation light beam 10 and to transmit the response light beam 11.
• the wavelength of the response beam 11 is indeed higher than that of the excitation beam 10 since the particles are fluorescent.
• the light response is expected at 670 nanometers, which imposes a cut-off wavelength of the dichroic filter of the order of 640 nanometers.
• This filter is also chosen so as to have a maximum transmission factor for the wavelength of the response beam 11.
• the dichroic filter is arranged at 45 ° of the incident excitation beam 10.
• the dichroic filter can thus direct, after reflection on the dichroic separation means 6, the excitation light beam 10 to the focusing means 5.
• These means 5 are able to direct the beam towards a focusing point 12 on the sample 2 through the confocal microscopy system 1.
• These means 5 are for that purpose constituted of an objective or a lens.
• the focusing means 5 consist of a microscope objective.
• This objective is for example an apochromatic objective having a magnification of 40x and a numerical aperture of 1, 2.
• This high numerical aperture makes it possible to collect an optimal light intensity in response to the excitation by the light beam 10. The focusing and collection efficiency are then made optimal.
• the focusing means 5 consist of a lens.
• This type of focusing means has a low numerical aperture, which does not make it possible to optimally exploit the additional effect obtained by the excitement means 14. Nevertheless, for a lower cost than a microscope objective, the low efficiency of focusing and collection are compensated by the means of exaltation 14.
• the focused excitation light beam 10 thus interacts with particles in solution in sample 2 according to a fluorescence phenomenon.
• each excited particle will emit fluorescence a light response at a wavelength greater than that of the excitation beam 10.
• Part of this light response is then collected by the focusing means 5 so as to form a light beam of answer 11.
• This response light beam 11 is sent to the detection means 8 through the respective focus 5, dichroic separation 6 and spatial filtering means 7.
• the cutoff wavelength of the dichroic filter is indeed chosen so as to transmitting the response light beam 11, whose wavelength is greater than that of the excitation beam 10.
• the spatial filtering means further comprise a set of lenses 7 ' and 7 '"enabling the focal plane of the focusing means 5 to be combined with the capture plane of the detection means 8.
• the spatial filtering means 7 also comprise an aperture 7 "of adjustable size, which opening is disposed along the axis 15 of the response light beam 11. It is optically conjugated with the focusing point 12 of the focusing means 5. It allows selecting a detection volume - or spatial filtering volume - around the focusing point 12, since light rays not coming from this spatial filtering volume do not pass through the opening 7 ". The dimensions of the detection zone are all the shorter as those of the opening 7 "are also so.
• the assembly 7 consisting of elements 7 ', 7" and 7' "thus forms a pinhole having a variable opening 7 placed in an intermediate image plane so that the microscopy system conjugates the focusing point 12 of the excitation beam 10 with the variable aperture 7 "of the pinhole camera.
• the spatial filtering means 7 do not comprise an opening 7 ", they may in fact be made up of other elements that make it possible to carry out spatial filtering, and the microscopy system thus implemented does not is more of a confocal type, these elements can be for example:
• the detection means their surface being small, of the order of a few tens of micrometers in diameter, or - the excitation beam itself, in the case of a two-photon excitation.
• the detection means 8 make it possible to measure the intensity of the response light flux 11 produced by the interaction of the excitation beam 10 on the sample 2 and collected by the microscopy system 1.
• these means 8 comprise electron amplification photodetectors.
• These photodetectors are advantageously photodiodes operating in avalanche or cascade - APD (avalanche photodiode, or "avalanche photodiode” in the Anglo-Saxon language). These photodetectors may also be photomultipliers.
• these detection means 8 comprise photodetectors with optical amplification, cooled CCD or CMOS cameras, for example with liquid air or Peltier element.
• These detection means 8 can operate in two reading modes.
• the detection is carried out according to a continuous regime, where the optical signal emitted by the targets is detected by integrating the signal for each pixel or group of pixels - in the case where the detection means comprise several pixels - of the detector. Radiometric measurements are then also possible between several pixels or groups of pixels.
• the detection is carried out according to a time regime where the detector integrates the signal over short time ranges with respect to the process to be analyzed. The information is then given by the analysis of this temporal trace.
• the signal from these detection means 8 is sent to the signal processing means 9.
• These means 9 advantageously comprise a counter and a correlator that allows to digitally process the received data.
• the counter realizes the recording of the value of the received fluorescence luminous intensity and the correlator performs the temporal analysis of the fluctuations of the received fluorescence luminous intensity. This analysis can be done at short and long times so as to obtain additional information on the particles in solution in sample 2.
• the autocorrelation function of the detected light intensity makes it possible to access the photophysical parameters of the emitters as well as the average number of molecules detected. At long times, this function gives information on the average residence time - diffusion time - of the molecules and their mode of diffusion through the spatial filtering volume.
• the excitation means 14 make it possible to modify the shape of the excitation light beam 10 so as to exalt the luminous flux and thus concentrate it in an analysis volume of smaller dimensions.
• the excitation means 14 are arranged in the path of the excitation light beam downstream of the support means 3 and upstream of the focusing point 12.
• exaltation means 14 have a strictly positive focal length and a refractive index greater than the refractive index of said sample 2. Under these conditions, the portion of the incident light beam 10 which passes through the excitation means 14 converges in a zone adjacent to said enhancement means 14. This zone is made narrower because their focal length is strictly positive and their refractive index is greater than that of the solution in the sample 2. The portion of the light beam 10 that does not pass through said means of exaltation 14 will interfere with the part that has passed through them according to a phenomenon of destructive interference.
• Most of the light intensity of the incident beam 10 is thus concentrated in a volume of longitudinal dimension of the order of half the wavelength and axial dimension of the order of the wavelength, so smaller than the diffraction limit, which makes it possible to reach a volume of analysis less than one-tenth of femtoliter.
• the response light beam 11 is directed in a privileged manner so that the light or re-emitted towards the means of exaltation 14.
• the excitation means 14 thus constitute a microstructured interface between the support means 3 and the focusing point 12 of the excitation beam 10, so as to reduce the dimensions of the analysis volume to be detected and to concentrate a luminous intensity thereon. superior to that without means of exaltation.
• the exaltation means 14 have micrometric dimensions, of the order of 1 to 5 micrometers, and a high refractive index, of the order of 1, 4 to 1, 6.
• the exaltation means 14 are not integral with the support means 3, are at least partially integral with said support means 3, or are entirely integral with said support means 3.
• the exaltation means 14 are integral with the support means 3.
• the exaltation means thus consist of a projection 14 'on the support means 3.
• This projection presents a strictly positive curvature so that the focal length of the thus constituted means of exaltation is strictly positive.
• the exaltation means 14 are not integral with the support means 3.
• the microstructured interface thus constituted of the exaltation means 14 can be manufactured by dispersing said means 14 on the glass substrate of the support means 3.
• the enhancement means 14 "consist of a convergent microlens.
• This lens of micrometric dimensions, advantageously has a high focal length so as to best focus the light flux from the excitation beam 10 in an area as close as possible to the lens, this area is thus all the more isolated from the rest of the sample 2 where other particles are likely to be excited.
• the exaltation means 14 "consist of a microdrop: the focal length of this micrometric drop advantageously has a high curvature in order to best concentrate the light flux from the beam excitation 10 in an area as close as possible to the drop.
• the excitation means 14 preferably consist of a microbead.
• This microbead is a dielectric ball of high refractive index and micrometric dimensions.
• This ball may be made for example of latex or polystyrene, whose index is 1, 6 and which also has the advantage of being more flexible than the glass so as not to be damaged during the manufacture of the microstructured interface.
• the microbead has the advantage of have very high curvatures, which makes it possible to optimally focus the light intensity of the excitation beam in a small analysis volume
• the diameter of a microbead is between 1 and 5 micrometers, and preferably of 2 micrometers.
• the exaltation means 14 may comprise both an integral part 14 'and a non-integral part 14 "of the support means 3.
• said enhancement means 14 are advantageously axially centered on the axis 15 of the excitation light beam 10. They are also advantageously centered longitudinally on the focusing point 12 of the excitation light beam 10. These centering means of excitation 14 with respect to the excitation light beam 10 make it possible to exalt said beam 10 optimally.
• the exciting means 14 are covered at least in part with at least one thin metal layer.
• the metal used for this layer is a metal selected from aluminum and noble metals, such as gold, silver, copper and nickel.
• the thickness of this layer is advantageously less than or equal to 30 nanometers in order to avoid having a too opaque layer which significantly reduces the transmission of the microscopy system.
• This thin metal layer may be for example a gold layer, of thickness equal to 20 nanometers, covering entirely or partially the means of exaltation.
• FIG. 3 represents a diagram of a fluorescence correlation spectroscopy analysis device according to a second embodiment of the invention.
• the confocal microscopy system comprises:
• a helium-neon laser source emitting at 633 nanometers, for which a light response resulting from an interaction with fluorescent molecules is expected at 670 nanometers
• a diode-pumped solid laser source emitting at 488 nanometers, for which a light response resulting from an interaction with fluorescent molecules is expected at 520 nanometers
• a conventional microscopy device comprising a microscope objective 23 and a lamella of glass 24 carrying a sample 25 containing the particles in solution
• a second band-stop dichroic filter 27 around 488 nanometers a pinhole camera 28 comprising an adjustable opening and two lenses arranged so as to spatially filter a detection volume around the focusing point of the beams coming from the two laser sources 20 and 21 ,
• a third high-pass dichroic filter 29 whose cut-off wavelength is between 520 and 670 nanometers so as to separate the fluorescent light responses resulting from the interaction of the particles with each of the laser sources 20 and 21,
• a separator cube 34 two avalanche photodiodes 35 and 36 respectively preceded by filters 37 and 38 for measuring the intensity of the fluorescence luminous flux at 670 nanometers collected by the microscopy system, and
• the spectroscopy device 30 makes it possible to analyze the spectral composition of the light over a spectral range of 500 to 700 nanometers for an excitation wavelength of 488 nanometers.
• the intensity of the emitted light is measured by a detector 31, which may be single-channel, for example of the photomultiplier type, or multichannel, for example of the CCD type. It is advantageously associated with the device 30 a high-pass filter 32 and a notch filter so as to improve the quality of the signal transmitted to the detector 31.
• Such an analysis device combining a fluorescence correlation spectroscopy analysis and a Raman spectroscopy analysis, thus makes it possible to obtain not only statistical and temporal information on the particles in solution, but also information on particles taken individually.
• the excitation means 14 of the excitation light beam 10 may be arranged in parallel.
• An exemplary embodiment is a microbead carpet disposed against the glass substrate 3. This microstructured microbead assembly makes it possible to obtain a large and thin surface analysis volume. Detection can also be done with a multi-pixel CCD or CMOS sensor.
• the particles to be analyzed can also have properties of chemiluminescence, bioluminescence, diffusion (Rayleigh or Hyper-Rayleigh), vibrational spectroscopy (spontaneous or stimulated Raman), thermal emissivity or reflection (case of granulometric studies). .

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