WO2020021154A1

WO2020021154A1 - Method for detection of marked structures

Info

Publication number: WO2020021154A1
Application number: PCT/ES2019/070533
Authority: WO
Inventors: Beatriz HERNÁNDEZ JUÁREZ; José Ricardo ARIAS GONZÁLEZ DE LA ALEJA; Héctor RODRÍGUEZ RODRÍGUEZ; María ACEBRÓN RODICIO; Francisco José IBORRA RODRÍGUEZ
Original assignee: Universidad Autónoma de Madrid; Fundación Imdea Nanociencia; Consejo Superior De Investigaciones Científicas
Priority date: 2018-07-27
Filing date: 2019-07-26
Publication date: 2020-01-30
Also published as: ES2745070B2; ES2745070A1

Abstract

The present invention relates to a method for detecting marked structures, to its uses and to a device designed to carry out the method.

Description

DESCRIPTION

METHOD OF DETECTION OF MARKED STRUCTURES Field of the invention

The present invention relates to a method for the detection of marked structures, their uses and an apparatus designed to carry out said method.

Background of the invention

Fluorescence microscopy is a basic tool for the investigation of biological samples. In it, the objects are illuminated by electromagnetic radiation of a certain wavelength and the image observed is the result of the electromagnetic radiation emitted by fluorophores that have absorbed the primary excitation and re-emitted a light with a different wavelength. Among the available techniques, those in which the excitation takes place in the near infrared biological windows (700-950 nm and 1000-1350 nm) are especially relevant, in which case the depth of penetration into the tissue can reach some millimeters

In particular, multifotonic microscopy (optical excitation by two or more photons) combines this characteristic with a resolution whose limit is the volume of laser focusing so it has become one of the most used fluorescence microscopy techniques. However, one of the most significant problems of multifotonic microscopy is the accelerated whitening of fluorophores due to the high photon fluxes (10 ²⁷ -10 ²⁹ cnr ² s ¹ ) to which they are subjected [Fischer M., et al . “Fluorescence quantum yield of Rhodamine 6G in ethanol as a function of concentration using thermal lens spectrometry”, vol.2614, n ° 260, pp 115-118] This effect, which is also irreversible, limits the use of this type of microscopy to Experiments of a few minutes duration. Likewise, the submission of biological specimens to high optical powers can lead to the destruction of irradiated cells (photo-toxicity) [Debarre, D. et al. "Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0-1.2 pm wavelength range, ”PLoS One, vol. 9, n ° 8, e.g. 104250, 2014] Quantum dots (PCs) are luminescent nanoparticles (PL) that have differentiated optical and electronic properties. For example, when they are illuminated they emit light at a very specific wavelength that depends on the size and other characteristics of said quantum point. These properties cause quantum dots to be adopted as fluorescent probes in biology and medicine for microscopy, detection and diagnosis.

The document Liu et al. [H. Liu, H. Maruyama, Vibration-assisted optical injection of a single fluorescent sensor into a target cell, Sensors and Actuators B: Chemical, 2015, 220, pp 40-49] describes the injection of a micrometric probe (5 microns in diameter) in a cell under the vibration of the focal point of optical tweezers. It is a complex probe that includes quantum dots and, under UV illumination, changes its z potential allowing it to be fixed to the surface of the cell membrane. To follow the progress of said probe through the cell membrane that is labeled with fluorophores, the document by Liu et al. uses the fluorescence resonance energy transmission (Fluorescence Resonance energy transfer or FRET) that occurs between the quantum dots of the probe and the membrane fluorophores. These quantum dots are excited by UV radiation. In the document Liu et al., Therefore the observation of the membrane, after applying UV radiation, is limited to an area of micrometric size where the adhesion of the probe has taken place. In addition, this technique can be invasive since the use of this radiation can cause damage to biological structures and since the probe penetrates the cell.

The FRET mechanism contemplates a transfer of non-radiative energy between donors and acceptors (which can be fluorophores), mediated by interactions between molecules or in general between dipoles, limiting the distances in which they take place. Therefore, FRET-based techniques are limited by the distances between donor and acceptor. On the other hand, in radiative transfers, the propagation of radiation emitted by an emitter is affected by absorption, emission or dispersion, and therefore, the distances at which it occurs can be much greater, since they do not depend on inter-molecular interactions or inter-dipoles. Consequently, the methods of detection of structures present problems such as low resolution and selectivity, limited measurement times due to degradation of the useful life of the markers and possible photo-toxicity on the sample, limitation in the area of study and modification of the samples. Therefore, new methods of detection of marked structures that solve some or all of the above limitations are necessary.

Brief Description of the Invention

The present invention provides a new method of detecting marked structures that allows both selectivity and resolution of said detection to be increased. Also, it allows to extend the measurement times without damaging the sample (due to a reduction in the irradiance on the sample). In addition, this method allows the study of different areas of the marked sample along the same measure without significantly disturbing said sample since it is a non-invasive technique.

Therefore, a first aspect of the invention relates to a method of detection of marked structures comprising the steps of:

i) provide:

- a structure marked with a luminescent marker, wherein said luminescent marker has an absorption spectrum;

- at least one luminescent nanoparticle (PL), where said at least one PL has an emission spectrum; wherein said emission spectrum of said at least one PL overlaps with the absorption spectrum of the luminescent marker of step (i);

- an optical clamp, wherein said optical clamp comprises a focused laser; wherein said focused laser has an emission in a range of wavelengths that does not overlap with the absorption spectrum of the luminescent marker of step (i);

ii) confine and simultaneously excite said at least one PL of stage (i) by means of the optical clamp of stage (i) giving rise to at least one excited and confined PL;

iii) optionally displacing the at least one excited and confined PL of stage (ii) by means of the optical clamp of stage (i); Y iv) exciting the luminescent marker of stage (i) by the radiation emitted by said at least one excited and confined PL of stage (ii) or (iii), giving rise to an excited luminescent marker; where said excited luminescent marker emits a luminescent signal.

A second aspect of the invention would be directed to the use of a luminescent marker in the method of detecting labeled structures of the present invention in any of its particular embodiments.

A third aspect of the invention would be directed to the use of a luminescent particle (PL) in the method of detecting labeled structures of the present invention in any of its particular embodiments.

A fourth aspect of the invention would be directed to the use of an optical clamp in the method of detection of marked structures of the present invention in any of its particular embodiments.

A further aspect of the present invention is directed to an apparatus designed to carry out the method of detection of marked structures of the present invention comprising:

i) means for trapping and rotating the marked structure of step (i); ii) the optical clip of step (i); Y

iii) a signal detector of step (iv) of the method of detection of marked structures of the present invention in any of its particular embodiments.

Figures

These and other features and advantages of the invention will become more clearly apparent from the detailed description that follows in a preferred embodiment, given only by way of illustrative and non-limiting example, with reference to the accompanying figures. .

Figure 1: (a) Scheme of a possible chain excitation or radiative transfer system in which a PL is excited by a process of absorbing two photons in the near infrared, 2Y _NIR , after which it emits a photon in the visible spectrum, Yvis _, which in turn excites a fluorescent marker (a dye molecule) that in turn emits a photon in the visible spectrum at a different wavelength from the previous one, and _VIS-, which will be detected as a light signal; (b) graph of emission intensity / absorption versus wavelength (nm) showing the overlap between the emission spectrum of PLs (colloidal quantum dots, continuous line PCs) and the absorption spectrum of a dye (dashed line) .

Figure 2: Experimental scheme in which an aggregate of PCs is optically trapped and moved by an optical clamp around a marked cell. The laser beam of the optical clamp, in the near infrared, simultaneously excites the PCs, whose remission allows to turn the marked structures of the cell in turn. This scheme is not to scale.

Figure 3: Transillumination images obtained by means of an optical microscope and emission spectra collected from an aggregate of optically trapped and excited PCs at different distances (a-d) from the surface of a marked cell.

Figure 4: (a) Experimental scheme in which an aggregate of PCs is optically trapped and excited within a solution of a dye and at a depth (distance L) relative to the inner surface that limits the bottom of a microfluidic chamber containing said dye (the scheme is not drawn to scale); and (b) emission spectra measured by trapping and exciting an aggregate of PCs at various distances L (depths) from the surface of said microfluidic chamber that serves as a container of the dye solution.

Figure 5: (a) Scheme of the overlap between the emission spectrum of two species of colloidal PCs, 1 and 2, which have a maximum emission at 530 and 580 nm respectively (continuous lines 1 and 2) and the absorption spectrum of a dye (dashed line), said overlap is represented as the shaded area below the curve. Emission spectrum measured for a solution of a dye into which an aggregate of type 1 PCs at different depths (distances L) and (c) an aggregate of type 2 PCs are trapped and excited (b). Figure 6: Scheme of an experimental device that allows the detection of marked structures formed by two lasers (L1 and L2) that are combined by a beam splitter cube (PBS) and directed by means of mirrors (M1 and M2) inwards of a fluorescence microscope. The combined laser beam is reflected in a dichroic mirror (DM) and focused through a microscope objective (O) on the sample (SH). A lamp (WL), a grid system (l & S) and a condenser (C) make up the lighting system of the experiment. Fluorescence from the sample is collected through a filter (F) and selectively sent to a spectrometer (S) or to a camera (VC) by various optical elements (FL, focusing lens; M3, mirror; P, prism; RL1 and RL2, relay lenses.

Detailed description of the invention

Unless stated otherwise, all the scientific terms used here have the meaning that is commonly understood by the person skilled in the art to which this description is directed. In the present invention, the singular forms include the plural forms unless otherwise indicated. In particular, the given pronouns (el, la, lo) or indeterminate (one, one, one) singular do not limit to a cardinal number and can refer to more than one element (for example, one, two, three or more) . This is particularly relevant in the present invention when referring, for example, to "a marked structure", "a luminescent marker", "an optical clamp" and "a focused laser".

The main object of the present invention is to provide a method of detection of marked structures comprising the steps of:

i) provide:

a structure marked with a luminescent marker, wherein said luminescent marker has an absorption spectrum;

at least one luminescent nanoparticle (PL), where said at least one PL has an emission spectrum; wherein said emission spectrum of said at least one PL overlaps with the absorption spectrum of the luminescent marker of step (i);

an optical clip, where said optical clip comprises a focused laser; where said a focused laser has an emission in a range of wavelengths that do not overlap with the absorption spectrum of the luminescent marker of step (i);

iii) optionally displacing the at least one excited and confined PL of stage (ii) by means of the optical clamp of stage (i); Y

iv) exciting the luminescent marker of stage (i) by the radiation emitted by said at least one excited and confined PL of stage (ii) or (iii), giving rise to the luminescent marker of stage (i) excited; where said excited luminescent marker emits a luminescent signal.

Step (i) The method of detection of marked structures of the present invention comprises step (i) of providing:

- at least one luminescent nanoparticle (PL), where said at least one PL has an emission spectrum; wherein said emission spectrum of said at least one PL overlaps with the absorption spectrum of the luminescent marker of step (i); Y

- an optical clamp, wherein said optical clamp comprises a focused laser; wherein said focused laser has an emission in a range of wavelengths that does not overlap with the absorption spectrum of the luminescent marker of step (i).

In the context of the present invention, the term "marked structure" or "marked structures" refers to a structure comprising one or more luminescent markers; said structure may be present in biological and non-biological specimens. When said structure is present in biological specimens, it may be marked by any of the luminescent markers commonly used for biological marking and known to the person skilled in the art. In a particular embodiment, said marked structure may be a cell or part of a cell such as a cell membrane. As a non-limiting example, said labeled structure can be a waterproofed Jurkat-T cell whose proteins have been unspecifically labeled with a fluorescent dye such as Alexa Fluor® 546.

In the context of the present invention, the term "luminescence" characterizes the property of an energy absorbing material (for example, in the form of an electromagnetic radiation, composed of photons) which then emits in the form of electromagnetic radiation.

In the context of the present invention, the term "luminescent marker" refers to a material, for example a functional group, compound or chemical composition, which is capable of absorbing energy in the form of electromagnetic radiation in a given range of wavelengths. and to re-emit electromagnetic radiation in a different range of different wavelengths; therefore, it is characterized by having an absorption spectrum and a characteristic emission spectrum. Non-limiting examples of luminescent markers are fluorescent markers, phosphorescent markers or combinations thereof. Preferably, the luminescent marker of the present invention is selected from fluorophores, chromophores and combinations of the foregoing.

In the context of the present invention, the term "fluorescent marker" is synonymous with fluorochrome, that is, a material, a functional group, compound or chemical composition, which is capable of absorbing energy in the form of electromagnetic radiation in a given range. of wavelengths and of emitting electromagnetic radiation in another range of wavelengths greater than the first (ie with less energy). Non-limiting examples of fluorochromes suitable for use in the present invention are any of the fluorochromes known to those skilled in the art, for example those appearing in the database of the website http: //www.fluorophores.tugraz. at / substance / a July 19, 2018 [Fluorophores.org is a user-driven platform for fluorescent dye data initiated by the Applied Sensor Group of the Institute of Analytical Chemistry at the Graz University of Technology in Austria] Preferably fluorochromes selected from : fluorescein and its derivatives such as 5-carboxyfluorescein, 6-carboxyfluorescein, 6- (fluorescein) -5- (and 6) -carboxamide-hexanoic acid and fluorescein isothiocyanate; dyes AlexaFluor® and its derivatives such as AlexaFluor 488®, Alexa Fluor® 546 or AlexaFluor 594®; cyanine dyes such as Dy2, Cy3, Cy5, Cy7; optionally substituted coumarin; R-phycoerythrin, allophycoerythrin and its derivatives; rhodamine, tetramethyl rhodamine, rhodamine 6G and its derivatives; Princeston Red; R-phycoerythrin conjugates; members of ficoliproteins and quantum dots.

In a more particular embodiment, the luminescent marker of step (i) is a fluorescent marker; preferably selected from the group consisting of AlexaFluor®, rhodamine and its derivatives; preferably it is a fluorescent marker selected from Alexa Fluor® 546, tetra-methyl-rhodamine and 6G rhodamine.

In a particular embodiment, the luminescent marker of step (i) has an absorption spectrum; wherein said absorption spectrum of the luminescent marker of step (i) overlaps with the emission spectrum of said at least one PL; preferably it overlaps at least 10% of the range; preferably in 60% of the wavelength range of the emission spectrum of said at least one PL; more preferably it overlaps in a wavelength range of 80%.

In a particular embodiment the luminescent marker of the invention has an absorption spectrum and an emission spectrum; preferably it has an absorption spectrum and an emission spectrum, in particular said marker can be excited and, once excited, emit a luminescent signal.

As a non-limiting example, the Alexa Fluor® 546 luminescent marker is a fluorescent marker whose maximum absorption of its absorption spectrum is around 546 nm and whose maximum emission of the emission spectrum is around 573 nm ( orange color seen through a conventional fluorescence microscope) and whose extinction coefficient is 104000 at the maximum emission in cnr ¹ M ¹ . For example, the excited Alexa Fluor® 546 marker results in a luminescent signal with a maximum centered around 573 nm

In the context of the present invention, the term "absorption spectrum" refers to the intensity of incident electromagnetic radiation that a material absorbs in a given wavelength range. In the context of the present invention, the term "emission spectrum" refers to the intensity of electromagnetic radiation that a material emits in a given wavelength range.

In a particular embodiment, the one structure marked with a luminescent marker of step (i) and the at least one luminescent nanoparticle (PL) are in a liquid medium; preferably in an aqueous or organic medium; more preferably in an aqueous medium; even more preferably in a phosphate buffered saline (PBS). In a more particular embodiment, the liquid medium is a culture medium.

In a particular embodiment, the one structure marked with the luminescent marker of step (i) and the at least one luminescent nanoparticle (PL) are located in a microfluidic chamber; preferably in a transparent microfluidic chamber in the visible and near infrared spectrum.

In a particular embodiment, the one marked structure of step (i) is present in biological and non-biological specimens; preferably in biological specimens; more preferably in cells. Non-limiting examples of suitable cells for the present invention are T lymphocytes, HL60, Jurkat, macrophages, HeLa (in suspension), also adherent cells such as HeLa, fibroblasts, MFC7, etc.

In an even more particular embodiment, the one labeled structure of step (i) is a labeled protein in a cell; preferably a nonspecifically or specifically labeled protein in a cell; more preferably a specifically labeled protein; even more preferably a protein specifically labeled with a luminescent marker.

In the context of the present invention, the term "luminescent nanoparticle (PL)" refers to a particle that comprises at least one of its dimensions in a size range equal to or less than 100 nm; which is capable of absorbing energy in the form of electromagnetic radiation in a certain range of wavelengths and of re-emitting electromagnetic radiation in another range of different wavelengths and that has an emission spectrum. In the context of the present invention it should be understood that the term "luminescent nanoparticle (PL)" or "nanoparticles Luminescent (PLs) "throughout the description and claims also includes the more specific meanings of" photoluminescent nanoparticle (PF) "or" photoluminescent nanoparticles (PFs) and also of "quantum dot (PC)" or "quantum dots ( PCs) ”

In a particular embodiment, the at least one luminescent particle of step (i) is at least one photoluminescent nanoparticle or at least one quantum dot (PC); preferably at least one quantum dot (PC); more preferably it is an aggregate of quantum dots (PCs).

In the context of the present invention, the term "photoluminescent nanoparticles" refers to nanoparticles of organic or inorganic nature capable of absorbing energy in the form of electromagnetic radiation in a certain range of wavelengths and of emitting electromagnetic radiation again in another range of different wavelengths (greater or lesser wavelengths; preferably longer). Non-limiting examples of photoluminescent nanoparticles are inorganic nanoparticles such as sulfates, phosphates and fluorides doped with rare earth ions, sulfide particles or Ag selenides; quantum dots; nanodiamonds; carbon nanoparticles (quantum dots carbon); and nanoparticles of organic nature as polymer particles doped with dyes; preferably nanoparticles of inorganic nature.

Non-limiting examples of photoluminescent nanoparticles are nanoparticles of inorganic nature doped with luminescent metal ions, preferably as lanthanide ions.

The inorganic nanoparticles included in the photoluminescent nanoparticles can be selected from known sulfates, phosphates and fluorides depending on the dopant to be incorporated. Since most photoluminescent dopants are di- or trivalent metal ions, it is preferred to use sulfates, phosphates or fluorides of non-luminescent di- or trivalent metal atoms such as group 2 metals (alkaline earth metals, such as Mg, Ca, Sr, or Ba), or from group 3 (Se, Y or La) or from group 13 (for example, Al, Ga, In or TI) or Zn. Regarding the type of photoluminescent metal ions to be incorporated as dopants included in the photoluminescent nanoparticles, there are no specific restrictions as long as they are capable of converting the absorbed photons into luminescent radiation. Preferably lanthanide ions are used as dopant metal ions of inorganic salts doped with luminescent metal ions. The dopant lanthanide ion (s) can be conveniently selected from Ce (item number 58), Pr (59), Nd (60), Sm (62), Eu (63), Gd (64), Tb (65) , Dy (66), Ho (67), Er (68), Tm (69), or Yb (70); preferably between Yb (70), Er (68), Tm (69) or Nd (60. In a particular embodiment, the preferred dopants are Er ³ ", Nd ³⁺ and Yb ³⁺ .

In the context of the present invention, the term "quantum dot (PC)" or "quantum dot (PCs)" refers to crystalline semiconductor particles that have tunable optical properties with the particle size. They comprise elements belonging to groups I l-VI, lll-V, or IV-VI of the periodic table.

In a particular embodiment, the at least one luminescent particle (PL) of step (i) is at least one quantum point (PC), preferably at least one quantum point (PC) comprising elements belonging to the groups in the table periodic I-VI, II-V, or IV-VI; more preferably at least one quantum dot (PC) comprising compounds selected from indium arsenide (InAs), indium phosphide (InP), cadmium sulphide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe) , lead sulfide (PbS), lead selenide (PbSe), lead telluride (PbTe), zinc sulfide (ZnS), zinc selenide (ZnSe), CdSeZnS, CulnSe, Cd3As2, CdsP2, C, as well as mixtures of The these compounds.

In a particular embodiment the at least one quantum dot (PC) of the present invention has a core-shell configuration, core-shell-shell, a configuration with alloyed elements, or a configuration in "giant quantum dots" form; preferably a core shell configuration (core-shell) or core-shell-shell (core-shell-shell). In the context of the present invention the term "giant quantum dot" refers to core-cortex systems where the crust is thicker than 10 atomic layers of material.

In a particular embodiment, the at least one luminescent particle of step (i) is a aggregate of quantum dots (PCs); particularly an aggregate of quantum dots (PCs) of CdSeZnS; preferably an aggregate of quantum dots (PCs) of coated CdSeZnS; more preferably an aggregate of quantum dots (PCs) of S1O2 coated CdSeZnS. In an even more particular embodiment, the addition of quantum dots comprises between 2 and 10,000 quantum dots; preferably between 2 and 1000 quantum dots.

In a particular embodiment, the at least one luminescent particle of step (i) is functionalized; preferably it comprises ligands on its surface; more preferably it comprises functional groups on its surface.

In a particular embodiment, the at least one luminescent particle of step (i) is encapsulated in a matrix; preferably an inorganic matrix; more preferably in an inorganic matrix formed by inorganic oxides; even more preferably selected from alumina, silicon oxide (S1O2), titanium oxide and combinations thereof; even more preferably silicon oxide (S1O2).

In a particular embodiment, the at least one luminescent particle of step (i) is at least one quantum dot (PC) encapsulated in an inorganic matrix; preferably at least one quantum dot (PC) of CdSeZnS encapsulated in S1O2.

The authors of the present invention have observed that, without being linked to a particular theory, using the quantum dots encapsulated in a matrix reduces the bleaching effect of said quantum dots, increasing the emission stability. Furthermore, it has been observed that the encapsulation increases the biocompatibility of said quantum dots and makes them dispersible in polar media. Finally, the encapsulation or matrix that covers the quantum dots or their aggregates favors radiative energy transfers over non-radiative ones.

The at least one luminescent nanoparticle (PL) of the marked structure detection method of the present invention has an emission spectrum; wherein said emission spectrum of said at least one PL overlaps with the absorption spectrum of the luminescent marker of the detection method of the present invention. In a particular embodiment said spectra overlap in at least 10% of their wavelengths; preferably at least 20%; more preferably at least 30%; even more preferably at least 40%. In a particular embodiment, the maximum of the emission spectrum of said at least one PL overlaps the maximum of the absorption spectrum of the luminescent marker of the detection method of the present invention.

The optical clamp of the method of detection of marked structures of the present invention comprises a focused laser; where said a focused laser has an emission in a range of wavelengths that does not overlap with the absorption spectrum of the luminescent marker of step (i). That is, the optical clamp of the method of detection of marked structures of the present invention is not capable of causing the luminescence emission of the luminescent marker of the present invention. In a particular embodiment, the optical clamp of the detection method of the present invention is not capable of exciting the luminescent marker of step (i).

In the context of the present invention, the expression "optical clamp" is synonymous with the expression "optical trap" as known by the person skilled in the art and refers to a focused laser that generates an attractive force on dielectric objects, which it allows to catch, confine or hold a particle and move or move it physically [Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S (1986). "Observation of a single-beam gradient force optical trap for dielectric particles". Opt. Lett. 11 (5): 288-290] In particular, said particle can be at least one luminescent nanoparticle (PL) or at least one quantum dot. Said optical clamp may alternatively be formed by combining two laser beams traveling in opposite directions (counterpropagators) in a common focus as described, for example, in the article [Smith SB, Cui Y., Bustamante C. Optical-trap forced transducer that operates by direct measurement of light momentum. Methods Enzymol. 2003; 361: 134-162]

In a particular embodiment, the focused laser of the optical clamp of step (i) of the present invention has a monochromatic emission in a range of wavelengths that does not overlap with the absorption spectrum of the luminescent marker of stage (i ). In a more particular embodiment, the focused laser of the optical clamp of step (i) of the present invention is not capable of generating luminescence of the marker by any optical process. In a particular embodiment, the range of lengths of The emission wave of the focused laser of the optical clamp of the invention is not included in the absorption spectrum of the luminescent marker.

In a particular embodiment, the optical clamp of step (i) comprises a monochromatic focused laser beam.

In a particular embodiment, the optical clamp of step (i) comprises a monochromatic, single mode, continuous and focused laser beam; preferably a monochromatic diode laser beam, single mode, continuous and focused.

In a particular embodiment, the optical clamp of step (i) comprises a femtosecond laser beam.

In a preferred embodiment, the optical clamp of step (i) comprises a continuous regime laser.

In a preferred embodiment, the optical clamp of step (i) comprises a highly focused laser; preferably highly focused by means of a high numerical aperture objective.

In a particular embodiment, the optical clamp of step (i) has a power between 300 and 100 mW; preferably between 200 mW and 120 mW; more preferably 150 mW.

In a particular embodiment, the optical clip of step (i) has a wavelength that is in a range between 650 nm and 950 nm; preferably between 700 and 900 nm; more preferably between 750 and 850 nm; more preferably 845 nm. In a more particular embodiment, the optical clamp of step (i) has an emission in a range of wavelengths between 650-950 nm that excites said at least one PL.

In a particular embodiment, the optical clip of step (i) comprises a photon flow of between 10 ²⁰ cnr ² s ^_1 and 10 ²⁶ cnr ² s ^-1 .

The authors of the present invention have observed that, without being linked to a In particular, the fact that the optical clamp of the method of detection of marked structures of the present invention is not capable of exciting the luminescent marker of the present invention makes it possible to increase the selectivity of the method and prevent the degradation of said markers.

Stage (ii)

The method of detection of marked structures of the present invention comprises step (ii) of confining and simultaneously exciting said at least one luminescent nanoparticle (PL) of step (i) by means of the optical clamp of step (i) giving rise to at least one excited and confined luminescent nanoparticle (PL).

In the context of the present invention, the term "confined" in relation to the luminescent nanoparticle of the present invention of steps (ii), (iii) and (iv) refers to the state in which the luminescent nanoparticle of the present The invention is "trapped" in the focused laser of the optical clip as it would be known by a person skilled in the art. Particularly, the confinement of the luminescent nanoparticle makes it possible to displace said particle by displacing said focused beam.

In the context of the present invention, the term "excited" in relation to a luminescent particle of the present invention of steps (ii), (iii) and (iv), refers to an excited state due to its interaction with radiation Electromagnetic focusing laser of the optical clamp as would be known by a person skilled in the art.

In a particular embodiment, the excitation of the at least one PL of stage (ii) is produced by absorption of the focused laser comprised in the optical clamp of stage (i) and the at least one PL of stage (i); preferably by a two photon absorption mechanism between the focused laser comprised in the optical clamp of stage (i) and the at least one PL of stage (i); more preferably by means of a two-photon absorption mechanism between the monochromatic, single-mode, continuous and focused laser comprised in the optical clamp of stage (i) and the at least one PL of stage (i).

In a particular embodiment, the excitation of the at least one PL of step (ii) is produced by a photon or multifotonic excitation mechanism; preferably multifotonic; more preferably by a two photon absorption mechanism.

In the context of the present invention, the expression "two photon absorption mechanism" refers to a mechanism in which a luminescent particle, such as a quantum dot or an aggregate of quantum dots, is capable of absorbing energy in form of photons or electromagnetic radiation in a given range of wavelengths of a certain energy and of emitting electromagnetic radiation again in another range of wavelengths different from energy greater than that of individual photons as is known to an expert in the matter. That is, a luminescent particle is capable of absorbing two low energy photons resulting in the emission of a higher energy photon than any of the photons initially absorbed. For certain types of luminescent particles, a high flow of photons is typically required, such as that produced in laser radiation for this type of mechanism to occur.

Stage (iii)

The method of detection of marked structures of the present invention optionally comprises step (iii) of displacing the at least one excited and confined PL of stage (ii) by means of the optical clamp of stage (i).

In a particular embodiment, the present invention is directed to a method of detection of marked structures comprising the steps of:

i) provide:

ii) an optical clamp, wherein said optical clamp comprises a focused laser; where said a focused laser has an emission in a range of wavelengths that does not overlap with the spectrum of absorption of the luminescent marker of step (i); confine and simultaneously excite said at least one PL of stage (i) by means of the optical clamp of stage (i) giving rise to at least one excited and confined PL;

iii) displace the at least one excited and confined PL of stage (ii) by means of the optical clamp of stage (i); Y

iv) exciting the luminescent marker of stage (i) by the radiation emitted by said at least one excited and confined PL of stage (ii) or (iii), giving rise to excited luminescent marker; where said excited luminescent marker emits a luminescent signal.

In the context of the present invention, the expression "displace" in relation to the at least one excited and confined luminescent particle of step (ii) by the optical clamp of step (i) of the detection method of the present invention, it refers to moving said particle to a determined position by means of the optical clamp of step (i). This movement is not performed only once but could be repeated. That is, it would be possible to displace the at least one excited and confined luminescent particle (PL) from stage (ii) by means of the stage's optical clip (i) at different points relative to the marked structure. Such displacement could consist of an individual displacement or a series of successive displacements. As a non-limiting example, Figure 3 of the present invention shows an aggregate of quantum dots that is "confined" and excited by an optical clamp, and how it is possible to move said aggregate into a liquid medium inside a chamber of microfluidic

In a particular embodiment, the displacement of stage (iii) occurs until the excited and confined PL is at a distance from the marked structure of stage (i) such that the radiation emitted by said at least one excited PL and confined excites the luminescent marker of said marked structure; preferably less than 10 microns; more preferably a distance between 10 microns and 1 nm; more preferably a distance between 1 microns and 1 nm. As a non-limiting example, Figure 3 of the present invention shows an aggregate of quantum dots that is "confined" and excited by an optical clamp. In said non-limiting example, it can be seen that, at a certain distance from said aggregate to the marked surface (a cell) luminescent signals of said marked structure are obtained (Figures 3c and 3d).

The method of detection of marked structures of the present invention comprises step (iv) of exciting the luminescent marker of step (i) by the radiation emitted by said at least one excited and confined PL of step (ii) or (iii ), resulting in excited luminescent marker; where said excited luminescent marker emits a luminescent signal.

In a particular embodiment, the at least one excited and confined PL of any of steps (ii), (iii) and (iv) emits electromagnetic radiation; preferably in a wavelength range that overlaps with the absorption spectrum of the luminescent marker of step (i); preferably in a wavelength range between 300 and 800 nm.

In a particular embodiment, the at least one excited and confined PL of any of stages (ii), (iii) and (iv) emits photons with an energy capable of exciting the luminescent marker of stage (i).

In a particular embodiment, the excitation of the luminescent marker of step (iv) is produced by a radiative transfer between the at least one excited and confined PL of any of stages (ii) or (iii) and (iv) and the marker luminescent stage (i).

In a more particular embodiment, the luminescent marker of stage (i) acts as an energy acceptor in the radiative transfer of stage (iv); in particular as a photon acceptor.

In a more particular embodiment, the at least one excited and confined PL of any of stages (ii) or (iii) and (iv) acts as an energy donor in the radiative transfer of stage (iv); in particular as a donor of photons.

In a more particular embodiment, the luminescent marker of step (i) and the at least one excited and confined PL of any of stages (ii) or (iii) and (iv) act as a donor-acceptor pair in the radiative transfer of stage (iv).

In the context of the present invention, the term "radiative transfer" refers to processes of interaction between the at least one excited and confined PL of any of steps (ii), (iii) or (iv) and the luminescent marker of step (i) where part or all of the electromagnetic radiation emitted by the at least one excited and confined PL is absorbed by the luminescent marker of stage (i) so that said luminescent marker passes into an excited state. In other words, a radiative process occurs between the at least one excited and confined PL of any of stages (ii), (iii) or (iv) and the luminescent marker of stage (i) through the emission and absorption of photons of a certain energy. Said process only occurs when the emission spectrum of said at least one PL overlaps with the absorption spectrum of the luminescent marker of step (i).

Without being linked to a particular theory, the authors of the present invention have found that by exciting in step (iv) of the detection method of the present invention the luminescent marker of stage (i) by the radiation emitted by said at less an excited and confined PL of step (ii) or (iii) problems associated with the use of high irradiance such as accelerated whitening of luminescent markers, such as fluorophores, and their degradation would be avoided. Furthermore, it has also been observed that by this method the degradation of the marked structure is reduced for relatively long measurement times, particularly when said structure is part of a biological specimen. Also in this way, the selectivity and resolution of said method is increased.

Without being linked to a particular theory, the authors of the present invention have found that when the at least one excited and confined PL of any of stages (ii), (iii) and (iv) and the luminescent marker are at distances greater than 10 nm a radiative transfer occurs between the at least one excited and confined PL of any of stages (ii), (iii) and (iv) and the luminescent marker allowing the luminescent marker to be excited. Thus, the detection of the present invention is not limited to small distances possessing other techniques such as the fluorescence resonance energy transfer technique (FRET). The person skilled in the art would know how to select the distances optimal between the excited and confined PL and the luminescent marker or the structure marked to study said structure. This distance is not limitative of the method since the confined PL can be displaced.

In a particular embodiment, steps (iii) and (iv) of the detection method of the present invention are repeated at least once; preferably between 2 and 100 times; more preferably between 2 and 50 times.

Without being linked to a particular theory, the authors of the present invention have found that by repeating steps (iii) and (iv) of the method of detection of marked structures of the present invention, different zones or different structures marked in The same experiment or measure. In addition, it is possible to study the sample for a longer time without observing that the sample is degraded.

In a particular embodiment, the excited luminescent marker of step (iv) emits luminescence; preferably radiation in a wavelength range between 400 and 650 nm.

In a particular embodiment, the excited luminescent marker of step (iv) emits a luminescent signal; preferably a fluorescent signal; more preferably a fluorescent signal formed by an emission of electromagnetic radiation in a wavelength range between 300 and 800 nm; preferably between 400 and 700 nm.

In a particular embodiment, the luminescent signal of step (iv) is detected by means of light detection; preferably by means of a spectrometer or a camera; more preferably by means of a camera.

In the context of the present invention the terms "spectrometer" and "spectrophotometer" are equivalent.

In a particular embodiment, the detection method of the present invention is applied in biological samples. In a more particular embodiment, the detection method of the present invention is applied to map cell membranes, biosensar membrane proteins or monitor electrical signals. In the context of the present invention, the term "biosensar" refers to using a method in a living being to obtain information from a biological process or structure. One skilled in the art would be able to adapt the method of the present invention to be able to biosensar.

Without being linked to a particular theory, the authors of the present invention have found that by optically exciting the marked structure to be detected by the method of detection of marked structures of the present invention, such contactless structures can be detected and subjected only to the Low irradiance of PCs, therefore non-invasively, and remotely.

Applications

A second aspect of the invention would be directed to the use of a luminescent marker in the method of detecting labeled structures of the present invention in any of its particular embodiments. The luminescent marker may comprise any of the features described in any of the particular embodiments of the present invention.

A third aspect of the invention would be directed to the use of a luminescent particle (PL) in the method of detecting labeled structures of the present invention in any of its particular embodiments. In a particular embodiment, said luminescent particle can be used individually or in conjunction with other luminescent particles of the same type. The luminescent particle may comprise any of the features described in any of the particular embodiments of the present invention.

A fourth aspect of the invention would be directed to the use of an optical clamp in the method of detection of marked structures of the present invention in any of its particular embodiments. The optical clip may comprise any of the features described in any of the particular embodiments of the present invention. Means to carry out the method

i) means for trapping and rotating the marked structure of step (i); ii) the optical clip of step (i) of the method of the present invention; and iii) light detection means of the luminescent signal of step (iv) of the method of detection of marked structures of the present invention; preferably a signal detector of step (iv) of the method of detection of marked structures.

In a particular embodiment, the apparatus designed to carry out the method of detection of marked structures of the present invention further comprises

(iv) a container, preferably a microfluidic chamber, and means for viewing said container, preferably an optical camera; more preferably a camera coupled to an optical microscope.

In a particular embodiment, the light sensing means of the apparatus of the present invention comprise a spectrometer.

In a particular embodiment, the light detection means of the apparatus of the present invention do not use spectral filters; preferably they do not use spectral filters to separate the signals from the different emitters.

The authors of the present invention have observed that the use of a spectrometer allows to reduce costs in the method of detection of marked structures in addition to allowing unequivocally observe the emission of PCs and fluorophores. In addition, the apparatus designed for the detection method of the present invention comprises a simpler design since it does not need to use spectral filters to separate the emission signals.

The use of a luminescent marker, the use of a luminescent particle (PL), the use of an optical clamp in the method of the present invention and the apparatus designed to Carrying out the method of the present invention comprises all the features described for the luminescent marker, the luminescent particle (PL), the optical clamp and the apparatus described for the method of detection of marked structures of the present invention in any of its embodiments. private individuals

Examples

The invention is described below by the following examples that should be considered as merely illustrative and in no case limiting the scope of the present invention.

Example 1: Detection of structures marked in cells.

In the present example, the detection of labeled structures in cells was studied.

As marked structures, waterproofed Jurkat-T cells were used whose proteins were unspecifically labeled with a fluorescent dye which in this case is Alexa Fluor® 546. Alexa Fluor® 546 has an absorption spectrum with a maximum around 546 nm, and a emission spectrum with a maximum centered around 573 nm.

On the other hand, luminescent nanoparticles (PL) were synthesized, specifically quantum dots (PCs) formed by nanocrystals of a CdSeZnS alloy encapsulated in S1O2 (ad hoc PCs) with a maximum emission at 540 nm following the method described in Acebron M. et to the. [M. Acebrón, JF Galisteo-López, D. Granados, J. López-Ogalla, JM Gallego, R. Otero, C. López, and BH Juárez, “Protective Ligand Shells for Luminescent Si0 ₂ -Coated Alloyed Semiconductor Nanocrystals,” ACS Appl. Mater. Interfaces, p. 150319103751001, 2015.]. S1O2 encapsulation of quantum dots favors radiative processes over non-radiative processes (ie FRET) by creating a “barrier” between 10 and 100 nm.

It should be noted that said dye / quantum point pair was selected since the maximum emission of said quantum point at 540 nm overlaps with the spectrum of absorption of the fluorescent dye used as a marker, the Alexa Fluor® with a maximum absorption around 546 nm. However, other dye / quantum dot pairs (or PL in general) that met the overlapping condition of absorption / emission spectra could also be used.

The cells with structures marked with said fluorescent dye were deposited in the bottom of a micro-fluidic chamber manufactured from two glass coverslips. Next, the PCs were dispersed in a calcium-free saline phosphate buffer (PBS) at pH 7.4 and injected into the micro-fluidic chamber. The PCs in the micro-fluidic chamber were both scattered as individual quantum dots and forming aggregates.

Optical tweezers (optical trap) formed by a continuous and focused single mode 845 nm diode laser operated at a power such that 150 mW reach the optical trap to trap, excite and displace both individual and aggregate PCs were used.

One such aggregate of PCs was trapped, excited and displaced with the optical tweezers described by the surroundings of the marked cell so that it excited different luminescent markers that in turn emitted a luminescent signal. Each of the radiative transfers of said process was as follows: the excitation of said aggregate of quantum dots was produced by a process of absorption of two photons in the near infrared, 2Y _NIR from the continuous and focused single-mode laser of the described optical clamp previously (emission at 845 nm). This two photon excitation process is that the quantum or aggregate of quantum dots is capable of absorbing two low energy photons resulting in the emission of a photon of greater energy than any of the photons initially absorbed. After the absorption of two 845 nm photons, the aggregate of PCs emitted more energetic photons in the visible spectrum, yvis _, at about 540 nm. These photons in turn excited the Alexa Fluor® 546 marker which, as we have already mentioned, has a maximum in its absorption spectrum around 546 nm. Said excited marker in turn spontaneously emitted a photon in the visible spectrum, around 573 nm, and yvis-, which was detected as a light signal by means of a spectrometer (Ocean Optics USB2000 +). When moving these points quantum trapped and excited to a new point, and the described radiative transfer repeated, luminescent signal was collected from other marked areas of the cell membrane.

Figure 1 presents a (a) scheme of a chain excitation or radiative transfer system in which a quantum dot is excited by a process of absorbing two photons in the near infrared, 2Y _NIR , after which it emits a photon in the visible spectrum, yvis _, which in turn excites a fluorescent marker (a dye molecule) that in turn emits a photon in the visible spectrum at a different wavelength from the previous one, yvis- _, which will be detected as a signal light. Figure 1b shows a graph of emission intensity / absorption versus wavelength (nm) showing the overlap between the emission spectrum of colloidal PCs (solid line) and the absorption spectrum of a dye (dashed line).

Figure 2 shows the experimental scheme of the method of detection of marked structures in which an aggregate of PCs is trapped by means of optical tweezers near the surface of a marked cell. The figure is not drawn to scale.

The experimental system used for the experimental embodiment illustrated in Example 1 modified the position of the microfluidic chamber with respect to the optical tweezers so that the aggregates of PCs could be placed in different relative positions with respect to the cell. In this way, aggregates of trapped and excited PCs were used by means of the optical clamp to excite various marked regions of the cell. Figure 3 shows transillumination images obtained by a camera (MTV-1802CB, DBS) coupled to an optical microscope (Zeiss Axiovert 135M) and emission spectra collected by a spectrometer. An aggregate of optically trapped and excited PCs was located at different distances (ad) from the surface of a cell marked with Alexa Fluor® 546. As can be seen in said figure, when the aggregate of PCs trapped and excited by an optical clamp is was approximately 10 microns from the labeled cell membrane, the emission spectrum collected corresponded to that of said aggregate whose maximum is centered at 540 nm (Figure 3a). It was observed that, as the aggregate of PCs moved to positions closer to the cell, the intensity of its emission spectrum was decreasing. When the aggregate of trapped and excited PCs was located at a submicron distance, a spectrum corresponding to the emission of the aggregate of PCs whose maximum is centered at 540 nm was obtained together with an emission of the luminescent membrane markers of Alexa Fluor® 546 with a maximum centered around 573 nm (Figure 3c). By placing said aggregate of PCs trapped and excited on the cell (although not in contact with said cell since they would remain fixed), that is, by forming the trap and collecting light through the cell, a spectrum corresponding to the emission of cell markers (Figure 3d).

This experiment demonstrated the possibility of optically exciting fluorophores present in a cell, that is, marked structures, by issuing an aggregate of trapped and excited PCs by means of optical tweezers. It also demonstrated the ability to solve based on the location of the trapped PCs with respect to the marked structure (in this case, the cell). Finally, it was also demonstrated that the method of detection of marked structures is selective to the detection of said marked structures. Also note that the markers do not emit as a result of their interaction with the optical clamp used.

Example 2: Detection of scattered markers in an aqueous medium.

In the present example, the detection of luminescent markers dispersed in an aqueous medium was studied. As a luminescent marker, tetramethyl rhodamine (TRUC) was used, a fluorescent dye with an absorption spectrum with a maximum around 557 nm and an emission spectrum with a maximum around 576 nm. Tetra-methyl rhodamine (TRUC) was dissolved in water to a concentration of 2.7 nM and said aqueous solution was injected into a micro-fluidic chamber.

Later, PCs (coated with silicon oxide (S1O2)) described in Example 1 were injected into said micro-fluidic chamber giving rise to individual and aggregated PCs in the aqueous medium. An aggregate of PCs was trapped and excited inside said medium by means of an optical clamp (see figure 4 (a)). Through movements of the icrofluidic chamber, the position of the aggregate of trapped and excited PCs with respect to the surface of said microfluidic chamber was modified. That is, said aggregate of trapped and excited quantum dots was located at different depths (variable distance L) from the surface of the chamber and the emission spectrum from the trap region was studied. To do this, the luminescence or fluorescence from the sample was collected through the trapping target and sent to a spectrometer. Figure 4 (b) shows several emission spectra measured by trapping and exciting said aggregate of PCs at various distances L (depths) from the surface of the microfluidic chamber that serves as a container for the TRUC solution.

It was observed that the signal from the PCs is lost as L increases (Figure 4 (b)), that is, as the PCs are excited more deeply within the solution, while the emission corresponding to the TRUC increases. This is due to the fact that in the process of radiative transfer, when increasing L, there is a greater probability that the photons emitted by the aggregate of confined and excited PCs are absorbed by a TRUC molecule, thus reaching less photons corresponding to said emission of the PCs added to the detector. It should be noted that it only produces TRUC emission when there are excited PCs in the medium. This experiment would therefore demonstrate that the excitation of the marker of the present invention takes place through a radiative transfer process.

Example 3: Effect of the overlap of the emission spectrum of the PCs and the absorption spectrum of the fluorescent marker.

In the present example, the effect of overlapping the emission spectrum of the PCs used and the absorption spectrum of the fluorescent marker was studied.

In the present example, as a luminescent marker, rhodamine 6G (Ro-6G according to its acronym in English) was used, a fluorescent dye with an absorption spectrum with a maximum around 530 nm and an emission spectrum with a maximum around 566 nm Ro-6G was dissolved in ethanol to reach a concentration of 2 mM. Said solution was injected into a microfluidic chamber. On the other hand, two types of quantum dots were used that were referred to as quantum dots of type 1 and type 2. Type 1: quantum dots encapsulated in silica with a maximum emission at 527 nm similar to those described in the Example 1. Type 2: quantum dots with a maximum emission at 580 nm were synthesized and encapsulated on silica following the method described in Acebrón M. et al. [M. Acebrón, JF Galisteo-López, D. Granados, J. López-Ogalla, JM Gallego, R. Otero, C. López, and BH Juárez, “Protective Ligand Shells for Luminescent Si0 ₂ -Coated Alloyed Semiconductor Nanocrystals,” ACS Appl. Mater. Interfaces, p. 150319103751001, 2015.].

Therefore, the emission spectrum of type 1 quantum dots overlapped with the absorption spectrum of the Ro-6G dye and while that of type 2 did not. Figure 5a shows a diagram of the overlap between the emission spectrum of two colloidal PCs of type 1 and type 2 (solid lines 1 and 2) and the absorption spectrum of a dye (broken line), said overlapping is represented as the shaded area below the curve.

Different emission spectra were measured for a sample comprising a Ro-6G solution in which an aggregate of type 1 PCs is trapped and excited (b) at different depths of the microfluidic chamber surface (distances L) such and as shown in Figure 5b. The same measurements were repeated for an aggregate of type 2 PCs shown in Figure 5c. The emission spectra obtained showed that when moving an aggregate of type 1 PCs a distance L in the transverse direction of the microfluidic chamber, a change in the emission spectrum from the same was observed (Figure 5b), as the distance increased L, decreases the signal corresponding to the emission spectrum of the aggregate of type 1 PCs in favor of the emission intensity of the Ro-6G (Figure 5b). However, this was not observed when handling the emitting PCs at 580 nm (Fig 5c) since, in this case, the light coming from the PCs leaves the microfluidic chamber without being absorbed by the Ro-6G, so that the emission spectrum does not change when L. is increased. In this way, the importance of overlapping the emission spectrum of the quantum dots used with the absorption spectrum of the dye or luminescent marker used to detect marked structures was demonstrated.

Example 4: Devices used.

Figure 6 shows a schematic of an experimental device. The device used to make examples 1-3 of the present invention was formed by two lasers (L1 and L2) which are combined by a beam splitter cube (PBS) and directed by means of mirrors (M1 and M2) inwards of a fluorescence microscope. The combined laser is reflected in a dichroic mirror (DM) and focused through a microscope objective (O) on the sample (SH). A lamp (WL), a grid system (l & S) and a condenser (C) make up the lighting system of the experiment. The sample under study was placed inside a microfluidic chamber manufactured from two glass coverslips that, in addition, could be moved three-dimensionally using micrometric screws.

The optical trap was implemented in the optical microscope by combining two continuous and focused single-mode infrared diode laser sources with a wavelength of 845 nm (L1 and L2) driven at a power such that 150 optical waves reach the optical trap. mW The objective of the microscope with which the laser is focused is high numerical aperture (NA = 1.2), immersion in water and corrected to infinity. The trap is formed at the focal point of the target, whose working distance is 0.7 mm. The target also picked up the luminescent emission in the trap area. The fluorescence from the sample, after passing through the dichroic mirror, was filtered through a low-pass spectral filter at 750 nm (F) and selectively sent to a spectrometer (S, Ocean Optics USB2000 +) or to a video camera (MTV-1802CB , DBS) (VC) using various optical elements (FL, focusing lens; M3, mirror; P, prism; RL1 and RL2, retransmission lenses). An integration time of 5 s was used for each spectrometer measurement.

The experiment was visualized in real time and through images obtained by trans-lighting (Figure 3), for which there is a 100 halogen lamp W and a low numerical aperture capacitor (NA = 0.3). Lamp light is blocked during spectrum acquisition.

Once the nature of the present invention has been sufficiently described, as well as a way of putting it into practice, it only remains to be added that, as a whole and its component parts, it is possible to introduce changes in form, materials and arrangement as long as said alterations are not substantially vary said invention.

Claims

1. A method of detection of marked structures comprising the steps of: i) providing:

iv) exciting the luminescent marker of stage (i) by the radiation emitted by said at least one excited and confined PL of stage (ii) or (iii), giving rise to the excited luminescent marker; where said excited luminescent marker emits a luminescent signal.

2. The detection method according to claim 1, wherein said luminescent marker is selected from fluorophores, chromophores and combinations of the foregoing.

3. The detection method according to any one of claims 1 or 2, wherein said at least one PL is encapsulated in a matrix.

4. The detection method according to any of claims 1-3, wherein said at least one PL is functionalized.

5. The detection method according to any of claims 1-4, wherein said at least one PL is at least one quantum dot (PC).

6. The detection method according to any of claims 1-5, wherein the focused laser of step (i) has an emission in a range of wavelengths between 650-950 nm that excites said at least one PL .

7. The detection method according to any of claims 1-6, wherein the excitation of the at least one PL of step (ii) is produced by a two photon absorption mechanism.

8. The detection method according to any of claims 1-7, wherein steps (iii) and (iv) are repeated at least once.

9. The detection method according to any of claims 1-8, wherein said at least one confined and excited PL of step (ii) emits luminescence; preferably radiation in a wavelength range between 400 and 650 nm.

10. The detection method according to any of claims 1-9, wherein the signal produced by the excited luminescent marker of step (iv) is a luminescent emission; preferably fluorescent in the range between 400 and 700 nm.

11. The detection method according to any of claims 1-10, wherein the luminescent signal of step (iv) is detected by a spectrometer.

12. The detection method according to any of claims 1-11, wherein said marked structure of step (i) is present in biological and non-biological specimens.

13. The detection method according to any of claims 1-12, wherein said method is applied in biological samples.

14. The detection method according to claim 13, wherein said method is applied to map cell membranes, biosensing membrane proteins or Monitor electrical signals.

15. Use of a luminescent marker in the method of detection of marked structures according to any of claims 1-14.

16. Use of a luminescent particle (PL) in the method of detection of marked structures according to any of claims 1-14.

17. Use of an optical clamp in the method of detection of marked structures according to any of claims 1-14.

18. Apparatus designed to carry out the method of detection of marked structures according to any of claims 1-14 comprising:

i) means for trapping and rotating the marked structure of step (i);

ii) the optical clip of step (i) of the method of detection of marked structures according to any of claims 1-14; Y

iii) a signal detector of step (iv) of the method of detection of marked structures according to any of claims 1-14.