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
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
• • 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
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
  • G01N21/553—Attenuated total reflection and using surface plasmons
  • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
• • 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
• • 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
  • G01N21/65—Raman scattering
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/06—Means for illuminating specimens
  • G02B21/08—Condensers
  • G02B21/082—Condensers for incident illumination only
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
• • 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
  • G01N2021/6463—Optics
• • 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
  • G01N2021/6463—Optics
  • G01N2021/6471—Special filters, filter wheel
• • 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
  • G01N2021/6491—Measuring fluorescence and transmission; Correcting inner filter effect
  • G01N2021/6493—Measuring fluorescence and transmission; Correcting inner filter effect by alternating fluorescence/transmission or fluorescence/reflection
• • 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

Definitions

• the present invention relates to the field of imaging for the chemical, biological or biochemical analysis of a sample.
• the samples are generally in the form of "spots" or (micro) drops of known substances (or analytes) deposited on supports such as microscope slides and on which optical analyzes are performed by making an image of the slide by the microscope. one or the other of the techniques mentioned below, possibly after or during the contacting of the sample with a substance to be analyzed (or of known composition, respectively).
• the invention relates, according to a first of its objects, to an imaging system for the chemical, biological or biochemical analysis of a sample, the system comprising
• an illumination device capable of emitting a monochromatic light beam
• microscopy system is well known to those skilled in the art.
• numerous developments have been made in microscopy in contrast-imaging techniques revealing certain properties of the imaged object, particularly in UV / visible reflectance / transmittance spectroscopy otherwise known as LSPR for "Localized Surface Plasmon Resonance". Or surface plasmon resonance; in fluorescence (in particular in MEF for “Metal-Enhanced Fluorescence”), as well as in Raman scattering (or SERS for "Surface-Enhanced Raman Scattering”).
• the present invention advantageously exploits at least some of these techniques in the field of biosensors.
• biosensors offer more efficient solutions than conventional membrane tests, especially when they can be used in configurations allowing a massively parallel measurement of several probe / target pairs on the same support.
• the high-throughput measurement of biomolecular interactions is an important issue in certain fields of application such as the diagnosis or the screening for the search for new ones. drugs.
• Fluorescence offers the best sensitivity, but has the disadvantage of having to mark targets or probes with fluorophore groups.
• Surface plasmon resonance has the advantage of being able to detect unmarked target-probe interactions, but has a lower sensitivity.
• the constraints related to excitation geometry make imaging difficult. It can advantageously be used in imaging mode by exciting surface localized plasmon resonances (LSPR) in structured metal layers in the form of islets of nanometric dimensions. The analysis is then done by means of a simple spectroscopic analysis of the variation of the reflection or the transmission of the layer in contact with a sample.
• LSPR surface localized plasmon resonances
• parallel LSPR analysis of fluorescence analysis can be used to detect the presence of non-specific adsorption on the sensor, which beyond a certain threshold may hide the probes and make the sensor insensitive. Such control is essential to maintain performance in applications in the area of control or monitoring.
• the LSPR used alone also has the disadvantage of not offering chemical discrimination.
• the light emitted should be collected at a solid angle as large as possible; the excitation must also be monochromatic and intense, but we must also analyze the wavelength distribution of the light emitted by the sample and more precisely the wavelength changes (Raman offsets) with respect to the exciting radiation.
• a dispersive device such as for example a diffraction grating
• An alternative is to use a holographic network, or more generally a spatial filtering device, and then to reconstruct images for each wavelength analyzed by means of elaborate image processing programs.
• the device according to the invention is essentially characterized in that the objective is configured for downstream of the sample:
• Transforming the re-emitted or scattered light beam into a quasi-parallel beam and in that it further comprises a device for selectively closing the collected light beam coming from the sample by said lens, preferably disposed in said focal plane ; which selectively allows the analysis of the sample according to at least two of the analysis techniques among the set comprising surface plasmon resonance, fluorescence, and Raman scattering as described later.
• the upstream and downstream are defined with respect to the direction of the light beam from the source to the detector.
• the selective shutter device comprises
• a diaphragm configured to be placed in the focal plane of said objective around the focusing point C, and
• the selective closure device further comprises at least one of an array comprising a high-pass filter and a band-pass filter.
• system further comprises, upstream of said objective, means for reflecting the light beam from the source of said beam to the support device of the sample.
• said reflection means comprise a mirror disposed near the optical axis, or a semi-transparent mirror placed in the optical axis, oriented at 45 ° ⁇ 2 ° with respect to the optical axis, and arranged so that to allow the observation of the sample in transmission or in reflection with minimal occultation.
• the device for selectively closing the collected light beam further comprises an electrical or mechanical switch, for selectively activating said diaphragm or said shutter.
• the illumination device comprises a white light source associated with a monochromator, and / or a monochromatic light source, tunable or not.
• the illumination device comprises an assembly of at least one optical fiber and an assembly of at least one unitary light source, possibly tunable, and in which the input of at least one fiber is capable of to be connected to at least one unitary source.
• the input of at least one fiber is connectable to a plurality of unit sources, the system further comprising a switch for connecting the input of the fiber to one of the unitary sources in a single source. Relative movement between the input of the fiber and the unit source connected to the input thereof.
• the switching between the sources can then be done without relative displacement, by electrically activating the chosen unit source, or with the aid of shutters placed at the output of each unit source.
• Such an embodiment may be advantageous when the unitary sources are LEDs or non-tunable lasers.
• the invention also relates to a method for chemical, biological or biochemical analysis of a sample, in a system according to the invention, comprising the steps of: illuminate a sample with a monochromatic light beam, and
• the method is essentially characterized in that it further comprises steps of:
• the method further comprises a step of superimposing said images.
• the method further comprises a step of filtering the light beam, downstream of the selective focusing.
• the method further comprises steps of
• the invention it is possible to easily compare images recorded by two or three of the techniques mentioned, in particular by simple superposition, thanks to a common optical column and a CCD detector for all these techniques, that is to say to couple different techniques within the same apparatus, without movement of the sample relative to the optical axis. Thanks to the invention, it is possible to carry out tests that make it possible to obtain information of a chemical or biochemical nature with or without marking the targets or probes, to correlate the information obtained by at least two independent techniques and to perform performance checks of real-time tests of media used for testing. These performances are obtained by allowing the coupling of different techniques within the same apparatus. Thanks to the invention, it is possible to perform imaging of a centimetric zone, with a resolution of the order of 10 ⁇ m, sufficient to image spots of the order of 100 ⁇ m in diameter.
• the present invention also allows the high-speed measurement of biomolecular interactions in real time, a fundamental issue in certain fields of application such as diagnosis or screening for the search for new drugs. Similarly, the chemical recognition measurement of Specific real-time targets are also needed to provide effective solutions for control or monitoring applications.
• Other characteristics and advantages of the present invention will emerge more clearly on reading the following description given by way of illustrative and nonlimiting example and with reference to the appended figures in which:
• FIG. 1 illustrates an embodiment of the system according to the invention in the LSPR configuration, with a reflectance light source and / or a transmittance light source, and
• FIG. 2 illustrates an embodiment of the system according to the invention in the MEF configuration, also with a reflectance light source and / or a transmittance light source.
• One of the principles of the present solution is to implement a single optical detection device for a plurality of possible analysis techniques among the set comprising the LSPR, the MEF and the SERS (see definitions above) .
• the optical detection device 20 comprises a lens or group of lenses f2 and a detector such as a sensor (or camera) CCD 21.
• the magnification of the optical system is determined once the dimensions of the CCD and the area to be explored.
• a lens fl (lens or lens array), described later and used for shaping the collected light beam from the sample 10 and incident on the detector 21.
• lens fl lens or lens array
• the zoom function can be provided by the lens fl, but in this case it is necessary to provide an adjustment of the distance between the point C and the lens, which is a little less advantageous.
• a cooled CCD camera so as to be able to use long time measurement for recording each image.
• the illumination of a sample 10 consists of a quasi-parallel incident light beam, with the most uniform intensity possible over the entire surface of the zone studied.
• the wavelength of this illumination, monochromatic, can however be variable or not, and the beam can illuminate the sample in two configurations, also called geometries:
• illumination can be achieved from the front or the back.
• an illumination from the front is slightly more favorable, because it leads to parasitic light (due for example to scattering by defects or dust, or to the fluorescence or Raman scattering of the substrate on which the analyzed sample is deposited and through which this sample is illuminated) significantly lower.
• the quasi-parallel shaping of the incident light beam for the illumination of a sample 10 can be carried out as follows.
• the light beam is derived from the illumination device 30_R. It is then focused, for example by a lens, on an inclined mirror 50, placed (in the detection column) a few millimeters from the optical axis YY and the focal plane FF, in this case higher, of the objective fl, i.e. close to the focus point C of the lens f1.
• the mirror 50 may be a mirror of diameter 2 mm whose center is located 3 ⁇ 1 mm from the optical axis.
• the objective fl transforms the beam into a quasi-parallel beam at the level of the sample 10.
• a semitransparent mirror can be placed in the optical axis YY, also close to the focal plane FF of the lens fl, that is to say near the focusing point C of the lens f1.
• the first arrangement requires working off the optical axis (more difficult settings, larger optical aberrations), but the second has the disadvantage of losing a factor T (lT) on the collected intensity (where T is the transmission coefficient of the mirror semitransparent), which is best avoided in fluorescence and Raman scattering.
• the optical axis, and therefore the optical column is vertical
• the light beam coming from the illumination device 30_R is horizontal
• the mirror 50 is inclined by approximately 45 ° ( 45 ° ⁇ 2 °).
• the inclined mirror 50 is the smallest possible (diameter of the order of 1 mm to a few mm), and the inclination of the beam relative to the optical axis is the lowest possible, given the need to prevent convergent C rays from intercepting said mirror 50.
• the light beam is derived from the illumination device 30_T. It is shaped quasi-parallel by unrepresented optical means and directed towards the sample from the rear, in almost normal incidence.
• the illumination device 30_R, 30_T may comprise a white light source (for example a xenon arc lamp, an arc or incandescent lamp) associated with a monochromator, or a source of light.
• a white light source for example a xenon arc lamp, an arc or incandescent lamp
• monochromatic light light-emitting diode, laser diode or laser
• the quasi-parallel beam shaping can be carried out by injecting light from the source into a fiber or bundle of optical fibers of moderate opening (advantageously less than or equal to 0.2) coupled to a lens.
• the uniformity of the illumination of the area to be explored is based on the homogeneity of the angular distribution.
• beam from the optical fiber Any nonuniformities may be corrected, either by a gradient filter or a suitable holographic filter interposed at the collimating lens, or by numerical correction at the level of the image analysis, these two correction techniques being equally applicable. to be combined.
• the parallel beam can be collimated to obtain illumination on a non-circular area, for example square or rectangular.
• the term "light source” means either a unit light source or a plurality of unit light sources, each unit source being able to emit on a common monochromatic wavelength or at a respective monochromatic wavelength.
• the objective fl comprises a lens or lens assemblies, of focal length f, providing at the level of the detector 21 a magnification f2 / f1 of the order of 1 (advantageously between 0.1 and 10). In order to satisfy the requirements of fluorescence, the opening of said objective fl is as large as possible (advantageously at least 1: 1.4).
• the objective fl is configured to focus the exciter light beam downstream of the sample, at a point C located in the focal plane (in this case higher) FF of said lens fl (which in the case of a single lens is located at the distance fl above this lens).
• the lens fl large aperture allows upstream of the sample to illuminate it in a quasi-parallel beam, normal or near-normal incidence, and downstream of the sample, on the one hand to focus the beam reflected at the point C located in the focal plane of said objective, and secondly to transform the scattered beam into a quasi-parallel beam, so that it can be optionally detected by the optical detection device 20 at a wide angle solid (for example, for an aperture 1: 1.4, a half-angle at the apex of 20 ° and a solid angle of 0.12 ⁇ steradians).
• the beam coming from the specular reflection is focused by the lens fl at the point C downstream of the sample, whereas the scattered beam (reemitted by the sample) is not focused but is transformed. in quasi-parallel beam.
• the light beam is reflected by the sample with a wavelength identical to that of the incident beam.
• the light beam is re-emitted or scattered by the sample at a wavelength different from the incident wavelength (excluding Rayleigh scattering).
• a device 70 for selectively closing the collected light beam (by the lens fl) is advantageously placed in the focal plane.
• This selective closure device 70 selectively enables the analysis of the sample according to at least two of the analysis techniques among the set comprising surface plasmon resonance, fluorescence, and Raman scattering, as described later.
• the device 70 for selectively closing the light beam comprises, for example:
• a diaphragm 71 configured to be placed in the focal plane of said objective around the focusing point C, so as to close off the unfocused part of the beam so that only the focused part is transmitted towards the detection device 20, and
• a shutter 72 preferably complementary to the diaphragm 71, configured to be placed at the focusing point C, so as to create an opaque zone on and around the point C, so as to close the focused part of the beam so that only the non-polar part focused is transmitted to the detection device 20, so that it can detect at a large solid angle.
• the device 70 for selectively closing the light beam may furthermore comprise at least one filter (no shown) from the set comprising a high-pass filter and a band-pass filter.
• the high-pass filter is implemented for the fluorescence detection, and the band-pass filter for the Raman scattering detection.
• the band-pass filter is a narrow band-pass filter, which makes it possible to obtain a good resolution in the determination of the Raman shifts, and advantageously placed at the level of the shutter.
• This can be done by using a narrow-band interference filter.
• the bandwidth of the filter is less than or equal to 3 nm.
• the filter diameter 50 mm reference 03 FIL 008 marketed by Melles Griot which has a bandwidth of 1 nm centered on a wavelength of 632.8 nm, allows the measurement of Raman offsets with a resolution of 25 nm. cm -1 .
• a filter blocking the wavelength of the light used for the excitation;
• parasitic radiation due to the diffusion of the exciting light by the surface of the sample 10 or by the defects or dust of the optical system is prevented.
• optical filters For example, it is possible to increase the rejection ratio of a band-pass filter for certain blocked wavelengths by superimposing a high-pass filter and / or a low-pass filter on it, or by superimposing a low-pass filter. and a high-pass filter limit the detection band of the fluorescence to better guard against parasitic signals.
• the filter (s) is (are) coupled to said shutter 72, and possibly integral with the movement thereof.
• the shutter 72 it is possible for the shutter 72 to be rotatable and a set of at least one filter to be rotatable as well, possibly integrally with the movement of said shutter 72.
• the diaphragm 71 and the shutter 72 thus make it possible respectively to select the type of analysis technique from the set comprising surface plasmon resonance, fluorescence, and Raman scattering.
• the selective closing of the light beam by the device 70 is implemented by switching between the diaphragm 71 and the shutter 72.
• the switching between the diaphragm 71 and the shutter 72 may be implemented by a mechanical switch, for example by rotational movement about an axis XX (FIG. 1), preferably parallel to the optical axis YY, by virtue of a rotating disk. Or we can also provide a translational movement, preferably in a plane parallel to the focal plane FF, for example by a sliding zipper.
• a mechanical switch for example by rotational movement about an axis XX (FIG. 1), preferably parallel to the optical axis YY, by virtue of a rotating disk.
• a translational movement preferably in a plane parallel to the focal plane FF, for example by a sliding zipper.
• the disk or the pull tab may also carry other optical functions (bright field imaging: wide diaphragm, dark field imaging: zone cache around point C, but no filter). Electrical switching may also be provided by a liquid crystal shutter device or an electrochromic device.
• the assembly comprising the device 70 for selectively closing the light beam and the mirror 50 (when it exists) is constructed in the form of a mechanically rigid block. It should be noted that the two sets (diaphragm 71 + possible filter and shutter 72 vs inclined mirror 50) can not be located, exactly and simultaneously, in the focal plane FF of the objective f1. In practice, an offset of a few millimeters is tolerable, given the focal depth of the lens fl and the diameter of the mirror 50, which can be slightly oversized.
• the illumination device 30 it is sufficient to adapt the illumination device 30 to select a length of light. particular wave of illumination of the sample 10.
• the illumination device 30 is capable of emitting a monochromatic light beam, variable or otherwise.
• the illumination device 30 may comprise an assembly of at least one optical fiber and an assembly of at least one unitary light source. It can be provided that at least one unit source is tunable, and / or that at least one unit source is a white source, the illumination device 30 then further comprising a monochromator.
• the input of each fiber may be connected to a respective unit source, or the input of a single fiber is switched, shifted, from unit source to unit source. Alternatively, it can be provided that the single fiber is fixed and the unit sources are switched (displaced) at the input thereof.
• the switching of light source can be carried out either with movement by moving the end of an optical fiber (single fiber whose input is moved from one source to another, or fibers each connected to a respective source the output of one of them being selected (selected) for illumination of the sample 10); either without movement for example by variable wavelength source, or white source coupled to a monochromator.
• optical fiber single fiber whose input is moved from one source to another, or fibers each connected to a respective source the output of one of them being selected (selected) for illumination of the sample 10
• variable wavelength source or white source coupled to a monochromator
• the light beam transmitted (reflected) by the sample 10 is collected through the diaphragm 71 placed at the convergence point C of the lens fl, the diaphragm for filtering the unfocused portion of the output beam of the lens fl, so that only the focused portion of the light beam reaches the detection device 20.
• the transmitted light beam (reflected) by the sample 10 is blocked by the shutter 72 (preferably complementary to the diaphragm 71), the shutter 72 for filtering the focused portion of the beam output of the lens fl, so that only the non-focused portion of the light beam reaches the detection device 20.
• the shutter 72 preferably complementary to the diaphragm 71
• the shutter 72 for filtering the focused portion of the beam output of the lens fl, so that only the non-focused portion of the light beam reaches the detection device 20.
• the light is collected on a large solid angle by the detector.
• This constraint makes it difficult to develop a system for direct comparison with reflectivity measurements for LSPR analysis.
• the present solution proposes instead to perform the detection of Raman scattering at a given wavelength, and to measure the Raman shifts by varying the excitation wavelength, which allows a Direct and simple comparison between LSPR, Raman and fluorescence images, and seems totally innovative. In particular, it is useful to be able to make such a comparison without processing the images, and having the ability to compare pixels with pixels having different contrasts and recorded by different techniques.
• the light source is monochromatic and tunable, so as to be able to measure different Raman offsets by choosing different wavelengths for the excitation.
• Raman images are thus recorded directly corresponding to the light scattered at the wavelength ⁇ _det selected by the narrow filter used for the detection, for an excitation wavelength from the light source X_exc.
• the principle retained has the further advantage of avoiding any parasitic contribution due to the Rayleigh scattering of the sample 10 or of the optical system.
• sources that are sufficiently intense and that have a precisely defined wavelength.
• lasers or laser diodes are used in preference to electroluminescent diodes.
• a source for example a titanium-type laser: sapphire, possibly equipped with a frequency doubling system according to the desired wavelength range for the excitation), or a source based on on an optical parametric oscillator (OPO).
• a tunable solid laser for example a titanium-type laser: sapphire, possibly equipped with a frequency doubling system according to the desired wavelength range for the excitation
• OPO optical parametric oscillator
• These sources can be continuous or pulse light sources.
• a pulsed source it is advantageous to have a pulse duration greater than or equal to 100 fs so as to maintain the monochromatic nature of the source, and the highest possible repetition rate.
• OPO-based commercial sources can be used that provide illumination in the form of pulses of a few ns carrying a few tens of mJ with a repetition rate of 10 Hz or more; such sources provide high instantaneous powers favorable to observing the Raman effect.
• this filter can be a holographic filter with high rejection power.
• the 53684 reference filter marketed by Oriel attenuates the intensity of the radiation at 632.8 nm by a factor greater than 10 A 6, with a blocking bandwidth of 28 nm, and makes it possible to detect higher Raman shifts. at 350 cm -1 .
• this filter being used in transmission on the excitation, it may be advantageous to use lower blocking power filters but having an excellent out-of-band blocking transmission so as to have a system more resistant to high current intensities.
• the B46-566 B46-566 Continuous Index Multilayer Filter marketed by Edmund Optics attenuates the intensity of the radiation at 632.8 nm by a factor greater than 10 A 3, with a slightly lower blocking bandwidth. less than 32 nm, which makes it possible to detect Raman shifts greater than 400 cm -1 . Given their high transmission outside the locking band, it is possible to use two or more of these filters in series to increase the rejection power.
• the rejection filter is advantageously arranged at the output of the source.
• the simple substitution of a set of filters and diaphragms interposed in the optical column at the focal plane of the objective fl makes it possible to switch from the reflectivity measuring arrangement to the fluorescence measurement or Raman scattering array.
• the system can also keep the basic imaging function (bright field / dark field) by adding an additional set of diaphragms. It allows the realization of tests in air or in contact with a liquid medium and in real time to perform kinetic measurements.
• the coupling of the different techniques within the same tests makes it possible to obtain chemical or biochemical information with or without marking the targets or probes, to correlate the information obtained by at least two independent techniques and to carry out performance checks. real-time tests of the media used for the tests.
• the system can therefore implement a method of chemical, biological or biochemical analysis of a sample 10, in which said sample is illuminated with a monochromatic light beam; several images of said sample are acquired by switching between at least two of the analysis techniques among the set comprising surface plasmon resonance, fluorescence, and Raman scattering, and superimposing said images.
• the necessary spectral analysis is performed by recording several images corresponding to several excitation wavelengths.
• a tunable source may be advantageous.
• the invention it is possible to focus the transmitted or specularly reflected beam, so that it can either be selected by passing it through a diaphragm (reflectance / transmittance) or masked by a shutter (fluorescence, diffusion Raman), while maintaining the possibility of imaging the studied surface.
• a diaphragm reflectance / transmittance
• a shutter fluorescence, diffusion Raman
• no dispersive analysis device or spatial filtering device is used for the Raman analysis on the detection system; on the contrary, only the light emitted in a narrow band of wavelengths selected by a filter is detected.
• the invention is not limited to the embodiments described above. It can be applied in other contexts.
• These encoded particles can be produced in large numbers at low cost with more than 10 distinct codes a6.
• These codes are read by measuring the reflectivity spectrum of the particle.
• the same probe is grafted onto all the particles of the same code, and then all the particles are put in contact with a sample to be analyzed containing targets marked with a fluorophore.
• the particles are then dispersed (by deposition and then evaporation) on the surface of a microscope slide, and molecular recognition is analyzed by measuring, under optical microscope, for each particle, its reflectivity spectrum and its fluorescence.

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