WO2010060454A1

WO2010060454A1 - Device for determining a fluorescence polarization anisotropy distribution in real time and related procedure for measuring in real time a temperature distribution of a fluid medium

Info

Publication number: WO2010060454A1
Application number: PCT/EP2008/064870
Authority: WO
Inventors: Romain Roger Quidant; Guillaume Frederic Marcel Baffou
Original assignee: Institut De Ciencies Fotoniques, Fundacio Privada; Institució Catalana De Recerca I Estudis Avançats
Priority date: 2008-11-03
Filing date: 2008-11-03
Publication date: 2010-06-03

Abstract

The present invention refers to a device for determining a fluorescence polarization anisotropy distribution in real time of a medium of interest (104). Said device comprises: a light producing means (102) to induce a fluorescence emission of the medium of interest (104), a sensing means (106) to measure the fluorescence intensity, an optical imaging means (105) which conjugates optically at least part of the medium of interest (104) with a photosensitive part of the sensing means (106), and an active polarizing means (103) which modulates periodically the fluorescence intensity measured by the sensing means (106). Additionally, the invention refers to a procedure for measuring in real time a temperature distribution of a fluid medium of interest (104). Said procedure comprises the steps of : incorporating fluorescent temperature nanoprobes in the medium of interest (104), inducing the fluorescent temperature nanoprobes to emit fluorescence, measuring the fluorescence using an optical imaging means (105) and an active polarizing means (103) modulating said active polarizing means (103) periodically, extracting a fluorescence polarization anisotropy from the modulated fluorescence intensity.

Description

DEVICE FOR DETERMINING A FLUORESCENCE POLARIZATION ANISOTROPY DISTRIBUTION IN REAL TIME AND RELATED PROCEDURE FOR MEASURING IN REAL TIME A TEMPERATURE DISTRIBUTION OF A FLUID MEDIUM

D E S C R I P T I O N

FIELD OF THE INVENTION

The field of the invention is nanotechnology . More precisely, the invention discloses a thermo-imaging technique operating at the sub-micrometer scale based on fluorescence polarization anisotropy measurements. At the same time, it is disclosed a device capable to obtain fluorescence polarization anisotropy measurements in real-time .

BACKGROUND OF THE INVENTION

Mapping the temperature at the nanoscale is not anymore only of fundamental interest since recent applications and needs in medicine, microelectronics and nanofluidics . The use of nanoparticles in medicine is one of the important challenges in nanotechnology at this time. Infrared light induced heating of gold nanoparticles could be at the basis of cancer cell therapeutics and drug and gene delivery. Heating biological systems in vivo requires caution, and a precise temperature monitoring is mandatory for safe and successful applications. Also in microelectronics, the transition to nanoscale devices driven by the Moore's law implies much higher current densities. The associated strong temperature increase is one of the limiting factors for down scaling. The ability to map the temperature at the nanoscale in microelectronics is foreseen to help understanding and designing low consuming devices. Finally, recent works on plasmonics forces arising in aqueous medium highlighted the possible effect of temperature induced fluid convection at the microscale around the heated plasmonics structures. Imaging the temperature profile and evolution at the microscale in fluids is still a challenge and may unravel a completely new physics.

Several techniques aiming at temperature mapping at the microscale already exist. Scanning Thermal Microscope, SThM, uses a composite sharp tip which consists of a thermocoupler or a thermoresistor to probe the temperature of the surface of a sample. The highest spatial resolution reached so far using a Scanning Thermal Microscope is 50 nm. However, using a scanning probe causes a slow readout rate and makes this technique suited only for surface science investigations. Furthermore, the necessary tip sample contact acts like a thermal bridge between the sample and the tip. For this reason, it is not evident that the true sample temperature is measured. Optics-based thermometry techniques have been developed more recently. The first family relies on Raman spectroscopy. This is a noninvasive far-field method which can render submicrometer spatial resolution. This technique is well suited for wide range temperature measurements, up to 1000⁰C. However detecting a Raman signal requires long exposure time, around 1 second per pixel, which makes this technique more suited for single point measurements. The second family of optical thermometry is based on molecular fluorescence. For some fluorophores like rhodamine the fluorescence intensity decreases as a function of the temperature. Mapping the fluorescence intensity can thus provide temperature map if the temperature-intensity calibration is known. This technique can be applied in dry or aqueous solution but can give rise to numerous artefacts since fluorescence intensity is not only temperature dependent. Such a method achieves resolutions down to around 1 μm and 0.03 to 3.5⁰C depending on the amount of signal averaging done. A recent paper reports the use of particles containing rare earth ions glued at the end of an atomic force microscope tip. Fluorescence spectra of rare earth ions based particles display a pronounced temperature dependence. This configuration is well suited for dry surfaces investigations like in microelectronics and allows the temperature mapping with a spatial resolution of 1 μm. Publication [Rob Zondervan, Florian Kulzer, Harmen van der Meer, Jos A. J. M. Disselhorst, and Michel Orrit; "Laser-Driven Microsecond Temperature Cycles Analyzed by Fluorescence Polarization Microscopy"; Biophysical Journal, Volume 90, April 2006, 2958-2969] discloses a procedure to obtain a temperature mapping. Said procedure is based on the measurement of the fluorescence anisotropy. However, said method is only applicable for a limited period of time and to a specific environment. It cannot be applied to biological or aqueous solutions and requires a few minutes to get an image. In order to measure the fluorescence anisotropy, the parallel and orthogonal fluorescence are measured, using one avalanche photodiode for the parallel component and a different avalanche photodiode for the orthogonal component. Both photodiodes must be aligned optically with a position accuracy of 1 μm. If there is a slight misalignment, some artefacts may be seen on the images obtained. Therefore, both photodiodes must be realigned on every experiment performed.

BRIEF DESCRIPTION OF THE INVENTION

The invention, on a first aspect, relates to a device for determining a fluorescence polarization anisotropy distribution in real time of a medium of interest exhibiting spatial, temporal, or combinations thereof variations in fluorescence polarization anisotropy .

The medium of interest comprises elements or particles that can emit fluorescence.

Said device comprises a light producing means to induce a fluorescence emission of the medium of interest, a sensing means to measure the fluorescence intensity arising from the medium of interest and induced by the light producing means, and an optical imaging means which conjugates optically at least part of the medium of interest with a photosensitive part of the sensing means.

Depending on the characteristics of the medium of interest, the light produced by the light producing means may be polarized or not.

In the case that the light producing means produces a non polarized light, this configuration is suited for providing information about the orientation of the particles that emit fluorescence. If the light producing means produces a polarized light, the information obtained is related to the molecular dynamics of the particles that emit fluorescence.

The light producing means are designed to induce the fluorescence emission on all the relevant portion of the medium of interest. The relevant portion of the medium of interest is considered as a unit and the measures are made on said unit at once. The medium of interest can be mounted on a translation stage to facilitate the selection of the said unit prior the measurements. The light producing means and the medium of interest do not need to have a relative movement during the measures. A single shot of the light producing means will excite all the medium of interest or the relevant portion thereof. The optical imaging means retrieves the fluorescence intensity information from the medium of information in such a way that said information can be treated and processed by electronic means. Therefore, it is possible to provide real-time information of the medium of interest.

Therefore, the information from the entire medium of interest, or from the relevant portion thereof, is obtained at the same time. It is not necessary to scan the medium of interest, since the light producing means induce the fluorescence emission on all the relevant portion of the medium of interest at the same time, and the optical imaging means retrieves such information, at the same time, from the medium of interest.

According to the present invention, the device further comprises an active polarizing means which modulates periodically the fluorescence intensity measured by the sensing means. The modulation of the fluorescence intensity measured by the sensing means, giving that the modulation pattern is known, provides means to obtain, with only one sensing means, the orthogonal fluorescence intensity component and the parallel fluorescence intensity component. Therefore, the alignment process is not necessary, since the device consists of only one sensing means. The active polarizing means permits to the sensing means to calculate the orthogonal and parallel components of the fluorescence intensity thanks to the modulation performed.

Once the fluorescence intensity components are known, the fluorescence polarization anisotropy is directly calculated. The fluorescence polarization anisotropy parameter has a clear relation with some important characteristics of the materials. It can be used to get some information concerning fluorophores orientation and fluorophores dynamics. This information can be used to measure order parameter in polymer science, microviscosity in fluids, protein-protein interaction in biology, antigen-antibody reactions in immunology, or local temperature. Information about any of these parameters may be obtained once the device of the invention calculates the fluorescence polarization anisotropy.

The device disclosed performs a non-invasive measurement, obtaining the fluorescence intensity. The accuracy obtained can be refined easily modifying the amount of signal detected by the sensor means. This may be done, for example, increasing the concentration of fluorophores in the medium of interest, increasing the exposure time of the sensor means or increasing the power of the light producing means.

On a possible embodiment of the device, the active polarizing means may be a rotary linear polarizer located between the light producing means and the medium of interest. Alternatively, the active polarizing means may be located between the medium of interest and the optical imaging means .

The sensing means may be selected from a CCD camera, a CID sensor, a CMOS sensor and the like. The sensing means must have the ability to acquire successive images at a high frequency, typically, with a pixel readout higher than 20 kHz therefore, must be based on electronic technology, being possible to transmit the information to a computer or any other electronic processing means to treat the data obtained and get the final results.

The invention, on a second aspect, relates to a procedure for measuring in real time a temperature distribution of a fluid medium of interest exhibiting spatial, temporal, or combinations thereof variations in temperature. Said procedure comprises the following steps .

Fluorescent temperature nanoprobes are incorporated in the medium of interest. A fluorescent temperature nanoprobe is a probe capable to emit a temperature dependent signal, whose size is on the submicrometric scale .

The fluorescent temperature nanoprobes will be mixed with the fluid, being possible to rotate in said fluid. Their orientations, therefore, are not fixed. The medium of interest is exposed to a light producing means, which induces the fluorescent temperature nanoprobes to emit fluorescence. Additionally, the procedure of the invention further comprises the steps of measuring the fluorescence arising from the medium of interest as a function of time using an optical imaging means and an active polarizing means, modulating said active polarizing means periodically a fluorescence intensity arising from the medium of interest, and extracting a fluorescence polarization anisotropy from the modulated fluorescence intensity recording by a sensing means using computer means based on synchronous detection.

The procedure provides information to calculate the temperature of a fluid medium of interest. The medium of interest must be fluid in order to the fluorescent temperature nanoprobes to rotate. However, the same procedure can be used to calculate the temperature of a solid. In this particular case, the procedure would be the same as disclosed previously, but the fluid medium of interest must be put in contact with the solid. Being in contact the solid and the medium of interest, eventually, a temperature equilibrium will be reached where the temperature at the fluid medium of interest corresponds to the temperature of the solid. The temperature measured on the medium of interest, thus, may be considered as the temperature of the solid. A possible example is the temperature mapping of an electronic circuit board, where said circuit board has been covered with a liquid medium of interest. The temperature mapping obtained for the medium of interest corresponds to the solid temperature mapping directly. The procedure disclosed provides a non-invasive method to measure the fluorescence polarization anisotropy. Depending on the concentration of fluorescent temperature nanoprobes, the intensity of the light produced by the light producing means and the exposure time of the sensing means, the temperature accuracy of the measures would be greater or smaller, being possible to modify said accuracy.

As explained before, as a result of the procedure, measurements of the fluorescence polarization anisotropy using an active polarizing means are obtained. Therefore, considering the modulation pattern and the fluorescence intensity measure, the parallel and orthogonal fluorescence intensity components can be calculated. Giving the relation existing between the parallel and orthogonal fluorescence intensity components and the fluorescence polarization anisotropy, once the first components are obtained, fluorescence polarization anisotropy can be calculated.

Moreover, the procedure may include the step of calculating the temperature from the fluorescence polarization anisotropy using a calibration data. There is a straightforward relation between temperature and fluorescence polarization anisotropy, not affected such relation by any other aspect or parameter of the procedure. Therefore, the temperature calculated or obtained refers exactly to the medium of interest, and no other consideration must be made to adjust the result. The temperature is exactly calculated.

The relation between the temperature and the fluorescence polarization anisotropy depends on the medium of interest and the temperature nanoprobe used. For each combination of parameters, there is a different relation between medium of interest and temperature nanoprobe. Said relation is constant in time, and, once it has been obtained can be applied with no need to recalculate it on every measurement experiment.

The active polarizing means may be a rotary linear polarizer located between the light producing means and the medium of interest. Alternatively, it may be located between the medium of interest and the optical imaging means .

The optical imaging means may comprise an objective and, at least one, lens. The sensing means may be selected from a CCD camera, a CID sensor, a CMOS sensor and the like.

The fluorescent temperature nanoprobes may be fluorescent molecules or proteins. The fluorescent molecules may be applicable in media of interest where the viscosity is relatively high. In those cases, the rotational correlation time of the fluorescent molecules would be of the same order of magnitude as their fluorescence life time. Therefore, the fluorescent molecule would be able to, once excited, rotate first and then emit fluorescence, being such actions in relation to the rotational correlation time and fluorescence life time ratio parameters, respectively.

In cases wherein the viscosity is lower, fluorescent molecules rotation may be too fast, therefore, the fluorescence polarization anisotropy measure may be zero. In these cases, bigger particles are needed. The constraint that has been stated in the previous paragraph, the rotational correlation time of the fluorescent particle must be of the same order of magnitude as its fluorescence life time. Same order of magnitude is defined in this invention as a rotational correlation time and fluorescence life time ratio between 0 . 0 1 and 1 0 0 .

Possible embodiments of the previously mentioned bigger particles are fluorescent proteins, fluorescent polymer beads or organic-inorganic hybrid nanoparticles . In these cases, the hydrodynamic volume is bigger, forcing a slower rotation induced by Brownian dynamics, resulting to a fluorescence polarization anisotropy measure greater than zero.

An organic-inorganic hybrid nanoparticle may be engineered as an inorganic core coated with a fluorescent shell. The fluorescent shell may consist of fluorescent molecules .

BRIEF DESCRIPTION OF THE DRAWINGS

To complement the description being made and in order to aid towards a better understanding of the features of the invention according to a preferred practical embodiment thereof, a drawing is attached as an integral part of said description, showing the following with an illustrative and non-limiting character:

Figure IA and IB show a schematic embodiment of the device of the present invention, where the light producing means directly emits light towards the medium of interest. On figure IA the light is polarized before it excites the medium of interest, while on figure IB the fluorescence emitted by the medium of interest is then polarized.

Figure 2A and 2B show a schematic embodiment of the device of the present invention, where the light producing means emits light towards a beam splitter or dichroic mirror that diverts the light to the medium of interest. On figure 2B the light is polarized before it excites the medium of interest, while on figure 2A the fluorescence emitted by the medium of interest is then polarized. Figure 3 shows the initial image obtained by the sensing means, the orthogonal and parallel fluorescence intensity components obtained for the previous image, the fluorescence polarization anisotropy calculated from the previous two parameters and the temperature map obtained.

PREFERRED EMBODIMENT OF THE INVENTION

In view of the discussed figures, a possible embodiment of a device for determining a fluorescence polarization anisotropy distribution in real time of a medium of interest (104) and the procedure for measuring in real time a temperature distribution of a fluid medium of interest (104) using said device according to the invention is disclosed. In the embodiment that is going to be described, the device permits to obtain a temperature map of a medium of interest (104), using the known relation that there exists between the fluorescence polarization anisotropy and the temperature. The same device may be used to obtain different parameters of the medium of interest (104), all of them based on the relation that the fluorescence polarization anisotropy may have with said parameters .

The medium of interest (104) chosen is a biological cell with uptaken gold nanoparticles . The metal structures, not represented in the figures, are heated with a light that excites their localized surface plasmons . Using the device of the invention, a temperature map of the biological cell will be obtained in real time. In order to obtain said temperature map, fluorescent temperature nanoprobes, fluorophores, are incorporated in the medium of interest (104) .

Other possible media of interest where the temperature can be mapped are, for example, a flat glass sample on which metal nanostructures are lying embedded in a liquid, or a microelectronic chip or some wire connections heated by an electrical current producing Joule effect. The three possible variants listed are not a limited number of possible media of interest. They are cited as examples.

Figures IA, IB, 2A and 2B show a schematic view of the device of the present invention, according to two possible embodiments. The device comprises a light producing means (102) to induce a fluorescence emission of the medium of interest (104), a sensing means (106) to measure the fluorescence intensity arising from the medium of interest (104) and induced by the light producing means (102) , an optical imaging means (105) which conjugates optically at least part of the medium of interest (104) with a photosensitive part of the sensing means (106), and an active polarizing means (103) which modulates periodically the fluorescence intensity measured by the sensing means (106) .

The light producing means (102) used to obtain the temperature map of the medium of interest (104) is polarized. A polarized light provides information about the fluorophores dynamics in the medium of interest

(104) . The dynamics of the fluorophores, associated with the temperature, will provide the relation to calculate the temperature.

The polarized light causes the fluorophores in the medium of interest (104) to emit fluorescence. The wavelength of the polarized light is selected to match the absorption wavelength maximum of said fluorophores.

The sensing means (106) measures the fluorescence intensity arising from the fluorophores of the medium of interest (104) and the optical imaging means (105) conjugates optically at least part of the medium of interest (104) with a photosensitive part of the sensing means (106) . The optical imaging means (105) is a CCD camera with NxM pixels. The optical part comprises of an objective and a lens.

On figure IA and figure IB the light emitted by the light producing means (102) directly goes through an active polarizing means (103), the medium of interest (104), the optical imaging means (105) to end up on the sensing means (106) . The difference between the devices represented on both figures is the position of the active polarizing means (103) . On figure IA the active polarizing means (103) is before the medium of interest

(104), according to the light path, and on figure IB it is after the medium of interest (104) . Figure IA corresponds to figure 2B and figure IB to figure 2A. The only difference is that the light from the light producing means (102) is diverted using a partially reflective mirror, a beam splitter or a dichroic mirror

(202) to illuminate the medium of interest from the other side . The active polarizing means (103) modulates periodically the fluorescence intensity measured by the sensing means (106) . The active polarizing means (103) may be a linear polarizer (103) spinning at a constant frequency around the optical axis of the optical system of the device. The active polarizing means (103) may be also a Pockels cell, i.e. a transparent crystal which modifies the polarization when a high voltage is applied. In this case, when the Pockels cells are used, no mechanical motion is required.

At a given time, t, since the optical imaging means (105) and the medium of interest (104) are optically conjugated, an optical image of the medium of interest

(104) will be formed by the optical imaging means (105) on the optical sensing means (106) which will then provide a map of the fluorescence intensity of NxM pixels.

It is considered, for simplification, that the active polarizing means (103) is a linear polarizer (103) and that it can rotate around the optical axis of the device. When the linear polarizer (103) is parallel to the excitation polarization light, provided by the light producing means (102) , the associated intensity map is maximum and equals the parallel fluorescence component, I₁₁, for each pixel. When the linear polarizer (103) is perpendicular to the excitation polarization, the associated intensity map is minimum and equals the orthogonal fluorescence component, I_τ, for each pixel.

In order to get these two maps in real time, the orientation of the linear polarizer (103) is modified periodically. The linear polarizer (103) is spinning at a constant angular frequency, ω, around the optical axis of the device. This induces a sinusoidal variation of the fluorescence intensity, s(t), as a function of time, t, on each pixel of the detector with an amplitude, A, equal to half the difference between the parallel fluorescence, I_N, and the orthogonal fluorescence, I_τ, components and a mean value, B, equal to half the sum of the parallel fluorescence, I_n, and the orthogonal fluorescence, I_τ: S(t) = A cos(ωt + φ) + B + noise(t) where noise(t) represents the readout or external noise. Additionally, the mean value of noise(t) is zero.

Since the frequency, (D, and the phase, φ, of the signal are perfectly known, they are the same frequency and phase of the active polarizer means (103), a synchronous detection can be applied the extract from the noise the signal of interest and get the amplitude, A, and the mean value, B. This can be done in real time using an appropriate electronic device. The electronic device shall calculate the amplitude, A, and the mean value, B, using the following formula:

A =(s(t) x cos(ωt + φ))

B =(s(t)> where (f(Λ)) denotes the mean value of any function f(λ) over time.

This procedure furthermore removes noise (t) since the mean value of noise (t) is zero.

This electronic device shall also provide the parallel and orthogonal fluorescence components, I_M and I_τ, and/or directly the fluorescence polarization anisotropy, according to the following formula:

1_{1 I} = B + A

I_T = B - A 2A r =

3B - A where r refers to the fluorescence polarization anisotropy .

The calibration linking the temperature, T, and the polarization anisotropy, r, can be determined previously using a different and separate setup, out of the scope of the invention. Using this calibration the map of the temperature, T, is also obtained in real time.

Figure 3 depicts process disclosed on the previous paragraphs. Firstly, the intensity of the medium of interest (104) is obtained. Considering that the intensity follows a sinusoidal pattern, the parallel and orthogonal fluorescence components, I|| and I_τ, are calculated. The fluorescence polarization anisotropy is then calculated and the temperature obtained directly, using the appropriate calibration data.

The efficiency of the procedure is directly related to the time scale of the rotational correlation time, τ_R, of the fluorescent temperature nanoprobe, and its fluorescent life time, τ_F- A much steeper variation of the polarization anisotropy, r, as a function of the temperature, T, resulting in a much higher sensitivity of the method, is observed when the rotation correlation time, τ_R, is of the order of magnitude of the fluorescent life time, τ_F- τ_F „ τ_R

This feature can be simply derived from differentiation of the Perrin's law:

where r₀ , called the fundamental polarization anisotropy, is the polarization anisotropy when the exciting light from the light producing means is linearly polarized and when the fluorophores are in a rigid medium and do not rotate, l/τ_R =0.

The time scales, rotation correlation time and fluorescent life time, τ_R and τ_F, are both dependent on the medium of interest (104) and the fluorescent temperature nanoprobe . While the fluorescent life time, τ_F, is more an intrinsic property of the fluorophore, the rotation correlation time, τ_R, can be expressed as follows :

where T is the temperature, η(τ) the dynamic viscosity of the medium, V the hydrodynamic molecular volume and k_B the Boltzmann constant.

In the procedure of the invention, the fluorescent temperature nanoprobe means a nanoparticle comprising fluorophores linked to a rigid medium. This configuration is intended to hinder the too fast free rotation the fluorophores would have alone in the solution. The hydrodynamic molecular volume, V in the previous equation, is here the volume of the nanoparticle, increasing the hydrodynamic molecular volume, the rotation correlation time, T_R, can be increased and adjusted to match the fluorescent life time, τ_F-

In case that the fluorophores are located on the outer part of a non-fluorescent rigid core, the hydrodynamic molecular volume of the nanoparticle is increased. A different alternative may be to embed fluorophores inside a rigid matrix. Alternatively, fluorophores may be packed together so that they constitute themselves the rigid medium. This would be the case for a fluorescent polymer bead. Additionally, the rigid medium can be a protein containing at least one fluorescent centre.

In view of this description and set of drawings, a person skilled in the art will understand that the embodiments of the invention that have been described can be combined in many ways within the object of the invention. The invention has been described according to several preferred embodiments thereof, but it will be evident for a person skilled in the art that many variations can be introduced in said preferred embodiments without exceeding the scope of the claimed invention .

Claims

C L A I M S

1.- A device for determining a fluorescence polarization anisotropy distribution in real time of a medium of interest (104) exhibiting spatial, temporal, or combinations thereof variations in fluorescence polarization anisotropy, said device comprises: a light producing means (102) to induce a fluorescence emission of the medium of interest (104), a sensing means (106) to measure the fluorescence intensity arising from the medium of interest (104) and induced by the light producing means (102), an optical imaging means (105) which conjugates optically at least part of the medium of interest (104) with a photosensitive part of the sensing means (106), characterised in that it further comprises: an active polarizing means (103) which modulates periodically the fluorescence intensity measured by the sensing means (106) .

2.~ The device according to Claim 1, wherein the active polarizing means (103) is a rotary linear polarizer (103) located between the light producing means (102) and the medium of interest (104) .

3.- The device according to Claim 1, wherein the active polarizing means (103) is a rotary linear polarizer (103) located between the medium of interest (104) and the optical imaging means (105) .

4.- The device according to any of the previous

Claims, wherein the sensing means (106) is selected from a CCD camera, a CID sensor, a CMOS sensor and the like.

5.- A procedure for measuring in real time a temperature distribution of a fluid medium of interest

(104) exhibiting spatial, temporal, or combinations thereof variations in temperature, said procedure comprises the steps of: incorporating fluorescent temperature nanoprobes in the medium of interest (104), exposing the medium of interest (104) to a light producing means (102) which induces the fluorescent temperature nanoprobes to emit fluorescence, characterised in that the procedure further comprises the steps of: measuring the fluorescence arising from the medium of interest (104) as a function of time using an optical imaging means (105) and an active polarizing means (103) modulating said active polarizing means (103) periodically a fluorescence intensity arising from the medium of interest (104), extracting a fluorescence polarization anisotropy from the modulated fluorescence intensity recording by a sensing means (106) using computer means based on synchronous detection.

6.- The procedure according to Claim 5, wherein it further comprises the step of calculating the temperature from the fluorescence polarization anisotropy using a calibration data.

7.- The procedure according to any of Claims 5-6, wherein the active polarizing means (103) is a rotary linear polarizer (103) located between the light producing means (102) and the medium of interest (104) .

8.- The procedure according to any of Claims 5-6, wherein the active polarizing means (103) is a rotary linear polarizer (103) located between the medium of interest (104) and the optical imaging means (105) .

9.- The procedure according to any of Claims 5-8, wherein the optical imaging means (105) comprises an objective and, at least one, lens.

10.- The procedure according to any of Claims 5-9, wherein the sensing means (106) is selected from a CCD camera, a CID sensor, a CMOS sensor and the like.

11.- The procedure according to any of Claims 5-10, wherein the fluorescent temperature nanoprobes are fluorescent molecules.

12.- The procedure according to any of Claims 5-10, wherein the fluorescent temperature nanoprobes are fluorescent proteins.

13.- The procedure according to any of Claims 5-10, wherein the fluorescent temperature nanoprobes is a fluorescent polymer bead.

14.- The procedure according to any of Claims 5-10, wherein the fluorescent temperature nanoprobes are organic-inorganic hybrid nanoparticles .

15.- The procedure of Claim 14, wherein the organic- inorganic hybrid nanoparticles consist of an inorganic core coated with a fluorescent shell.

16.- The procedure of Claim 15, wherein the fluorescent shell consists of fluorescent molecules.