WO2010018557A1

WO2010018557A1 - Temperature sensor based on raman scattering for use in molecular diagnostics

Info

Publication number: WO2010018557A1
Application number: PCT/IB2009/053597
Authority: WO
Inventors: Cornelis R. Ronda; Jan F. Suijver
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2008-08-14
Filing date: 2009-08-14
Publication date: 2010-02-18

Abstract

The present invention relates to a method for temperature measurement based on Stokes and anti-Stokes Raman scattering and a molecular temperature sensor probe comprising Raman active compounds. The method comprises the steps of introducing a molecular sensor probe comprising a Raman active compound to a sample, irradiating the sample with light, detecting the intensities of the Stokes and anti-Stokes Raman emission lines of the molecular sensor probe in the sample, calculating the ratio of the intensities of the Stokes and anti-Stokes Raman emission lines of said molecular sensor probe, and finally assigning this ratio to a temperature. The invention also relates to the use of compositions comprising a Raman active compound in methods of treatment and diagnostical methods.

Description

Temperature sensor based on Raman scattering for use in molecular diagnostics

FIELD OF THE INVENTION

Subject of the present invention is a method for temperature measurement based on Raman scattering and a temperature sensor for the use in molecular diagnostics.

BACKGROUND OF THE INVENTION

The determination of physical parameters such as temperature is desired in many technical and medical fields. In molecular medicine such measurements are preferentially done on the (sub)cellular level. Due to the fact that currently available temperature sensors are all much larger (e.g. several hundreds of micrometers) than the typical sizes that are relevant for molecular medicine (typically in the 1 micrometer range), the temperature determination is difficult.

However, (local) temperatures can be measured by exploitation of the Raman effect (see e.g. WO 94/25861 and US 5,755,512).

The process of Raman scattering is schematically illustrated in Fig. 1, together with a typical emission spectrum. The most important point of Raman scattering is that a typical Raman spectrum in addition to the central Rayleigh band comprises two distinct emission bands: the Stokes (lower energy with respect to the excitation light) and anti-Stokes (higher energy) contributions. These differences in energy are related to the different vibrational levels involved. A typical Raman spectrum is symmetric in terms of energetic position, though not in terms of absolute intensities relative to the Rayleigh band. The intensities of the Raman bands are only dependent on the number of molecules occupying the different vibrational states when the process began. In other words: the intensities of the Raman bands are only dependent on the temperature of the material in combination with fixed materials properties. The ratio between the lower and higher energy levels is given by a Boltzmann distribution (equation 1):

— N₁ ^L = exp υ (equation 1)

N₀

where

N_Q: amount of atoms in the ground state

N_j : amount of atoms in the higher vibrational state ΔE_V: energy difference between the ground state and the higher vibrational state k: Boltzmann's constant T: temperature in Kelvin

This implies that the Stokes spectrum is more intense than the anti-Stokes spectrum, and the ratio between them is a direct measure for the temperature of the material.

The Raman effect is to be clearly distinguished from processes as fluorescence and/or luminescence. For the latter processes, the incident light is completely absorbed (into a real excited state) and the system can go to (various) lower states only after a certain resonance lifetime. For Raman scattering there is no real excited state involved (only a virtual one) and the process is essentially instantaneous. The result of both processes, however, is comparable: a photon with the frequency different from that of the incident photon is produced and the molecule is brought to a higher or lower energy level. But the major difference is that the Raman effect can take place for any frequency of the incident light. In contrast to the fluorescence effect, the Raman effect is therefore not a resonant effect. In WO 94/25861 (Pitt et al.) a method is described for measuring thermal conductivity of a solid sample, particularly a diamond, by employing temperature measurements based on the ratio of Stokes and anti-Stokes Raman lines.

US 5,755,512 (White) describes an apparatus for remotely sensing the temperature of a semi-conductor wafer by measuring the intensities of Stokes and anti-Stokes Raman lines.

SUMMARY OF THE INVENTION

This invention relates to a method for the measurement of the local temperature in a sample, comprising the steps of (a) introducing a molecular sensor probe comprising a Raman active compound to the sample or a desired portion of the sample, (b) irradiating the sample or the desired portion of the sample with light of (a) wavelength(s) of from 300 nm to 3 μm, (c) detecting the intensities of the Stokes and anti-Stokes Raman emission lines of the molecular sensor probe in the sample, (d) calculating the ratio of the intensities of the Stokes and anti-Stokes Raman emission lines of said molecular sensor probe, and (e) assigning said ratio to a temperature.

Also within the scope of the invention is a composition comprising a Raman active compound for the use as a diagnostic agent for the measurement of the local temperature or a temperature distribution in a sample or portions thereof, wherein said composition is introduced to said sample and wherein the ratio of the intensities of its Stokes and anti-Stokes Raman emission lines is determined upon irradiation with light of (a) wavelength(s) of from 300 nm to 3 μm thereby determining the temperature.

The present invention also concerns a composition comprising a Raman active compound for the measurement of the local temperature or a temperature distribution in a sample or portions thereof, wherein said composition is introduced to said sample and wherein the ratio of the intensities of its Stokes and anti-Stokes Raman emission lines is determined upon irradiation with light of (a) wavelength(s) of from 300 nm to 3 μm thereby determining the temperature. Also within the scope of the present invention is the use of the compositions of the invention for the determination of a local temperature or a temperature distribution in a mammal or a dead body of a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 : Left: Schematic description of the Stokes and anti-Stokes Raman scattering processes. Right: Typical emission spectrum showing the three spectral components.

Fig. 2: Example of the temperature dependence of the Stokes (+220 cm^" 1) and anti-Stokes (-220 cm^" 1) Raman bands. The large peak at 0 cm^"1 is the Rayleigh scattering peak and can be ignored. This figure shows the Raman signal from the transverse optical phonon in GaSb at 3 different temperatures.

Fig. 3: Temperature dependence of the ratio of Stokes and anti-Stokes Raman emission. This figure directly shows the Boltzmann behavior the ratio as was described in the equation above.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention also relates to a composition comprising a Raman active compound for the use as a diagnostic agent for the measurement of the local temperature or a temperature distribution in a sample or portions thereof, wherein said composition is introduced to said sample and wherein the ratio of the intensities of its Stokes and anti-Stokes Raman emission lines is determined upon irradiation with light of (a) wavelength(s) of from 300 nm to 3 μm thereby determining the temperature. This also includes the use of a composition comprising a Raman active compound for the use as a diagnostic agent for the measurement of the local temperature or a temperature distribution in a sample or portions thereof, wherein said composition is introduced to said sample and wherein the ratio of the intensities of its Stokes and anti-Stokes Raman emission lines is determined upon irradiation with light of (a) wavelength(s) of from 300 nm to 3 μm thereby determining the temperature, for the preparation of a diagnostic agent.

Preferably, the composition or the diagnostic agent is used in methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practiced on the human or animal body.

In particular embodiments, these therapeutical and diagnostic methods are selected from the group consisting of determination of temperature or temperature distribution during minimally invasive treatments, during radiation treatments, or during magnetic resonance treatment, determination of temperature or temperature distribution in a cooled body before heart-surgery, and determination of temperature or temperature distribution for the detection of enhanced metabolistic activity.

Preferably, the sample is a biological sample. More preferably, the biological sample is selected from the group consisting of a tissue, a living organism, an organ, a cell, a sub-cellular fragment, a living or dead human or animal body or parts thereof. Said Raman active compound comprises preferably at least one group with high electron density. Groups with high electron density, which are very suitable for Raman spectroscopy have charges which are delocalized and for this reason can be easily displaced. This is the origin of their large polarisability. The delocalized electron density is larger than 0 e/A^ and preferably larger than 0.20 e/A^. In preferred embodiments of the invention, said Raman active compound comprises at least one aromatic group or is an ion with at least one lone electron pair or is a carboxylic acid or is selected from the group consisting of M_mXθ3 or M_nYC>4, wherein M is selected from the group consisting of Na, K, Li, Mg, Ca and all other metals of the first and second group, X is selected from the group consisting N, C, Cl, Br and I and Y is selected from the group consisting of Si, Ge, Nb, Hf, Ta, Nb, and W and wherein m and n are 1 or 2. M_mXθ3 may preferably be selected from the group consisting of NaNO₃,

KNO₃, Na₂CO₃, K₂CO₃, MgCO₃ and LiNbO₃, and M_nYO₄ may be selected from the group consisting OfNa₂SiO₄, K₂SiO₄, Na₂GeO₄, YTaO₄ and K₂GeO₄ The carboxylic acid may be branched or unbranched, may contain preferably more than one carboxyl group, may be further substituted with -OH or -OR or other groups with a high electron density, such as -F^" or -O^2" and may have a chain length of from C_j to C_2Q. The carboxylic acid may for example be selected from the group Of CH₃COOH, HOOCCOOH, CFH₂COOH, CF₃COOH, H00CCH₂C00H, HOOCCOCOOH, HOOCCF₂COOH.

The Raman active compound may alternatively comprise molecular anions with a high local charge density. Such anions may for example be selected from the group consisting Of (VO₄)^3", (WO₄)^2" and [Fe^iπFe^π(CN)₆]^".

In some preferred embodiments of the invention, the Raman active compound is immobilized on the surface of a particle or is comprised in a particle. Such a particle has a diameter of from 5 nm to 50 μm. Preferably, said particle is substantially spherical or disk shaped.

A particle preferably comprises a metal selected from the group consisting of Cu, Ag and Au for Surface Enhanced Raman scattering (SERS). These materials are chosen, because the plasmon frequencies, which lead to SERS are located in a suitable wavelength range (VIS to near IR, i.e. wavelengths of around 300 nm to around 3 μm).

In some particular embodiments, the particle comprises a selective targeting moiety, e.g. an antibody or functional fragment thereof.

The temperature to be measured according to this invention is preferably within the range of from 25 ⁰C to 45 ⁰C.

The Raman emission may be based on a linear Raman effect or on a non-linear Raman effect. Light irradiation and detection are performed through optical fibers in some embodiments of the invention.

The methods according to the present invention are in some particular embodiments of the invention in vitro methods. Also within the scope of the invention is a composition comprising a Raman active compound for the measurement of the local temperature or a temperature distribution in a sample or portions thereof, wherein said composition is introduced to said sample and wherein the ratio of the intensities of its Stokes and anti-Stokes Raman emission lines is determined upon irradiation with light of (a) wavelength(s) of from 300 nm to 3 μm thereby determining the temperature. The characteristics of such a composition are the same as already described herein above.

The present invention also relates to the use of any of the above-described compositions for the determination of a local temperature or a temperature distribution in a sample. As already discussed herein above, the sample may be a biological sample.

Preferably, the biological sample is selected from the group consisting of a tissue, a living organism, an organ, a cell, a sub-cellular fragment, a living or dead human or animal body or parts thereof.

In some particular embodiments, the temperature or temperature distribution is determined during minimally invasive treatments, during radiation treatments, or during magnetic resonance treatment.

In another particular embodiment, the temperature or temperature distribution is determined in a cooled body before heart-surgery.

In yet another preferred embodiment the temperature or temperature distribution is determined for the detection of enhanced metabolistic activity.

As already stated above, the temperature to be measured is preferably within the range of from 25 ⁰C to 45 ⁰C.

The Raman emission may be based on a linear Raman effect or on a non-linear Raman effect. The light irradiation and detection may in particular embodiments be performed through optical fibers.

The terms "molecular sensor probe" and "molecular sensor" relate in the context of the present invention to a composition comprising at least one Raman active substance and can be incorporated into a sample, e.g. at a defined position in the sample, to probe the temperature in the sample or at the defined position in the sample by emitting Raman scattered light upon irradiation with a suitable wavelength.

The terms "Raman active compound", "Raman scattering material" and "Raman active substance" are used synonymously herein and relate in general to a molecule, compound or composition that is able to inelastically scatter photons according to the Raman effect. Unlike elastically scattered photons (Rayleigh scattering), Raman scattering result in scattered photons having a frequency different from the frequency of the incident photons and distinguishable Stokes scattering (molecule absorbs energy) and anti-Stokes scattering (molecule loses energy) lines. To obtain Stokes and anti-Stokes Raman lines with an intensity ratio of at most a factor of 100, the frequencies of the phonons involved should not exceed about 1000 crrf1 for measurements at about room temperature. This does not result in a strong selection criterion, for this reason preferred materials can be selected based on other criteria: a large cross-section for Raman processes and - where necessary - a compatibility with human bodies (e.g. not poisonous).

As Raman processes rely on a change in the polarisability, which in turn relies on the possibility to change the charge distribution, covalent materials and materials with large anionic groups are very suitable candidates. All these materials have distributed electron clouds. Such materials are, for example, aromatic organic compounds, materials containing ions with lone electron pairs (e.g. Bi^⁺, Sb^⁺, Sn^⁺), carboxylic acids, materials from the class MXO₃ (e.g. X = N, C, Cl, Br, I) or MYO₄ (e.g. Y = Si, Ge,Hf, Ta, Nb, W). In both cases M represents a metal ion. Examples are: NaNO₃, KNO₃, Na2CO₃, K₂CO₃, MgCO₃, Na₂SiO₄, K₂SiO₄, Na₂GeO₄, K₂GeO₄, YTaO₄ and LiNbO₃.

The Raman effect can be enhanced by using radiation that is absorbed in the material (Franck-Condon enhancement) or by using vibronic enhancement (a lattice vibration coupling two electronic excited states of the material). This does not result in a change of the

Stokes/anti- Stokes intensity ratio but may in some cases facilitate the measurements.

The Raman effect can also be surface-enhanced, i.e. by depositing Raman active moieties on e.g. small disks or spheres of Cu, Ag or Au, an amplification of the Raman effect of up to 10^ is encountered. This can be due to electrons in the metal surface being excited into an extended surface electronic excited state called a surface plasmon resonance.

Molecules in close proximity to the surface experience a very large electromagnetic field.

Vibrations normal to the surface are enhanced most strongly. Another cause of such a surface-enhancement may be the formation of a charge-transfer complex between the metal surface and the deposited molecule. The electronic transitions of many of such charge transfer complexes are in the visible range, so that resonance enhancement occurs. Examples are, e.g. particles comprising Au, or Ag covered with e.g. (VO4)^3", (WO4)^2", [FeFe(CN)g]^~ groups.

Preferably, the Raman scattering material involved is a nano- or micrometer sized material. Such materials may be easily functionalized, to result in specificity to a desired target location in the patient (e.g. cancerous cells). Due to the limited size and functionalized nature, the phosphor can be delivered at the desired location (reports have been published that prove that nanocrystals can even be delivered inside a cell), where the molecular temperature sensor can be read out locally through optical techniques.

In molecular medicine, the means and methods according to the present invention can be applied to locally measure temperature at the cellular, or even sub-cellular, level. Application may be found in a wide range of (future) medical procedures, including minimally invasive procedures. Common to all these treatments is that the temperature of the tissue to be treated changes. Examples may include, without limiting the scope of the invention, the measurement of the local temperature during radiation treatments to prevent too intense treatments, the measurements of the local temperature during magnetic resonance treatments to prevent too intense treatment, measuring the local temperature in tissue, e.g. to detect tissue with enhanced metabolistic activity, and measuring the temperature of any part of the cooled body of patients before heart-surgery to adjust the optimum temperature at selected body parts.

Optical excitation and detection of the Raman lines can be done through optical fibers. Thermal contact between the measuring unit and the molecular sensor is not required. In this way, the temperature of very small regions can be measured, only limited by the physical size of the molecular sensor. Application on nano-particles enables nano- thermometers.

In case of radiation treatment, the treatment itself might result in Raman scattering in the molecular sensors applied. In such a case, only detection is needed without separately exciting the molecular sensor.

A detector may comprise two (or more) photodiodes. The wavelength of the light to be detected by each of the photodiodes may be selected by e.g. interference filters. In a particular embodiment, the wavelength region can be determined using exchangeable interference filters. Microelectromechanical systems (MEMS) technology, together with μ- gratings, in some embodiments may even allow wavelength selection. Using up-conversion principles, three-dimensional ("depth") information may be obtained by using focused IR light. However, depth resolution is limited by absorption of light by the sample, e.g. tissue. Other applications of the means and methods of the present invention, not related to molecular medicine, are based on the fact that the invention allows local determination of the temperature even in real time.

The invention can also be used in criminology. By measuring the temperature distribution in dead bodies, the time of death of the body can be estimated quite accurately. The means and methods of the present invention may also be used to measure the temperatures and temperature distributions in real time in running engines, rotating tires and the like, in a large temperature range and therefore may be used in engine or tire development.

Typical steps of the method according to the present invention include: introducing the molecular sensor probe at the desired position in the sample, illuminating the material at a suitable wavelength, measuring the spectral data in the relevant spectral range, determining the ratio Ij/L^ of the two relevant spectral contributions (i.e.: the intensities of the Stokes and the anti-Stokes emission lines); either through integration of (a part of) the spectra, or through determination of the intensities at predetermined spectral positions, using the fact that this ratio follows the Boltzmann distribution of equation 2:

ΔE

I₁ 11₂ = exp υ (equation 2) kT

I_j : intensity of the Stokes Raman line L^: intensity of the anti-Stokes Raman line

ΔE_V: energy difference between the ground state and the higher vibrational state k: Boltzmann's constant T: temperature in Kelvin

Thus, the Raman effect can be used as an estimation of temperature (see e.g. Fig 2 and 3). A "non- linear Raman effect" in the context of the present invention relates to, e.g., Hyper Raman scattering (Hyper Raman spectroscopy), Inverse Raman spectroscopy, Raman induced Kerr effect, Coherent Anti-Stokes Raman scattering (Coherent Anti-Stokes Raman spectroscopy, CARS) and Photoacoustic Raman spectroscopy. In "Hyper-Raman spectroscopy" a non-linear effect in which the vibrational modes interact with the second harmonic of the excitation beam is exploited. This requires the observation of vibrational modes which are normally "silent", e.g. the 750 cm^~l mode of CCl₄.

In "Coherent Anti-Stokes Raman scattering" two laser beams are used to generate a coherent anti-Stokes frequency beam, which can be enhanced by resonance.

With the means and methods according to the present invention, temperature differences of substantially less than 1 ⁰C in samples (e.g. tissues, bodies) can be resolved.

In one embodiment, local temperatures are measured in a patient during radiation treatments (e.g. during cancer therapy or for cancer therapy) to prevent too intense treatments.

In another embodiment, local temperatures are measured in a patient during magnetic resonance treatments to prevent too intense treatment.

"Too intense treatments" in the context of these embodiments relate to treatments in which tissue or organ damaging temperatures are locally reached, e.g. temperatures above around 40 ⁰C.

In yet another embodiment, the local temperature in a biological tissue is measured, e.g. to detect tissue with enhanced metabolistic activity. "Enhanced metabolistic activity" in this context relates to deviating or conspicuous metabolic activities that produce a higher temperature than under normal circumstances or in the surround tissue. Such higher temperatures are e.g. 1-2 ⁰C higher than the normal tissue or body temperature (i.e. 36 - 37°C in a healthy human) or 1-2 ⁰C higher than the surrounding tissue.

An alternative embodiment relates to measuring the temperature of any part of the cooled body of patients before heart-surgery to adjust the optimum temperature at selected body parts. "Optimum temperatures" in this context are from 29 to 33 ⁰C, preferably 30 to 32°C, more preferably around 31°C. In addition, Raman spectroscopy can be used in achieving optimum re-warming of patients, also locally. This is known to be beneficial for patient recovery.

The present invention also relates to a device for performing the described methods and for the described uses. Such a device comprises a (tunable) light source, a light delivery means (e.g. an optical fiber), a wavelength-selective device (e.g. a grating) and a photo-detector. The photo-detector may for example comprise two (or more) photodiodes. The components are standard optical components and are known to a skilled person. The wavelength of the light to be detected by each of the photodiodes may be selected by e.g. interference filters. In a simple approach, the wavelength region can be determined, using exchangeable interference filters.

The following example describes the invention in greater detail but is not limiting to this invention:

Example 1: Obtaining Stokes and Anti-Stokes lines in Raman scattering

To obtain Stokes and Anti-Stokes lines with an intensity ratio of at most a factor of 100, the frequencies of the phonons involved should not exceed about 1000 cm^"1 for measurements at about room temperature. This does not result in a strong selection criterion, for this reason preferred materials can be selected based on other criteria: a large cross- section for Raman processes and compatibility with human bodies (e.g. not poisonous).

As Raman processes rely on a change in the polarisability, which in turn relies on the possibility to change the charge distribution, covalent materials and materials with large anionic groups are used. All these materials have distributed electron clouds.

Such materials are: - Aromatic organic compounds

Materials containing ions with lone electron pairs

Carboxylic acids

Materials from the class MX03 (X = N, C, Cl) or MY04 (Y = Si, Ge). In both cases M represents a metal ion. Examples are: o NaNO3, KNO3 o Na2CO3, K2CO3, MgCO3 o Na2SiO4, K2SiO4 o Na2GeO4, K2GeO4 o LiNbO3 The Raman effect is enhanced (this does not change the ratio but makes the measurements much easier) by using radiation that is absorbed in the material (Franck- Condon enhancement) or by using vibronic enhancement (a lattice vibration coupling two electronic excited states of the material). The Raman effect is also surface enhanced. By depositing Raman active moieties on spheres of Cu, Ag or Au, an amplification of the Raman effect of up to 109 is encountered. This can be due to:

Electrons in the metal surface are excited into an extended surface electronic excited state called a surface plasmon resonance. Molecules in close proximity to the surface experience a very large electromagnetic field. Vibrations normal to the surface are enhanced most strongly

The formation of a charge-transfer complex between the metal surface and the deposited molecule. The electronic transitions of many of such charge transfer complexes are in the visible, so that resonance enhancement occurs.

Claims

CLAIMS:

1. A method for the measurement of the local temperature in a sample, comprising the steps of a. introducing a molecular sensor probe comprising a Raman active compound to the sample or a desired portion of the sample; b. irradiating the sample or the desired portion of the sample with light of (a) wavelength(s) of from 300 nm to 3 μm; c. detecting the intensities of the Stokes and anti-Stokes Raman emission lines of the molecular sensor probe in the sample; d. calculating the ratio of the intensities of the Stokes and anti-Stokes Raman emission lines of said molecular sensor probe; and e. assigning said ratio to a temperature.

2. A composition comprising a Raman active compound for the use as a diagnostic agent for the measurement of the local temperature or a temperature distribution in a sample or portions thereof, wherein said composition is introduced to said sample and wherein the ratio of the intensities of its Stokes and anti-Stokes Raman emission lines is determined upon irradiation with light of (a) wavelength(s) of from 300 nm to 3 μm thereby determining the temperature.

3. The composition according to claim 2 for the use in methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practiced on the human or animal body.

4. The composition according to claim 3, wherein the methods are selected from the group consisting of determination of temperature or temperature distribution during minimally invasive treatments, during radiation treatments, or during magnetic resonance treatment, determination of temperature or temperature distribution in a cooled body before heart-surgery, and determination of temperature or temperature distribution for the detection of enhanced metabolistic activity.

5. The method according to claim 1 or the composition according to any one of the claims 2 to 4, wherein the sample is a biological sample.

6. The method according to any one of the claims 1, 5, or the composition according to any one of the claims 2 to 4, wherein said Raman active compound comprises at least one aromatic group or is an ion with at least one lone electron pair or is a carboxylic acid, or is selected from the group consisting of M_mXθ3 or M_nYC>4, wherein M is selected from the group consisting of Na, K, Li, Mg, and Ca, X is selected from the group consisting N, C, Cl, Br and I, and Y is selected from the group consisting of Si, Ge, Nb, Hf, Ta, Nb, and W and wherein m and n are 1 or 2.

7. The method or the composition according to claim 6, wherein M_mXθ3 is selected from the group consisting of NaNO₃, KNO3, Na₂CO₃, K2CO3, MgCθ3 and LiNbO₃.

8. The method or the composition according to claim 6, wherein M_nYOzJ is selected from the group consisting of Na₂SiOzJ, K₂SiOzJ, Na₂GeOzJ, YTaOzj and K₂GeOzJ.

9. The method according to any one of the claims 1, 5 or the composition according to any one of the claims 2 to 4, wherein the Raman active compound comprises anions selected from the group consisting of (VO^^", (WO_zj)^- and [FeHlFeH(CN)^]^".

10. The method according to any one of the claims 1 or 5 to 9 or the composition according to any one of the claims 2 to 9, wherein said Raman active compound is immobilized on the surface of a particle or is comprised in a particle.

11. The method or the composition according to claim 10, wherein said particle has a diameter of from 5 nm to 50 μm.

12. The method according to any one of the claims 1 or 5 to 11, wherein the method is an in vitro method.

13. A composition comprising a Raman active compound for the measurement of the local temperature or a temperature distribution in a sample or portions thereof, wherein said composition is introduced to said sample and wherein the ratio of the intensities of its Stokes and anti-Stokes Raman emission lines is determined upon irradiation with light of (a) wavelength(s) of from 300 nm to 3 μm thereby determining the temperature.

14. The composition according to claim 13, wherein said Raman active compound comprises at least one aromatic group or is an ion with at least one lone electron pair or is a carboxylic acid or is selected from the group consisting of M_mXθ3 or M_nYC>4, wherein M is selected from the group consisting of Na, K, Li, Mg and Ca, X is selected from the group consisting N, C, Cl, Br and I and Y is selected from the group consisting of Si, Ge, Nb, Hf, Ta, Nb and W and wherein m and n are 1 or 2.

15. The composition according to claim 13, wherein M_mXθ3 is selected from the group consisting OfNaNO₃, KNO₃, Na₂CO₃, K₂CO₃, MgCO₃ and LiNbO₃.

16. The composition according to claim 13, wherein M_nYθ4 is selected from the group consisting of Na₂SiO^ K₂SiO^ Na₂GeO^ YTaθ4 and K₂Geθ4.

17. The composition according to any one of the claims 13 to 16, wherein said

Raman active compound is immobilized on the surface of a particle or is comprised in a particle.

18. The method or the composition according to claim 17, wherein said particle has a diameter of from 5 nm to 50 μm.

19. Use of a composition according to any one of the claims 13 to 18 for the determination of a local temperature or a temperature distribution in a sample.

20. The use according to claim 19, wherein the sample is a biological sample.

21. The use according to claim 19, wherein the temperature or temperature distribution is determined in a cooled body before heart-surgery.

22. The use according to claim 19, wherein the temperature or temperature distribution is determined for the detection of enhanced metabolistic activity.