ES2329963B1 - PROCEDURE FOR LIGHTING AND DATA ACQUISITION FOR OPTICAL TOMOGRAPHY AND SYSTEM FOR APPLICATION. - Google Patents

Abstract

Procedure for lighting and acquisition of data for optical tomography and system for its application.

The invention is based on the use of a double scanning system in which two optical systems are used decoupled so that one conducts the light that serves to illuminate or excite fluorophores inside the sample by generating a set of point sources on the surface and the other contains the light emitted by the object - well be light from diffusion or scattering processes or light from fluorescence - for another set of points that act as emitters also located above the surface. Both systems optics dynamically fit the topography of the interface to maintain lighting and registration conditions for all points on the interface. For this it is used well an auxiliary point source, either the source itself lighting / excitation together with an auxiliary spot photodetector additional or the previous modifying point to point systems optics to maximize the recorded intensity.

Description

Procedure for lighting and acquisition of data for optical tomography and system for its application.

Object of the invention

The present invention relates to a system of lighting and data acquisition, specially designed for optical tomography

The object of the invention is to provide a Improved and non-invasive practical implementation of the technique of optical tomography with respect to previous systems.

The invention falls within the scope of medical application technologies and research in biomedicine

Background of the invention

There are two modalities of optical tomography in highly diffusing media (or high scattering ): diffusion optical tomography and fluorescence optical tomography. The first is based on the use of radiation corresponding to optical wavelengths for three-dimensional localization and quantification of absorption or diffusion regions in biological tissues. The fluorescence optical tomography modality also uses light but, in this case, in order to locate three-dimensionally and quantify distributions of fluorescent molecules (fluorophors or fluorochromes) in biological tissues.

Compared to other tomography systems, the use of electromagnetic radiation at frequencies Optics does not produce the harmful effects associated with the use of ionizing radiation, such as that used in other systems of tomography - for example, x-rays in CT - and has been proposed as Breast cancer screening or monitoring technique Constant cerebral hemodynamics in newborns.

Fluorescence optical tomography, in addition to the previous advantage, allows three-dimensional monitoring of biological processes related to gene expression, presence and activation of molecules or nanoparticles, drug monitoring, etc. even in laboratory animals " in vivo ". There is intense research in the improvement of the capabilities of fluorescent markers to reduce their toxicity, functionalization, widening of the spectral frequencies of excitation / emission, transport, etc. In the short term, the appearance of harmless fluorescent markers that allow this technique to be used in various human applications is predictable.

Despite the advantages indicated, the use of light also has drawbacks associated with the characteristics of the propagation of this type of radiation in the tissues that make it difficult to extract information about the environment. The propagation, unlike what happens in transparent media such as air or liquids, is characterized by the absorption and existence of numerous diffusion processes ( or scattering ) causing the photon trajectory to change direction in many ways randomized until emerging from the sample where they can be detected. In these media, the propagation of light is physically modeled using diffusion equations; unlike in transparent media where deterministic models are used using the principles of wave or geometric optics.

Very schematically, the practical implementation of optical tomography techniques involves the illumination of tissue and diffused light analysis. Both the source of lighting as the light analysis device should be, if the system is non-invasive, necessarily external, which forces to keep in mind that the objects of interest (for example, a biological sample) are constituted by means, in general, highly diffusers (tissues) surrounded by non-diffusing regions (the air or fluids), that is, the existence of two regimes of Spread in the problem. Without going into details, this fact requires registering and including the interface surface in the physical model used to interpret the data; model which justifies, in turn, the computational method of reconstruction tomographic

The scheme of the data acquisition process consists of illuminating a point on the interface surface, using pulsed or continuous light, and measure the intensity of the light that emerges at another point with a photodetector. Repeating this operation for different points, both lighting and record, a data matrix is built that is used in mathematical tomographic reconstruction algorithms of the regions absorption, in the case of diffusion optical tomography, or distribution of fluorescent emitters, in optical tomography of fluorescence.

Traditionally, the registration scheme of previous data, and the solution of the problem of regime change propagation, has been implemented using optical fibers in interface contact at one end and alternately coupled to light emitters or photodetectors on the other.

The use of optical fibers in contact It has several drawbacks. On the one hand, you have to couple and keep the extremes stable during the measurement procedure of optical fibers on the surface, for example, in contact with the skin. Also, the density of fibers that can be packaged to function as emitters or detectors, it imposes a limit on the density of lighting and registration points compromising the density of the data matrix and, as a consequence, the resolution of the tomography system. Finally, it is necessary to use, prior  or after the placement of the fibers, a method independent to determine the surface topography of contact that can incorporate errors if there are discrepancies between this measure and the surface situation when they were obtained Intensity data

Recently, presented as a breakthrough in fluorescence optical tomography, systems have been proposed experimental and contactless methods, that is, without requiring couple optical fibers to the fabric. In these systems, it is included, in the physical model, the propagation in air of the light emitted by a object and a CCD camera is used in air; but nevertheless, it is still necessary to keep in touch the sources and determine surface topography with an additional system of photogrammetry and establish and register control points for Secure the fit.

Description of the invention

The lighting and data acquisition system for optical tomography that the invention proposes, solves, in a way fully satisfactory, the problem described above in the different aspects commented. In particular: simplify the procedure, does not compromise data density and integrates the registration of the surface topography with the system of lighting or registration.

For this, the invention is based on the use of a double scanning system in which they are used two decoupled light beams replacing the optical fibers mentioned above, so that, one is used to illuminate the shows in a set of points on the interface surface, for example, air-tissue; and the other is formed by the light emitted from another set of points also located above the surface. The light contained in this last beam for each point emitter is analyzed with a photodetector. Being stored in memory of a computer, these measures are subsequently used to tomographic reconstruction

To generate light sources on the surface of interface, homogeneous and punctual (of small extension), the beam lighting should focus on different points of the surface.

In order that the photodetector signal is corresponds to the light emitted from a specific area - of extension corresponding to the image that the optical recording system generates the effective (or sensitive) area of the photodetector - on the interface surface, this optical system must be adjusted to that the conjugate image plane of the sensitive surface of the photodetector through the system, match the surface of interface at that location or, equivalently, the interface surface is focused on the sensitive surface of the photodetector for each point.

The focus adjustment, both in the lighting beam and in the beam corresponding to the optical recording system, will depend on how the transport or relay of the electromagnetic field is implemented from the source to the surface and from the surface to the detector. It can be done, for example, by moving a lens on a translation unit, controlled change in the length of the optical system, increasing distance between the optical system and the surface or any other opto-mechanical or opto-electronic means of phase change in the beams.

In the case of using an optical relay system with lenses and, within the different possibilities that this option admits, using the displacement of one of them as an adjustment method, a specific source is involved in the system that is recommended - which may be constituted by a laser, a relatively high numerical aperture lens, and an opaque pinhole or mask with a small aperture- through which the interface surface is illuminated using a pair of lenses interposed between both elements, so that, by means of a divider beam between the two previous lenses and an additional lens that collects the light returned by the tissue (or, equivalently, emitted by the interface surface) and reflected in the splitter, is recorded by a photodetector with active area such that it results intrinsically punctual or that it is punctual by virtue of the use of an additional pinhole .

This scheme serves to build a system of spot lighting on the interface. By varying the distance between the lens closest to the surface and the latter, different intensity values are obtained in the detector, the maximum intensity corresponding to the best focus position of the point source on the interface or, equivalently, the better focus of the surface on the spot photodetector. Setting this distance, the active area of the detector is conjugated optically with the surface, which will be illuminated with a fountain of light that is replica of the point source modified by the diffraction of the optical system. The photodetector has in this case an auxiliary character since its use is limited to the system setting optical lighting.

The optical recording system that is recommended is made up of components equivalent to the previous ones, including a point lighting source. The above procedure for finding the best focus also applies in this case. By moving the lens closest to the interface, you will find, at any given time, a position that will produce the maximum intensity that will correspond to the situation in which the optical recording system is focused. By setting this position and turning off the source, the photodetector will measure the intensity corresponding to the flow of light emitted by the object - generated by a lighting source in a different position on the interface surface generated by the lighting beam - through an area in the extension interface surface corresponding to the equivalent area, in accordance with the increases in the system, of the pinhole opening in the interface plane with the limitation in space frequency acquisition imposed by the numerical opening of the system. In this case, the point source has an auxiliary nature since its use is limited to the adjustment of the optical registration system.

It should be taken into account that the accuracy in the parameter setting of optical conjugation systems -o what is equivalent, the accuracy in determining the interface surface- depends on the size of the pinhole and the numerical opening In the practical implementation of the method that the proposed invention should consider this fact and take into account that high accuracy in topography can compromise, depending on the specific application, the flow detectability that crosses the interface if the same point detector is used and optical system for both tasks: optics adjustment by light reflected at the interface and analysis of the flow transmitted to through the interface. Given this, it is recommended as alternative use two point photodetectors with different sensitive areas in the registration beam, an auxiliary dedicated to adjustment of the optical parameters of conjugation with the interface and another to the detection of flow transmitted through it. At lighting system, if necessary, two sources of different extension: a specific auxiliary dedicated to adjustment of the optical parameters of conjugation and another of different extension to form an effective lighting source (for the fluorescence excitation or as a source of diffused light) on  the interface

The above-mentioned method of maximizing the flow of energy that passes through a pinhole to adjust the illumination beam and register locally with the interface surface is valid for other alternative focus variation systems.

So much to get to focus the beam of lighting such as the optical system (or beam) for two sets of points, not necessarily equal, on the surface, the previous systems are complemented by a generic scanning mechanism; for example, by entering in the beam Tilt angles with moving mirrors. Data density achievable will depend on the angular resolution of the systems introduction of inclination and diffraction.

The incorporation of the previous mechanism of Sweep to focus adjustment system, allows topography it is codified in the adjustable parameter - in the previous case, the lens position - which can be stored in the memory of a computer and is congruent with the lighting and registration of information of the flow of light emitted by the surface of interface.

In the mode of reflection, the invention proposes also the simplification of the system through the possibility of combination of optical lighting and registration systems in only one, after the introduction in each of them of the necessary focus corrections associated with their respective coordinates over the interface by, for example, a divisor of beam or a dichroic mirror.

The system can be further simplified from Several ways. A first is to avoid using the auxiliary source and limit the focus setting for a set of points on the interface only of the lighting beam (or avoid using a auxiliary photodetector limiting the focus setting for a set of points on the interface only of the register beam incorporating an auxiliary source). The topographic information of the interface surface obtained in that process can be used to adjust the optical recording system (or lighting) to those same points or others, extrapolating over the values obtained. This implementation entails the perfect characterization of the entire system which is recognized difficult in practice.

A second possible simplification, taking into note that the referred photodetectors are not used in any case simultaneously, it may consist of using only one, incorporating the necessary optical components to carry the light emitted from points on the surface to a photodetector common while the beams are properly sealed.

The previous option consisting of using only the illumination beam (or the record one) to find the focus parameters, it is not possible if the tomography system operates in transmission; the auxiliary elements that serve to adjust the optical systems to the surface must necessarily decouple and duplicate by accessing each one of the surfaces of entry and exit of the light. As before, and since the two photodetectors are not used simultaneously, could be reduced to one in practice in the two modalities of diffusion and fluorescence.

The invention, regarding the procedure and lighting method is concerned, it is valid even if it were it is necessary to modulate lighting sources over time, such as occurs in the generation of photon density waves in diffusion optical tomography.

Also, the invention is valid, as regards procedure and method of adjusting the optical recording system, regardless of the type of light flow analysis transmitted through the interface surface, either the measure of total intensity or other characteristics such as variations in intensity over time, tests that involve different wavelengths, angular spectrum analysis etc.

Description of the drawings

To complement the description that is being performing and in order to help a better understanding of the characteristics of the invention, according to a preferred example of practical realization of it, is accompanied, as part member of that description, a set of drawings where, with illustrative and non-limiting nature, what has been represented next:

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Figure 1.- Schematically shows the general principle of the lighting and registration system for Optical tomography proposed.

Figure 2 shows, schematically, a valid system such as lighting or data acquisition system for optical tomography using a relay configuration using lenses, performed in accordance with the object of the present invention, representing a first example of implementation practice of the proposed method of adjusting any of the two systems to the interface surface.

Figure 3.- Shows a scanning subsystem incorporated into the system of the previous figure and illustrating a second principle of the procedure for both the optical system of lighting as registration.

Figure 4.- Shows a possible implementation practice of the invention applied in optical tomography mode reflection where the modules are explicitly shown lighting and registration.

Figure 5.- Shows a possible implementation practice of the invention applied in optical tomography mode transmission.

Figure 6.- Shows, finally, a scheme detailed of a possible practical implementation of the invention specially designed for fluorescence optical tomography in reflection mode using moving mirrors in planes conjugates

Preferred Embodiment of the Invention

In view of the figures outlined you can observe how the process of the invention is based on the use of a double scanning system in which they are used two beams of light decoupled so that one is used to illuminate the sample at a set of points on the surface, and the other, collects the light emitted by the object for another set of emitting points also located on the surface.

As shown in Figure 1, to achieve different sources of light on the interface, homogeneous and of small extent, a point source (S), remote and external, is conjugated (or, equivalently, transported) with a optical system, (SO1), with different points of the interface surface, (I), this being generic and of unknown topography a priori , sequentially, modifying the optical system (SO1), and actively, once automating the process of Adjust with a computer. Similarly, in order to ensure that the signal from a point photodetector (FD), remote and external to the interface surface, corresponds to the flow of light emitted through multiple small areas over the interface (I), the Sensitive area of the detector is optically sequentially and actively conjugated to different points on the surface using the optical system (SO2). For the adjustment of the optical systems, the use of an auxiliary point detector (FD ') is also recommended for the adjustment of the system (SO1) using the light reflected back at the interface; and the use of an auxiliary point source (S ') for system adjustment (SO2) using the detector (FD) and the light reflected back from the auxiliary source (S'). A system is also provided, either for the displacement of the lighting and registration subsystems, or for the change of the optical axes, for the point-to-point interface scan. The invention includes the possibility that the sensitive area of the photodetector (FD) is adequate to obtain some precision in the function of adjusting the optical system (SO2) using reflected light but not for the function of analyzing the light passing through the interface If the flow is too weak. In that case, it is foreseen, either the dynamic change of the sensitive area of the photodetector (FD) according to each function, or the possible use of a second photodetector (FD '') with different sensory area also conjugated with the interface through the optical system (SO2). Similarly, the light source (S) may be of suitable extension for the optical system adjustment function (SO1) but inadequate for the function of forming light sources of a certain extent on the interface. In this case, it is foreseen, either the dynamic change of the source extension (S) for each function, or the possible use of a second source (S '') of different extension also conjugated with the interface through (SO1 ).

For both the optical lighting system, (SO2), and the recording system, (SO2), there may be different designs of conjugation or relay ( relay ) of the electric field of light and different methods of adjusting these systems for arbitrary points of an interface surface of generic and unknown topography a priori . In the event that the optical conjugation systems are based on the use of lenses in a relay configuration, there are different possibilities of active adjustment: by moving one of the lenses on a translation unit, through controlled change in the length of the optical system through the translation of mirrors or through the use of any other optoelectronic device for the introduction of a controlled phase change.

As an example, in the particular case that the active adjustment is carried out by moving a lens, the positions of the translation unit are found using a system such as the one schematically represented in Figure 2, in which a point source is placed. at point (S1), (in the figure constructed using a laser (L), a high numerical aperture lens, (L1), and a pinhole (opaque mask with a small aperture) in the plane (P1)). This source will produce a maximum intensity in the plane (P2) using the optical system consisting of the lenses (L2) and (L3); the latter with axial displacement capability. The light reflected by the interface is collected again with the lens (L3), is subsequently reflected in a beam splitter, (DH), crosses an additional lens (L4) and a pinhole located in the plane (P3). A photodetector (FD) with a certain effective (sensitive) area is placed behind the pinhole, collecting the light that is capable of passing through it. The pinhole- photodetector system fulfills the function of a point detector referred to previously, constituting the pinhole area, the effective area of the photodetector. With this configuration, only in the case that the interface surface, (I), coincides with the plane (P2), will a maximum be obtained in the signal generated by the photodetector. Given any distance between the plane (P2) and the surface (I), a certain intensity value will be obtained in the photodetector (FD). The lens adjustment procedure (L3) consists in its axial displacement until the maximum intensity value in the photodetector (FD) is obtained. This position of the lens (L3) allows an image of the point source to be formed in (S 1) in (S'1) being (S 1 ') on the interface surface (I). Keeping the lens (L3) in that position, if the laser (Lem) is turned off and there is light flow through the interface (I) due to the existence of an alternative light source, the intensity in the photodetector (FD) corresponds to the flow of light emitted by the object through a small area on the extension interface surface corresponding to the equivalent area (according to the optical system increases) of the pinhole opening in the plane (P2) on the interface surface (I), with the limitation of spatial frequencies imposed by the numerical aperture of the optical system.

The procedure described in the section above, based on maximizing the signal in a point detector, is valid for other optical conjugation adjustment options of photodetectors and sources with the system interface surface of lighting or registration.

Continuing with the example of optical relay system or optical conjugation by means of lenses, moving one of them, both in order to focus the illumination beam and the recording system for different points on the surface, the system of Figure 2 is complemented according to described in Figure 3. The extension consists in adding a generic mechanism, (SC), which introduces a controlled angle of inclination into the beam so that different points ((S1 '), (S2''), etc. can be illuminated. successively on the interface Figure 3 shows the process of adjusting the position of the translation unit for different points on the surface, following the strategy defined above to locate the lens positions (L3), in accordance with the surface topography The values obtained for lens displacement (L3) can be stored in the memory of a computer associated with those of system angle variation ( SC) and encode the surface topography, which, as indicated above, is necessary for tomographic reconstruction. Once the lens (L3) is set for a position on the interface, for example (S1 '), if the laser is turned off (Lem), and there are alternative light sources, the photodetector signal (FD) will be related to the corresponding intensity to the flow emitted through the area on the extension interface surface (I) corresponding to the image of the active area of the photodetector ( pinhole area) over the interface centered at the point (Sr). This process is repeated for other points (S2 ') by changing the angle introduced by the system (SC).

The complete lighting and registration system is represented in Figure 4 and consists, in essence, of duplicating the system of Figure 3. The lighting system originates from the laser (Lex) that generates a point source at (S2); the system (SC2) introduces an angle such that the image of the source is generated at the point (S2) in the position (S2 '). The signal produced by the photodetector (FD2) serves to find the correct displacement of the lens (L9) so that (S2 ') is placed on the interface (I). On the other hand, the laser (Lem) produces a point source at (S1) which, for a given angle introduced by (SC1), will be optically transported to the point (S1 '). The photodetector (FD1) is used to adjust the lens (L4) so that (S1 ') matches the interface (I). While Lex remains on and SC 1 and SC2 do not change their status, the laser (Lem) is turned off. In this situation, coinciding with that shown in Figure 4, (S2 '), it acts as a source of excitation / point illumination on the interface, registering the photodetector (FD1), the emerging light of the point (S1') on the interface ( renamed (S3) in figure 4) conjugated with the point (S3 ') on the pinhole in the plane (P2). The process: change of state in (SC1), (SC2), lasers (Lex) and (Lem), on; lens adjustment (L9) and (L4) using the signals provided by (FD1) and (FD2); laser (Lex) on and laser (Lem) off; intensity recording measured by the photodetector (FD1); it is repeated for different points on the surface (I), building the data set that, together with the topographic information collected in the same process, are used to solve the tomographic reconstruction problem.

From the establishment of a scheme of distribution of sources and detectors in the transverse plane, in where data density is mainly conditioned by the angular resolution of the systems (SC1) and (SC2), all Previous processes can be controlled automatically with a computer in such a way that both the lighting system and the one of registry, adapts of active form to the surface of interface whatever this is.

If the system destination is the mode of diffusion optical tomography, the beam splitter would be used, (DH). In fluorescence optical tomography, it can be used instead of the beam splitter, a dichroic mirror, (ED), and / or a filter spectral, (F), to block the arrival at the light detector with the excitation frequency generated by the laser (Lex). In this last  case, the laser frequency (Lem) must be such that the light pass through the dichroic mirror (ED) and filter (F). One option alternative is to use a single laser, (Lex), dividing the beam into two parts using a new beam splitter, so that one of the beams generate sources in (S1) and the other in (S2). In diffusion optical tomography, only one laser is necessary since only one wavelength is at stake and the process of turning off the laser, indicated above, is replaced by the closure of a shutter; with a single laser, in optical tomography of fluorescence, it would be necessary to incorporate into the system Electromechanically ED and / or (F) components.

In principle, the use of the source generated with the components (Lem), (L1), (P1) and (L2) in Figure 4 can be completely removed using the source in (S2) to, in a preliminary scan generated with SC2 and proceeding with the method described of active adjustment of the L9 lens, determine, point a point, the topography of the surface with respect to a plane of reference. This information would be used in a second stage to adjust the lens (L3) for each flow record point emerging from the interface (I) corresponding to different angles entered by the system (SC2) corresponding to each point of lighting (S2 ') (it would be unnecessary then also the component (DH)). Although the system is simplified with this option, This strategy may have counterparts associated with the calibration of the systems (SC1) and (SC2) and the fact of dividing the process of acquisition in two parts: a preliminary, in which the area of interest at the interface with the excitation beam and it focus values are stored in the computer memory and, a second, in which the excitation and registration is carried out using the saved values, trusting that, in the time interval no inconsistencies in the data are introduced between the two stages if the stability of the object is not assured.

On the other hand, and with reference also to the Figure 4, taking into account that the photodetectors (FD1) and (FD2) they are not used in any case simultaneously, it is possible further simplify the system, incorporating the optical components necessary to carry the emitted light from points (S2 ') and (S3) to a common photodetector, sealing the beam coming from (S2 ') when light from (S3) is being registered.

If the system is used in transmission instead in reflection should be modified as outlined in figure 5. The systems that adjust the optical system to the surface must be doubled, accessing each one of the surfaces (I1) and (I2). In the case of optical tomography of fluorescence a spectral filter (F) is used to select the emission wavelength and block the excitation. In spite of which, in the figure, shows two lasers, (Lex) and (Lem), could be used only by dividing the beam into two parts, as already indicated above. In the same way the two detectors (subsystems: (L5-P2-D1) and (L10-P4-D2)) could be reduced to one, both in the case of optical tomography and tomography fluorescence optics

A more concrete example is that shown in Figure 6, in which the system is used in fluorescence tomography for the location and three-dimensional quantification of fluorochromes, (FLs), embedded in a diffuser object, (O), which can be a ex-vivo or in-vivo tissue. A laser with a wavelength corresponding to the fluorochrome excitation, (Lex), serves to generate a point light source in the plane (P1 '). The lens (L1 ') focuses the laser beam on a pinhole of adequate size to pass the maximum intensity and spatially filter the beam. The lenses (L2 ') and (L3') form an image of the point on the plane (P2 ') ((P1') and (P2 ') are conjugated planes). (GS1 ') is a galvanometric mobile flat mirror whose angle is electronically controlled and that introduces an angle in the horizontal plane into the beam. (L3 ') and (L4') combine the plane containing the axis of rotation of (GS1 ') with the axis of perpendicular rotation of another moving mirror of similar characteristics to the previous one, (GS2'), which deflects the beam perpendicularly to the drawing plane and insert a variable angle into the beam. The mirror (M1 ') brings the beam back to the horizontal plane and the lenses (L4') and (L5 ') conjugate the plane (P2') over (P3 '). The lens system (L6 ') and (L7') combine the plane (P3 ') over (P4'), the latter coinciding with the interface surface. The lens (L6 ') is mounted on a computer controlled motorized translation unit (TS). The mirror M2 and the dichroic mirror DM match the axis of L6 'with that of L7.

Some of the light is reflected in the interface following the path backwards, firstly reflected in the dichroic mirror (DM) and then, through the system, reflected in the beam splitter (DH '). The light continues after this reflection through the lens (L8 ') that combines the plane (P4) over the plane (P5') where a pinhole is placed. Behind, a photodetector collects, with the help of the L9 'lens, the intensity of the light that is capable of crossing the pinhole .

For each angle of the mirrors (GS1) and (GS2), corresponding to predefined transverse coordinates in the plane (P4), the translation unit moves longitudinally while measuring the intensity collected by the photodetector The longitudinal offset value, corresponding to the maximum intensity, it is stored in memory of the computer, associated with the value of the transverse coordinates. Equivalently, with this process the coordinates are defined of a position vector in space for a point on the surface.

Also, for a particular angle of the mirrors (GS1) and (GS2) - once the adjustment process of the position of the lens (L6 '), already having a point source of light on the surface for the excitation of fluorophors in the inside the middle - the moving mirrors (GS1 ') and (GS2') rotate a determined angle associated with transverse coordinates Preset The light from the laser (Lem) is used for lens adjustment (L6) by movement of the unit translation (TS). For this, the light reflected in the interface that now crosses the dichroic mirror (DM) and is detected with the photodetector (FD) after crossing the filter spectral (F). Then the laser (Lem) turns off and then proceeds to acquire and store on a computer the intensity data of the fluorescence light emerging from the surface - capable of crossing also (DM) and (F) - associating it with the excitation and emission coordinates on the surface.

The procedure, automatically controlled by a computer, repeated for different coordinates for the source of excitation (angles of GS1 'and GS2') and, for each of them, different coordinates of the emission point (angles of (GS1) and (GS2)) thus building the data matrix necessary to use a tomography reconstruction algorithm that will output the localization and quantification of fluorochromes in the medium.

Claims (11)

1. Procedure for lighting and data acquisition in optical tomography, characterized in that it has the ability to illuminate the interface surface, (I), which separates media of different optical characteristics, (M1) and (M2), by optical conjugation of a light source considered punctual, away from the interface and immersed in the first medium (S); and also because information is acquired from the flow of light that crosses the interface from the second medium, (M2), in the direction of the first, (M1), by means of a photodetector considered punctual, (FD), away from the interface, immersed in the first medium, optically conjugated with an area considered punctual on the interface surface; having provided automatic and active adjustment of the parameters of the optical system necessary to perform the optical conjugation dynamically with different points of the interface surface, both of the optical system corresponding to that of the light source, (SO1), as the optical system corresponding to the photodetector, (SO2); having planned for the aforementioned adjustment use an auxiliary point source, (S '), additional or not to the previous one, and an auxiliary point photodetector, (FD'), additional to the previous one or not, carrying out a search procedure by means of the Variation of the parameters of the optical conjugation systems, (SO1) and (SO2), between lighting sources, (S) and (S '), interface, (I), and photodetectors, (FD) and (FD') , in order to maximize the detected intensity of the light reflected in the interface (I); having also planned, carry out the above procedure for two sets of points, one of them being the one containing the lighting points of the interface and the other points at the interface of characterization of the flow of light through it; having planned for this the use of a double scanning system, which works by introducing, at the same time, changes in the optical lighting systems, (SO1), and registration, (SO2), independently, and acting simultaneously, of in such a way, that the first one allows generating point sources in different positions on the interface surface (I) and optically conjugated with the point external light source (S) and, optionally, static and immersed in the first medium and corresponding to the first set of points, and the second to obtain flow information, using an external point detector (FD), optionally static and immersed in the first medium, optically conjugated with different areas considered point coincident with the interface surface corresponding to the second set of points. 2. Lighting system and data acquisition for optical tomography, according to the procedure of claim 1 characterized in that, optionally, two point sources with different extension are used in the lighting system, dedicating one (S '') to the function of adjustment of the optical conjugation system (SO1) and the other (S) to the function of generating a point illumination source on the interface (I), or a single point source (S) capable of dynamically changing its extension according to each function 3. Lighting and data acquisition system for optical tomography, according to the procedure of claim 1 characterized in that, optionally, two point photodetectors with different sensory area are used in the registration system, one (FD '') being dedicated to the adjustment of the Optical conjugation system (SO2) and another (FD) to the analysis of the light transmitted through the interface (I), or a single point photodetector (FD) with the ability to dynamically change its sensitive area according to each function. 4. Illumination and data acquisition system for optical tomography, according to the procedure of claim 1, characterized in that, in the adjustment of the optical conjugation parameters, a point light source at the point (S1) participates as the core of the system. ), in the plane (P1), a pair of lenses (L2) and (L3), so that they generate a conjugate source with respect to the previous one at the point (S'1) in the plane (P2), disposing between lenses, (L2) and (L3), of a beam splitter (DH), which directs the reflected light towards a lens (L4), after which a point photodetector (FD) is placed in the plane (P3), locating after the lens (L4), forcing a lens position (L3) or, in general, a phase introduced into the light by this component, which produces the maximum value signal in the photodetector (FD) associated with the situation in which the source at point (S'1) coincides with the interface surface (I). 5. Lighting and data acquisition system for optical tomography, according to the method of claim 1, characterized by incorporating into the system a mechanism, (SC), provided with means capable of introducing controlled angles into the light so that it can look for the value of the necessary parameters to optically combine different points of the interface with the photodetector and the source in fixed positions in the space. 6. Lighting and data acquisition system for optical tomography, according to the method of claim 1, characterized in that, in reflection mode, in the optical system, or beam, lighting and in the optical system, or beam, recording , optionally, two generic and independent optical subsystems, (SC1) and (SC2), are used to introduce angles in the light to sweep different points of the interface surface and two generic and independent phase modification optical subsystems in each of the beams that allow to adjust the optical conjugation of sources and detectors to the topography of the interface surface, prior to the combination of the illumination and registration beams by means of a beam splitter (DH) or dichroic mirror (ED) in a single system optical optic, (L5), located afterwards - in the direction of the lighting beam - and prior to the interface (I). 7. Lighting and data acquisition system for optical tomography, according to claim 6, characterized in that, optionally, replacing the beam splitter (DH) or dichroic mirror (ED) near the front interface (I1), incorporates a lens ( L6) next to the posterior interface (I2) to which an optical system of similar characteristics is associated to that associated with the lens (L5). 8. Lighting and data acquisition system for optical tomography, according to the procedure of claim 1 and following, characterized in that the scanning and recording scanning systems consist of devices that introduce angles in planes conjugated with the plane corresponding to the pupil of input / output of the optical system that finally focuses the illumination light and first collects the light emitted by the interface surface, and because the focus adjustments for different points of the interface surface are made by modifying the optical systems between the planes where they introduce the angles and the entrance / exit pupil of the optical system that focuses the illumination light and first picks up the light emitted by the interface surface. 9. Lighting and data acquisition system for optical tomography, according to the procedure of claim 1 and following, characterized in that the scanning systems consist of mirrors with rotational capacity whose rotation axes are located in conjugated planes between them and also with the plane corresponding to the entrance or exit pupil of the optical system that focuses the illumination light and collects the light emitted by the interface surface. 10. Illumination and data acquisition system for optical tomography, according to the procedure of claim 1 and following, characterized in that the point sources and / or the point photodetectors consist of non-point sources and / or photodetectors modified by virtue of the use of lenses that focus the light on pinholes . 11. Procedure for the illumination and acquisition of data in optical tomography, according to the procedure of claim 1 and following, characterized in that a preliminary scan of the interface surface is performed using a point source and a point photodetector and the topographic information obtained on The surface through the search for the maximum intensity in the reflected light is used in a next stage to adjust the recording system point by point and, optionally, if it is timely, the lighting system.