EP1774287A2 - Method and system for optical detection of nano-objects in a light refracting medium - Google Patents

Abstract

The invention concerns a method and a system for optical detection of nano-objects in a light-refracting medium. It consists in illuminating (A) a nano-object (n-oi) and the light-refracting medium with a heating coherent periodically amplitude-modulated electromagnetic wave (HB(O)), to generate a specific temperature and refractive index profile in the vicinity of the nano-object, and with a probe coherent (PB) electromagnetic wave to generate an emerging electromagnetic wave (EPB(O)) comprising at least one component beat modulated in amplitude at the modulation frequency of the heating coherent wave; detecting (C) on the emerging wave (EPB(O)) the intensity component beat modulated in amplitude, to isolate and represent said nano-object in the light-refracting medium. The invention is useful for detecting nano-objects in an industrial, physiological or intracellular light-refracting medium.

Description

Method and system for optically detecting nano-objects in a refractive medium

The invention relates to a method and a system for optical detection of nano-objects in a refractive medium.

In the technical field of nano-sciences, in which the dimension of the objects is essential and characterizes the latter, methods or processes making it possible to visualize these objects are essential.

The optical techniques used for this purpose at present are based on the phenomenon of luminescence. Fluorescent molecules have been studied, highlighted and are commonly used in the field of life sciences.

The aforementioned fluorescent molecules, however, allow only short observation times, due to the phenomenon of photo whitening.

The development of more intense and stable luminescent objects has partially remedied the aforementioned drawbacks, but at the expense of the introduction of a significant flicker phenomenon.

Another technique uses the absorption properties of objects.

At the temperature of liquid helium, isolated molecules were first detected by an absorption technique from the high quality factor of the phonon line zero which has a very large absorption section, some 10 ^{". 11} cm ² , at resonance.

Isolated ions or atoms in RF (radio frequency) traps or high quality factor cavities were detected by absorption of a probe beam.

In general, particles having a large absorption section and short time intervals between successive absorption events are likely to be successfully subjected to detection by absorption methods.

More recently, a method of detecting such particles has been developed for metal particles. Nanometer-sized metal particles, excited in the vicinity of their plasmon resonance by an electromagnetic wave, have a relatively large absorption section, of the order of 8 ^× 10 ^-14 cm ² for a particle of 5 nm in diameter. , and a fast electron-phonon relaxation time, of the order of one pico-second. Due to the fact that the luminescence phenomenon of these particles is extremely low, almost all the electromagnetic energy absorbed is converted into heat. The rise in the induced temperature causes a variation of the local refractive index. The aforementioned more recent method employs a polarized light interference contrast technique to detect this local thermal photo effect and to display images of gold particles of 5 nm diameter have been obtained, with a ratio of signal to noise of the order of 10.

For a more detailed description of the above-mentioned method, reference may be made to the article entitled Thermal Imaging of Nanometer-Sized Metal Particles Among Scatterers published on August 16, 2002, Volume 297, pp 1160-1163, Science by David Boyer, Philippe Tamarat, Abdelhamid Maali, Brahim Lounis, Michel Orrit, Publisher: American Association for the Advancement of Science. The aforementioned method gives satisfaction.

However, implementing an interferometric method, it involves the implementation of precision installations, including bulky and heavy optical banks, necessary for precision, for the conservation of phase relationships between laser beams, necessary for the implementation evidence of the phenomenon of interference.

This type of installation appears not conducive to easy implementation of industrial facilities, or even laboratories.

The object of the present invention is to overcome the drawbacks of the techniques of the prior art, in particular in terms of simplification of the necessary installations, with a view to achieving substantially facilitated industrialization.

Another object of the present invention is furthermore the implementation of a method and system for the optical detection of nano-objects, by local thermal photo detection, having a significantly increased sensitivity compared to the method of said interferometry thermal photo detection, the signal-to-noise ratio can be improved in a ratio 100, and the size of the detectable particles may be between 1 and 2 nm. The process for the optical detection of nano-objects in a refractive medium, object of the present invention, is remarkable in that it consists at least in illuminating at least one of the nano-objects and the refracting medium by means of a first wave IEC 60050 - International Electrotechnical Vocabulary - Details for IEV number 845-02-35 Electrical and magnetic data / Electromagnetic compatibility and modulated refractive index modulated refractive index variation in temperature and refractive index a zone in the vicinity of this nano-object, jointly illuminating this nano-object by means of a second coherent electromagnetic wave constituting a probe wave, to generate, by diffusion of this probe wave in this zone by the temperature profile and a refractive index, an emerging probe wave having at least one amplitude-modulated intensity component per beat at the modulation frequency of the first electromagnetic wave, detecting on the emerging electromagnetic wave this intensity component modulated in amplitude by beat, which allows to highlight and to represent this nano-object in the refractive medium.

The device for optical detection of nano-objects in a refractive medium, object of the present invention, is remarkable in that it comprises at least one illumination resource of at least one of these nano-objects and the medium refracting to means of a first coherent electromagnetic wave modulated periodically in amplitude, so as to cause an absorption of electromagnetic energy and to induce a modulated refractive index variation of this refracting medium by temperature rise, according to a determined temperature profile and of a refractive index, in a zone in the vicinity of this nano-object, a resource of joint illumination of this nano-object, by means of a second coherent electromagnetic wave constituting a probe wave, to generate, by diffusion of this probe wave in this zone by the temperature and refractive index profile, an emerging probe wave having at least one intensity component amplitude modulated by beat at the modulation frequency of the first electromagnetic wave, and a detection module, on this emergent wave, of this intensity component modulated in amplitude by beat, which allows to highlight and to represent this nano-object in the refractive medium. The method and device of the invention are applicable to the detection of absorbent or metallic nano-objects in an industrial or physiological refractive medium, with specific applications for the localization and representation of these nanoparticles in these media, in the field of physical chemistry, the sciences of matter and the detection of bio¬ molecules labeled in a physiological or intracellular medium.

They will be better understood on reading the description below and on the observation of the drawings, in which:

FIG. 1 represents, by way of illustration, a flowchart, in the form of block diagrams, of the essential steps for implementing the method that is the subject of the present invention;

FIG. 2a represents, by way of illustration, an exemplary implementation of an optical nano-object detection device according to the subject of the present invention; FIG. 2b represents, by way of illustration, a representation of an aggregate image of 67 gold atoms (1.4 nm) obtained by optical detection, thanks to the implementation of the method that is the subject of the present invention;

FIG. 2c represents a histogram of the amplitude of the signal detected for a given number of intensity peaks of the image represented in FIG. 2b;

FIG. 3a represents the variation law of the detected signal as a function of the modulation frequency;

FIG. 3b represents the law of variation of the absorption cross-section as a function of the size, the radius, of the nano-objects; FIG. 4a represents the dependence law of the detected signal as a function of the modulation frequency applied to the amplitude of the electromagnetic heating wave;

FIG. 4b represents the dependence law of the detected signal as a function of the power of the electromagnetic heating wave; FIG. 5a represents the fluorescence image of a preparation of

Colloidal CdSe / ZnS containing quantum boxes and excited by an electromagnetic heating wave with very low power density;

FIG. 5b represents, by way of comparison, the image of the same preparation, obtained thanks to the implementation of the process which is the subject of this in which the quantum boxes are detected and highlighted.

A more detailed description of the process for the optical detection of nano-objects in a refractive medium, object of the present invention, will now be given in connection with FIG.

With reference to the above-mentioned figure, a plurality of nano-objects designated n_θj are considered in a refractive medium. The aforementioned nano-objects may be suspended in a liquid refractive medium, for example, placed on a transparent slide, as will be described later in the description.

^• The method of the invention is noteworthy in that it comprises a step A to illuminate at least one of nano-objects and n_θj of the refracting medium, using a first periodically modulated electromagnetic wave, denoted HB (Ω) in amplitude, so as to cause an electromagnetic energy absorption of the refractive medium and to induce a modulated variation of the refractive index of the latter by temperature rise.

The amplitude modulation of the first electromagnetic wave refers to an intensity modulation at a modulation pulse, denoted Ω.

As a result, the aforementioned illumination causes a determined profile of temperature and refractive index in a zone in the vicinity of the nano-object considered n_θj.

The method according to the invention also consists in illuminating, in step B, the nano-object considered n_θj by a second coherent electromagnetic wave, the second coherent electromagnetic wave mentioned above being intended to constitute a probe wave.

The above-mentioned probe wave makes it possible to generate, by diffusion of the latter, in the zone considered by the temperature and refractive index profile, an emergent probe wave denoted EPB (Ω) comprising at least one intensity component. amplitude modulated by beat at the modulation frequency of the first electromagnetic wave.

It is understood, in particular, that because of the modulated variation of refractive index of the refractive medium, the probe wave PB is subjected to a phenomenon of diffusion or dispersion. The method which is the subject of the invention then consists of a step C, of detecting on the emerald probe wave EPB (Ω) the amplitude intensity component modulated by beat.

It is thus understood that the above-mentioned detection makes it possible to highlight and to represent the nano-object n_Oj in the refractive medium according to an image I _n- Oj by the variation of light intensity of the emerging probe wave.

EPB (Ω), which has a maximum in the vicinity of the nano-object considered in the neighborhood area of the latter.

The combination of the physical processes involved by the method object of the present invention is explained below. A considered nano-object introduced into a homogeneous refractive medium, when the latter is illuminated by the first amplitude-modulated coherent electromagnetic wave, behaves as a point source of heat with a thermal power of the form: Phβat. [1 + cos (Ωt)] (1)

In this relation Ω is the modulation pulse of the first coherent electromagnetic wave and P _hea t is the average power of the coherent electromagnetic wave absorbed.

The aforementioned point thermal source generates a temporal modulation of the refractive index in the vicinity of the nano-object considered according to a spatio-temporal profile given by the relation:

In the aforementioned relation: r denotes the distance to the center of the nano-object n_θj; n denotes the refractive index of the refractive medium;

denotes the partial derivative of the refractive index as a function of temperature, of the order of 10 ^-4 / K;

Rt _h - J ^2κ / ^ _n ' ^a length or radius characteristic of thermal diffusion, K designating the thermal conductivity of the refringent medium and C its specific heat volume. Under these conditions, the probe beam propagating in the zone in the vicinity of the illuminated particle, and of course in the corresponding refractive medium of modulated refractive index, according to the corresponding refractive index profile, generates an electromagnetic wave diffused probe having frequency offset frequency sidebands of the amplitude modulation frequency value of the first coherent electromagnetic wave.

The medium refractive variable refractive index in the zone near the nano-obj ^'and considered causes, by a phenomenon of heterodyning between the incident probe beam PB reflected or transmitted and the coherent electromagnetic probe broadcast wave, beat frequency at the modulation pulse Ω, which can be detected by a conventional detection technique.

Accordingly, the detection of the beat amplitude modulated intensity component can be performed on the emerging probe wave reflected by the refracting medium or on the emerging probe wave transmitted by the refractive medium.

The above-mentioned conventional type detection techniques may advantageously comprise a synchronous detection, this detection process making it possible to reduce the noise level, and consequently to optimize the signal-to-noise ratio of the signal obtained, following the detection of the amplitude modulated intensity component per beat.

In general, it is indicated that the method which is the subject of the present invention applies to the optical detection of nano-objects, whether these nano-objects are absorbent or metallic, such as aggregates of gold atoms, and it will be described later in the description.

When the nano-objects are metallic, as well as, for example, when metal particles of diameter of a few nanometers are used to ensure the labeling of cells in an intracellular medium, the wavelength of the first coherent electromagnetic wave is chosen at a value close to the wavelength of the plasmon resonance of the nano-object under consideration.

In any case, whatever the type of nano-object subjected to detection, in accordance with the method which is the subject of the present invention, the second An electromagnetic wave constituting a probe beam is focused in the area in the vicinity of the nano-object in a manner similar to the focusing of the first electromagnetic wave.

Tests were carried out under the conditions below. Metallic nano-spheres, gold nanospheres, were diluted in an aqueous solution of PVOH (2% by weight) and deposited on a microscope slide by the so-called "Spincoating" technique. The surface density obtained, less than 1 .mu.m.sup.- ² , was sufficiently low that on average there was less than one particle per Airy spot at the focal point of the coherent electromagnetic waves.

The first coherent electromagnetic wave at 532 nm was amplitude modulated at a frequency of about 1 Mhz. The aforementioned wavelength, 532 nm, is located in the vicinity of the plasmon resonance of the gold nanospheres. The latter under these conditions strongly absorb the electromagnetic energy of heating and behave accordingly as a source point at the origin of a local temperature variation modulated at the frequency Ω / 2τr.

In addition, in order to have a refracting medium that can be modeled from the point of view of its physical properties, a layer of silicone oil (30,000 mPa.s) of refractive index n = 1, 4 was deposited on the sample.

Under these conditions, the temperature profile in the zone in the vicinity of each nano-object is spherically symmetrical and is characterized by the aforementioned length R _th , mentioned above. The superposition to the electromagnetic heating wave of the 633 nm probe coherent electromagnetic wave and their focusing in the vicinity of a gold nanosphere, that is, typically at a distance less than the length or radius thermal diffusion Rt _h , allows to introduce a disturbance on the propagation of the probe beam through the fluctuations modulated at the Ω pulse of the refractive index of the medium, induced by the heating produced by the nano-object. Thus, the emergent probe wave consists of the interaction of a DC component with a very low intensity component modulated at the Ω pulse resulting from the diffusion of the EPB probe beam by the temperature profile in the vicinity of the nanoparticles. object. The aforementioned tests made it possible to map samples comprising nano-objects of size 1 nm and thus to characterize the dependence of the detected signal on the various test parameters, such as heating power, modulation frequency, size of each nano-object, for example.

A more detailed description of a device for optical detection of nano-objects in a refractive medium, in accordance with the subject of the present invention, will now be given with FIG. 2a.

As shown in the above-mentioned figure, the device for the optical detection of nano-objects in a refractive medium, object of the present invention, comprises at least 1 illumination resources of at least one of the nano-objects n_θj and of the refractive medium by means of a first coherent electromagnetic wave HB modulated periodically in amplitude at the Ω pulse. This makes it possible to induce an electromagnetic energy absorption and to induce a modulated refractive index variation of the refractive medium by raising the temperature, according to a determined temperature and refractive index profile in an area in the vicinity of the nano- object considered, as previously mentioned in the description.

The device which is the subject of the invention furthermore comprises resources 2 for jointly illuminating the nano-object n_θj considered by means of a second coherent electromagnetic wave PB constituting a probe wave, to generate in the zone in the vicinity of the nano-object object n_θj by diffusion of this wave in this zone, by the aforementioned temperature and refractive index profile, an emergent probe wave EPB (Ω) comprising at least one amplitude intensity component modulated by beat at the frequency of modulation of the first electromagnetic wave.

Finally, the device that is the subject of the invention comprises at least detection resources 3 on the emergent wave EPB (Ω) of the intensity component modulated in amplitude by beat. This procedure makes it possible to highlight and to represent the nano-object in the refractive medium according to an image of the refractive medium considered.

As represented in FIG. 2a above, the illumination resource 1 by means of the first coherent electromagnetic wave comprises at least one generator 1o of a first laser beam constituting the first coherent electromagnetic wave to form a heating wave. The laser generator 1 ₀ may advantageously consist of a Nd: Yag laser generator doubled at 532 nm. The illumination resource 1 further comprises a focusing device, denoted I ₂ , of the first laser beam constituting the first electromagnetic wave on the nano-objects in the refractive medium.

The modulation of the first to the pulsation Ω heating laser beam is performed by means of an acousto-optic modulator 1 ₀₃ which is controlled by a control generator, designated 3o in Figure 2a. This generator delivers acousto-optic modulator control signals at the Ω pulse. The acousto-optic modulator is a conventional type modulator and, for this reason, will not be described in detail.

As furthermore shown in FIG. 2a, the resource 2 for joint illumination of the nano-object under consideration via a second electromagnetic wave PB constituting the probe wave comprises at least one generator 2o of a second laser beam constituted for example by a laser

HeNe to form the probe wave.

The second laser beam generated by the laser generator ₂₀ is then focused on the nano-object considered in the refractive medium under the conditions below, represented in FIG. 2a.

The modulated heating wave HB (Ω) and the probe wave PB are superimposed by means of a dichroic mirror 1 i on a common optical path formed between the aforementioned dichroic mirror and a single common objective. ie by the lens 1 ₂ previously mentioned in the description constituting the focusing device.

Finally, the first laser beam HB is polarized rectilinearly because of the emission of the latter in a direction P perpendicular to the sheet of FIG. 2a for example. The second laser beam constituting the probe wave is linearly polarized on emission by the laser source ₂₀ in a second direction P 'contained for example in the plane of the sheet of FIG. 2a.

The polarization direction of the probe wave emitted by the laser source ₂₀ is then transformed into a circular polarization PC in a plane orthogonal to the sheet of Figure 2a, through a λ / 4 blade designated ₀₂₂ in the aforementioned figure. The aforementioned circular polarization is transmitted by the dichroic mirror. After focussing on the sample SA represented in FIG. 2a, transmission of the emerging EPB (Ω) probe wave, the circular polarization is substantially conserved and the transmission by the λ IA blade, referenced 2o2, of the probe wave EPB (Ω) makes it possible to restore a rectilinear polarization of the latter in a third direction P "orthogonal to the second polarization direction P 'of the initial probe wave PB.The second and third directions are thus contained in a plane orthogonal to the sheet of Figure 2a, as shown in the above figure.

A polarization separator device 2i formed by a polarization separator cube then makes it possible to restore the only emergent probe wave EPB (Ω) polarized in a direction P "orthogonal to the plane of the sheet of FIG. 2a constituting the third aforementioned direction. polarization splitter device ₂₀ i is placed on the optical path common to the emergent polarization wave polarized rectilinearly in the third direction P "and the polarized probe wave in the second direction P ¹ .

The device according to the invention as represented in FIG. 2a finally comprises detection resources 3, which comprise at least one photodiode 3i receiving the emergent probe wave EPB (Ω). The above-mentioned photodiode is advantageously constituted by a low-noise photodiode, the reception of the emergent probe wave EPB (Ω) being furthermore effected by means of a high-pass type filter 3-ιo. The detection resource further comprises, as shown in the aforementioned figure, a detection synchronization module receiving a detection signal delivered by the photodiode 3i and a synchronous detection control signal of the periodic amplitude modulation of the first beam. laser.

For this purpose, the modulation signal generator 3o delivers a signal representative of the periodic modulation Ω to a detection synchronization device 32, which makes it possible to ensure the synchronous detection of the signals delivered by the photodiode 3i for visualization by a system The synchronization device 32 may be constituted by a synchronized amplifier for example. Finally, it is indicated that the assembly constituted by the focusing device 1 ₂ and of course, the dichroic mirror 1 i, the laser generators 1 ₀ and 2o and the acousto-optical modulator optical components I ₀₃ , dichroic mirror I ₁ , blade 4/2 ₀ 2 polarization splitter cube ₂₀ i, high-pass filter 30 and photodiode 3-i are fixed.

To obtain a two-dimensional image or, where appropriate, a plurality of two-dimensional images constituting sections of a three-dimensional object, the sample SA, constituted by the preparation previously described, is advantageously placed on a three-dimensional piezoelectric table, for example, symbolized by the triad OXYZ.

A justification of the operating mode of the device object of the present invention, in accordance with the method previously described in the description, will now be given below.

An estimate of the detected signal measured by synchronous detection was obtained by using the scattering theory in a variable dielectric medium, to calculate the electromagnetic field scattered by the refractive index profile obtained in the vicinity of each nano-object and defined by relation 2 previously mentioned.

The beat at the pulse Ω between the reference probe wave PB and the scattered probe wave leads to a beat energy S at the synchronous detector which satisfies the following relationship and comprises two terms in quadrature:

s = ^" fc L ^(Ω M ^{fit) +} g _k ^(Ω W ^Q) ] 0 ⁾

Cλ

In the preceding relation a denotes a form factor close to unity; lin _c denotes the intensity of the incident second coherent electromagnetic wave, that is to say the laser beam constituting the probe wave;

P _rβf denotes the power of the second electromagnetic wave constituting the emergent probe wave, reflected in the experimental conditions; f _k (Ω) and gι _< (Ω) are two dimensionless functions that depend on the modulation pulse and the thermal diffusivity of the refractive medium. For low modulation pulses, the characteristic heat diffusion length or radius R _th is greater than the focusing diameter of the probe beam or wave and the component f _k (Ω) / Ω in phase with the modulation applied to the heating beam is preponderant. However, for a sufficiently high modulation frequency, such that R _th "Φ where Φ denotes the diameter of the focusing spot of the probe wave, the quadrature component gι _< (Ω) / Ω becomes preponderant and decreases as 1 / Ω.

The amplitude of the demodulated signal output from the synchronous detection device 3 ₂ of Figure 2a, verifies the relationship:

In the previous relation, the sign ≈ designates the proportionality of the demodulated signal S _d em and ls (ïf) denotes the square of the amplitude of the signal of

beat given by the previous relation 3. The variations of f _k (Ω) / Ω and gk (Ω) / Ω are represented in FIG. 3a for K / C = 2 1 CT ⁸ m ² / s.

The aforementioned previous relation 4 has made it possible to evaluate the energy of the beat component at the detector.

A nano-object consisting of a gold particle of 1.4 nm in diameter has an absorption section of 5 × 10 ^-15 cm ² at 532 nm and absorbs a thermal power P _he at _t = 10 mW, when It is illuminated by a laser beam of intensity 2 MW / cm ² .

For an incident probe beam with a power Pj _nc = 70 mW, for a modulation frequency Ω / 2π = 800 kHz and for a reference power P _ref = 100 μW, the relation (4) above indicates a beat power S _d em equivalent to 5 mW. After calibration of the detection chain, the measured effective value was substantially equal to 2 mW, in agreement with the predicted theoretical value.

FIG. 4a represents the evolution of the detected signal as a function of the frequency Ω / 2π of the modulation of the heating wave. The dashed curve represents the evolution of the component of the signal detected in phase with the modulation of the heating wave. The dashed line represents the evolution of the signal component detected in quadrature phase with the modulation of the heating wave. The solid curve represents the total amplitude of the detected signal.

FIG. 4b represents the linear dependence of the signal as a function of the thermal power Pheat expressed in milliwatts. The amplitude of the signal is given in relative amplitude a.u. for "absolute unit". An additional increase in thermal power is not followed by saturation but leads to fluctuations in the amplitude of the detected signal, and, if necessary, to irreversible damage to the particle. With reference to FIG. 2b, it is indicated that the method and the device that are the subject of the present invention make it possible to detect nano-objects formed by metal particles of dimension as small as 1.4 nm in diameter with a SNR signal-to-noise ratio. > 10.

In practice, a probe wave at 720 nm Ti: Sa laser and a heating beam at 532 nm Nd: Yag laser doubled, whose intensity was modulated at a pulse corresponding to a frequency between 100 kHz and 15 MHz by the acousto-optic modulator, have been implemented.

The lens used was a 100x objective, Zeiss, nA = 1.4. The power of the heating beam was in the range of 1 mW to 3.5 mW depending on the size of the nano-object to be detected.

An image of the SA sample was formed by moving the aforementioned sample relative to the first and second fixed focused beams through the 2D table.

SA samples were prepared, as previously mentioned, from a solution of gold particles of diameter 1, 4 nm, 2 nm, 5 nm, 10 nm, 20 nm, 33 nm, 20 nm, 33 nm or 75 nm.

FIG. 2b gives a three-dimensional representation of a heterodyned photothermal image, obtained in accordance with the implementation of the method and device that is the subject of the invention, for aggregates of gold atoms of 1.4 nm.

The image revealed the absence of background noise introduced by the substrate, which indicates that the detected signal comes only from the absorbent objects in the sample, that is to say aggregates of gold particles. These were detected with a relatively low thermal power, of the order of 3.5 nW, and a remarkable signal-to-noise ratio, greater than 10.

The histogram of the signal amplitude for 272 intensity peaks of the image of FIG. 2b and shown in FIG. 2c shows that the luminous intensity amplitude peaks emanate from the nano-objects themselves.

The capture section of the aforementioned aggregates is at most of the order of 10 ^'15 cm ² section comparable to that of good fluorophores or nano-crystals CbSe / ZnS. Their relaxation time is very short.

On the contrary, luminescent semiconductor nanocrystals or fluorescent molecules have emission relaxation times of the order of one nanosecond, which makes the latter difficult to detect by absorption.

However, for relatively high excitation intensities, the semiconductor nano-crystals no longer exhibit a luminescence phenomenon. The method and the device, objects of the present invention, allow the detection of nano-objects whose emission relaxation time is short.

FIG. 5a represents the fluorescent image of quantum dots of

Colloidal luminescent CdSe / Zns, whose emission peak is at 640 nm, excited by the heating beam at a very low intensity of the order of 0.1 kW / cm ² . The blinking behavior characteristic of the single quantum dot emission is visible on the above-mentioned image.

On the contrary, in FIG. 5b, there is shown an image of the same region obtained thanks to the implementation of the method and device that is the subject of the present invention, at an excitation intensity of 5 MW / cm ² , for which quantum boxes are no longer luminescent.

The two images are correctly correlated, which guarantees that the amplitudes of intensity in the image of FIG. 5b, obtained thanks to the implementation of the method which is the subject of the present invention, are really due to individual quantum boxes. The latter no longer exhibit a blinking phenomenon. Particularly noteworthy, initially non-fluorescent quantum boxes, i.e. absent from Figure 5a, are now detected and shown in Figure 5b.

Finally, it is indicated that for the implementation of the method and device that are the subject of the present invention, applications in the field of biology or Life sciences, the increase in temperature on the surface of nano-objects is an important parameter.

In a current configuration, a nano-object formed by a gold particle of 5 nm in diameter can be detected with a signal-to-noise ratio> 100, for a heating power of 1 mW.

Under these conditions, the local temperature increase can be estimated at 4 K in an aqueous solution.

Due to the fact that temperature decreases as the inverse of distance and that most life science element or organelle detection applications do not require such a high signal-to-noise ratio, it is here indicated that the method and device objects of the present invention can detect and represent by electronic image very small gold particles or nano-objects, while induced local warming can be made substantially less than 1 K, above the temperature average of the sample.

Finally, with regard to the simplification of the device according to the invention as represented in FIG. 2a, vis-à-vis the device represented in the article mentioned in the description, it can be observed that the unnecessary telecentric device is suppressed. . This is because in the method and device of the present invention, the interaction between the reference scattered probe wave and the modulated scattered probe wave generating the beat modulated amplitude component at the Ω pulse occurs in the area in the vicinity of the nano-object considered.

On the contrary in the device described in this article, interference interaction, and not beat, occurs in the Wollaston prism and the detector, which implies the imperative maintenance of phase relationships between split beams and therefore the use telecentric system, unlike the method and device objects of the invention.

Claims

1. Process for the optical detection of nano-objects in a refractive medium, characterized in that it consists at least of: illuminating at least one of said nano-objects and said refractive medium by means of a first coherent electromagnetic wave modulated periodically in amplitude, so as to cause an absorption of electromagnetic energy and to induce a modulated variation of the refractive index of said medium refracting by elevation of temperature, according to a determined profile of temperature and refractive index, in a zone in the vicinity of said nano-object; - Illuminate said nano-object jointly by means of a second coherent electromagnetic wave, constituting a probe wave, for generating, by diffusion of this probe wave in said zone by said temperature and refractive index profile, a wave emerging probe having at least one intensity amplitude modulated component per beat at the modulation frequency of said first electromagnetic wave; detecting on said emergent probe wave said intensity component modulated in amplitude by beat, which makes it possible to highlight and to represent said nano-object in said refractive medium. 2. Method according to claim 1, characterized in that the operation of detecting said amplitude modulated intensity component by beat is performed on the emerging probe wave reflected by said nano-object and said refractive medium. 3. Method according to claim 1, characterized in that the operation of detecting said beat modulated intensity intensity component is performed on the emerging probe wave transmitted by said refractive medium. 4. Method according to one of claims 1 to 3, characterized in that the operation of detecting said amplitude modulated intensity component by beat is performed by synchronous detection. 5. Method according to one of claims 1 to 4, characterized in that for a metal nano-object, the wavelength of the first wave coherent electromagnetic is close to the plasmon resonance wavelength of this nano-object. 6. Method according to one of claims 1 to 5, characterized in that said second electromagnetic wave is focused in said area in the vicinity of said nano-object. 7. Apparatus for optical detection of nano-objects in a refractive medium, characterized in that it comprises at least: means for illuminating at least one of said nano-objects and the refractive medium by means of a first coherent electromagnetic wave modulated periodically in amplitude, so as to cause an absorption of electromagnetic energy and to induce a modulated variation of refractive index of said refractive medium by temperature rise, according to a determined temperature and refractive index profile, in a zone in the vicinity of said nano-object; means for jointly illuminating said nano-object by means of a second coherent electromagnetic wave, constituting a probe wave, for generating, by diffusion of this probe wave in said zone by said temperature profile and refraction, an emerging probe wave having at least one beat modulated intensity intensity component at the modulation frequency of said first electromagnetic wave; means for detecting, on said emergent wave, said amplitude modulated intensity component by beat, which makes it possible to highlight and represent said nano-object in said refractive medium. 8. Device according to claim 7, characterized in that said illumination means by means of a first coherent electromagnetic wave comprise at least: means generating a first laser beam constituting said first coherent electromagnetic wave constituting a heating wave; means for focusing said first modulated laser beam on said nano-objects in the refractive medium. 9. Device according to one of claims 7 or 8, characterized in that said means for jointly illumination of said nano-object by means of a second electromagnetic wave comprise at least: means generating a second laser beam constituting said probe wave; means for focusing said second laser beam on at least one of said nano-objects in the refractive medium. 10. Device according to claims 8 and 9, characterized in that said heating wave and said probe wave are superimposed by means of a dichroic mirror, on a common optical path formed between the dichroic mirror and a single common objective constituting said focusing means. 11. Device according to claim 10, characterized in that for a detection on the emerging probe wave of amplitude intensity component modulated by flapping, by reflection, it further comprises: rectilinear polarizing means of the first laser beam in a first direction; rectilinear polarizing means of the second laser beam in a second direction, said probe wave being polarized rectilinearly in said second direction; rectilinear polarizing means of the emerging probe wave in a third direction orthogonal to said second direction; polarization separator means placed on the optical path common to said emergent polarization wave polarized rectilinearly in said third direction and to said polarized probe wave in said second direction, said separator means making it possible to deliver the single wave of emerging probe polarized rectilinearly in said third direction. 12. Device according to one of claims 9 to 11, characterized in that said detection means comprise at least one photodiode receiving said emergent probe wave. 13. Device according to claim 12, characterized in that said detection means further comprise a detection synchronization module receiving, on the one hand, a detection signal delivered by said photodiode, and, secondly, a synchronous detection control signal of the periodic amplitude modulation of the first laser beam.