WO2017114985A1 - Integral microscope, uses of same and integral microscopy system - Google Patents

Integral microscope, uses of same and integral microscopy system Download PDF

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Publication number
WO2017114985A1
WO2017114985A1 PCT/ES2016/000135 ES2016000135W WO2017114985A1 WO 2017114985 A1 WO2017114985 A1 WO 2017114985A1 ES 2016000135 W ES2016000135 W ES 2016000135W WO 2017114985 A1 WO2017114985 A1 WO 2017114985A1
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sample
micro
microscope
images
microscope according
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PCT/ES2016/000135
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Spanish (es)
French (fr)
Inventor
Manuel Martinez Corral
Genaro Saavedra Tortosa
Emilio Sanchez Ortiga
Ana Isabel LLAVADOR ANCHETA
Jorge SOLA PICABEA
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Universitat De València
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Publication of WO2017114985A1 publication Critical patent/WO2017114985A1/en

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements

Definitions

  • the present invention concerns, in general, in a first aspect, an integrated microscope !, comprising an ordered arrangement of micro-lenses through which images of a sample from different perspectives are captured, and more in particular to a microscope in which the micro-lenses are arranged in the pupil plane, or Fourier, of the microscope.
  • a second aspect of the invention concerns an integral microscopy system, which comprises the microscope of the first aspect and a system for processing the acquired images.
  • the invention also concerns a number of uses of the microscope of the first aspect for various applications.
  • Integral microscopy has as its main characteristic its ability to record 3D information from thick samples without the need to perform more than one single Shooting or shooting These systems are based on the simultaneous capture of a whole series of perspectives (vertical and horizontal) of the sample, which is achieved by placing an "array" or orderly arrangement of micro-lenses in front of the sensor (CCD or CMOS).
  • Fig. 1 a diagram of a conventional integral microscope ( ⁇ Mic) is shown, that is to say in its classical configuration, where the sample Re is arranged in the object plane Pe, and the microscope comprises an Oc objective with a corresponding diaphragm of aperture From and, outside the Oc lens, a Le tube lens is arranged so that its object focus coincides with the aperture diaphragm From.
  • ⁇ Mic integral microscope
  • the microscope comprises an Oc objective with a corresponding diaphragm of aperture From and, outside the Oc lens, a Le tube lens is arranged so that its object focus coincides with the aperture diaphragm From.
  • the sensor A set of micro-images containing a mues ⁇ reada version of! radiation map (the radiation map contains information on the energy, position and orientation of the light rays emitted by the sample).
  • the radiation map has 4 dimensions (2 spatial (r. Y) and two angular (, ⁇ ))
  • this document only shows, to simplify, a two-dimensional cut! ( ⁇ , ⁇ ) of it.
  • a schematic version of the radiation map captured with the scheme of Fig. 1 is shown in Fig. 2.
  • each column represents the pixels recorded by each micro-image, while if the information from the rows is extracted and grouped, different views of the sample can be generated from equidistant positions in the aperture diaphragm.
  • the present invention concerns an integral microscope comprising, in a manner known per se:
  • the arranged arrangement of micro-lenses is arranged in the pupillary plane defined by the opening diaphragm, that is to say in the piano of Fourier.
  • the information captured by a classic iMic working with an "array" of N micro-lenses and P pixels per micro-lens is the same (although ordered in a different way) than the one captured according to the microscope of the first aspect of the present invention, that is, placing P lenses on the pupillary plane, with N pspixels per lens.
  • the difference is that in the Fourier plane you can directly insert an "array" of micro-cameras (each micro-camera captures a different elementary image), which results in a very significant increase in resolution (in this case the total number of pixels is the result of multiplying the number of pixels of a micro-camera by the number of cameras).
  • the microscope of the first aspect is configured so that the elementary images acquired by said image acquisition means contain a sample version of the sample's radiation map.
  • each of said image sensors comprises N photosensitive elements, or active pspixels, so that the resolution obtained with the microscope is equal to N x P, where P is the number of micro-lenses.
  • the image acquisition means comprise an ordered arrangement (or "array") of image sensors, each of them arranged, according to said optical path, after one of the micro-lenses of the ordered micro arrangement -glasses.
  • the image acquisition means comprise a single image sensor (CCD, CMOS, etc.) comprising M photosensitive elements, or active psxels, grouped into a plurality of contiguous regions, each of which it comprises N pixels and is arranged, according to said optical path, after one of the micro-lenses of the ordered arrangement of rnicro.
  • each of said image sensors is part of a respective micro-camera without a lens.
  • said image sensor arrangement and said micro-lens arrangement are grouped together forming a micro-camera arrangement, where each of the micro-cameras comprises an image sensor and a micro-objective with a micro-lens.
  • said micro-camera arrangement comprises a support in which the micro-cameras are properly aligned, said being configured, sized and arranged support behind the opening diaphragm, so that the micro-tooth arrangement is inserted into the opening of the opening diaphragm that defines the pupil piano.
  • the microscope proposed by the first aspect of the invention can operate under ambient light, this is not the most common application or the one that offers the best results, so that, according to a more usual and efficient embodiment, the microscope of the The first aspect of the invention comprises lighting means that include at least one incoherent light source, configured and arranged to illuminate the sample.
  • the microscope of the present invention can also be used to acquire images of external samples to itself, but such use is also not the most common nor does it offer results as good as that related to acquiring images of an internal sample under the microscope.
  • the microscope of the first aspect of the invention comprises a sample holder.
  • the microscope of the invention comprises a support structure in which they are mounted, in the order defined by the aforementioned optical path: the lighting means, the sample holder, the optical system and the means of image acquisition.
  • the Optical system and the image acquisition means are mounted on the support structure in a displaced and guided manner in approximation / distance with respect to the support for the sample, to focus on the sample and / or different parts of it.
  • the microscope of the invention comprises a housing that houses said support structure with all the elements mounted therein, or that constitutes said support structure.
  • the microscope of the first aspect of the invention comprises, for one embodiment, an electronic system in connection with at least the means of image acquisition to control them to acquire images simultaneously in a single shot.
  • said electronic system is also connected to the lighting means to control them so that they emit incoherent light at least during said single shot of the image acquisition means.
  • the electronic system comprises internal processing means (ie, local) adapted to process the acquired images to perform at least one of the following tasks, based on the three-dimensional information of the sample contained in the acquired images;
  • the electronic system comprises means of communication, via cable or wireless (for example Bluetooth®), for sending the acquired images to an external (ie, remote) processing system adapted to process the images acquired to perform at least one of the following tasks, based on the three-dimensional information of the sample contained in the Images:
  • an external (ie, remote) processing system adapted to process the images acquired to perform at least one of the following tasks, based on the three-dimensional information of the sample contained in the Images:
  • the present invention also concerns, in a second aspect, an integral microscopy system, comprising: - the microscope according to the first aspect of the invention; Y
  • a processing system for example installed on a computer, "lablef, or I: Smartphone ! ) : adapted to process the images acquired by the microscope to perform at least one of the following tasks, based on the three-dimensional information of the ia Sample contained in the images:
  • the system proposed by the second aspect of the invention comprises at least one 3D printer communicated with the processing system to at least receive from the same profiling geometry information related to said depth map or three-dimensional matrix, and adapted to print a three-dimensional model of the sample based on the information received.
  • a third aspect of the present invention concerns a use of the microscope according to the pnmer aspect for hyo-mediein applications, including the screening of biological samples (potentially linked to some pathogen) in situ, analyzing the relief of the sample or a section In depth of the same, This can be very useful, for example, for the detection in situ of possible infections. Obviously, laboratory use for the same applications is also possible.
  • a fourth aspect of the invention concerns a use of the microscope according to the first aspect for profilometry applications, such as quality control of parts or components (such as microelectronic and semiconductor components, testing of micro-lenses and infraocular lenses) , and forensic science.
  • profilometry applications such as quality control of parts or components (such as microelectronic and semiconductor components, testing of micro-lenses and infraocular lenses)
  • forensic science e.g., forensic science.
  • the applications of profilometry of opaque microscopic samples in environments far from a laboratory are really advantageous. This can be very useful, for example, for the immediate verification of possible imperfections of An industrial part, the use of the microscope of the first aspect of the invention in laboratories is also contemplated, with e! same rigor as outside the laboratory
  • the data obtained from the perfiometric measurement can be used to build 3D models with a 3D printer.
  • Figure 2 shows a schematic version of the radiation map captured by the microscope of Figure 1.
  • Figure 3a shows, schematically, the microscope proposed by the first aspect of the invention, for an exemplary embodiment
  • Figure 3b shows a schematic version of the radiation map captured by the microscope of the first aspect of the present invention, according to the scheme of Figure 3;
  • Figure 4 shows, in its view (a) micro-images of a sample obtained with the iMic microscope of Figure 1, and view (b) an enlarged view of a few of said micro-images;
  • Figure 5 shows paths with different perspectives calculated for the iMIc from the micro-images of Figure 4;
  • Figure 8 shows two reconstructions of the sample at two depths, for the Mic of Figure 1, from the micro-images of Figure 4;
  • FIG 7 shows two views with different perspective captured directly with the microscope proposed by the first aspect of the invention, also called FiMic, according to the scheme of Figure 3a;
  • Figure 8 shows two reconstructions of the sample at two depths, for the FiMic, that is to say for a microscope proposed by the first aspect of the invention, according to the scheme of Figure 3a;
  • Figure 9 shows another embodiment of the microscope proposed by the first aspect of the present invention, according to a prototype illustrated schematically;
  • Figure 10 shows different distributions of micro-cameras on a support thereof, included in the microscope proposed by the first aspect of the invention, according to different embodiments. Detailed description of some embodiments
  • the microscope proposed by the first aspect of the present invention provides a new configuration or design in which the capture of the radiation map does not occur in the microscope image plane, but not in the papillary or Fourier piano.
  • This new design which the present inventors have called Fourier Integral Microscope (F ⁇ ⁇ c) presents in its final design, according to preferred embodiments, a simple and compact structure.
  • Figure 3a shows, schematically, the microscope proposed by the first aspect of the invention, for an exemplary embodiment, for which it comprises;
  • an objective O with an aperture diaphragm D configured and arranged to receive light from a sample R (located in the object plane Pe), according to a path OR; when said sample R is illuminated with incoherent light;
  • Figure 3b shows the radiation map obtained with the scheme of Figure 3a.
  • the information captured now is essentially Sa same as in the case of the conventional ⁇ Mic (see Figure 2), but with a much more compact architecture
  • the second, and not least, advantage of this second design is that the perspectives of the Sample (columns of the new radiation map) are captured directly and with a resolution that is no longer limited by the size of the micro-lenses, but by e! of the image sensor pixels. This design allows to reconstruct images of 3D samples with much better resolutions than those achieved so far in integral microscopy
  • Figures 7 and 8 show the images obtained with the F ⁇ M ⁇ c, that is, with the microscope of the present invention, according to the scheme of Figure 3a.
  • the sample used is the same as in the case of! Me, the region observed is different.
  • Figure 7 two perspective views of the sample are shown and in Figure 8 reconstructions corresponding to two depths are shown.
  • the distance between the furthest and the closest plane in the reconstructions of Figure 8 is also 18 microns.
  • FIG. 9 Another example of embodiment of! Is illustrated in Figure 9.
  • Microscope proposed by the first aspect of the present invention according to a prototype for which the microscope includes a support structure formed by two parts S1, S2 splitting each other according to a vertical direction (according to the position illustrated in Figure 9), of guided approach / distance way through a bar G, on which the part S 1 is mounted in a guided way through a stripper element 8 (manually or automatically operated)
  • a microscope objective 4 is mounted which includes an aperture diaphragm D : and an array or array of C micro-cameras, supported properly distributed on a Cs support, where each of the C micro-cameras includes an image sensor 3 of N active pixels (CCD, CMOS, etc.) and a lens 1 with a micro-lens 2 , the support S1 being mounted on the diaphragm D (According to the position illustrated in Figure 9) so that the two are arranged in the pupillary piano thereof.
  • a rechargeable battery 8 is also mounted for the power of a local electronic system (not shown) of the microscope.
  • the entire assembly illustrated in Figure 9 is housed inside a housing, in order to provide a compact device, similar to a compact camera, for use outside the laboratory. .
  • FIG 10 different distributions of micro-cameras C are shown on a support Cs thereof, included in the microscope proposed by the first aspect of the invention, according to different embodiments.
  • the distributions are adapted to the shape and size of the diaphragm D in which the micro-lenses 2 of the micro-cameras C must be inserted. Hexagonal distributions with 7 micro-cameras (view (a)) or 19 micro-cameras ( view (b)), as well as rectangular distributions with 12 micro-cameras (view (c)) and 24 micro-cameras (view (d)).

Abstract

The invention relates to a microscope comprising: an objective lens (O) with an aperture diaphragm (D), which are configured and arranged to receive light from a sample (R), according to an optical path, when the sample (R) is illuminated with incoherent light; an ordered arrangement of micro-lenses (2) disposed in the pupil plane defined by the aperture diaphragm (D); and image acquisition means configured and arranged to receive the light that passes through each of the micro-lenses (2), to acquire images of the sample (R) from different perspectives simultaneously. The system comprises the microscope of the invention and a system for processing the images acquired by same. The uses relate to biomedicinal uses and profilometric uses.

Description

Sector de la técnica La presente invención concierne, en general, en un primer aspecto, a un microscopio integra!, que comprende una disposición ordenada de micro-lentes a través de las cuales se captan imágenes de una muestra desde diferentes perspectivas, y más en particular a un microscopio en el que ias micro- lentes se disponen en el plano pupiíar, o de Fourier, del microscopio.  TECHNICAL FIELD The present invention concerns, in general, in a first aspect, an integrated microscope !, comprising an ordered arrangement of micro-lenses through which images of a sample from different perspectives are captured, and more in particular to a microscope in which the micro-lenses are arranged in the pupil plane, or Fourier, of the microscope.
Un segundo aspecto de la invención concierne a un sistema de microscopía integral, que comprende el microscopio del primer aspecto y un sistema de procesamiento de las imágenes adquiridas. La invención también concierne a una serie de usos del microscopio del primer aspecto para diversas aplicaciones. A second aspect of the invention concerns an integral microscopy system, which comprises the microscope of the first aspect and a system for processing the acquired images. The invention also concerns a number of uses of the microscope of the first aspect for various applications.
Estado de la técnica antenor En la actualidad el probiema de obtener imágenes tridimensionales (3D) de muestras microscópicas se resuelve, en general, realizando numerosas capturas tras un proceso de escaneo axial (como en el caso del "Ught-sheet microscope" [1 ]-|3], o dei microscopio por iluminación estructurada [4]) o de barrido 3D (como en el caso del microscopio confocal [5]-[6] o, para aplicación en perfííomeíría, el perfiiómetrc confocal [7]). Una alternativa a estas técnicas, pero que no requiere de realización deí esca seo de la muestra es la microscopía hoiográfsca digital [8]-f10]. Esta técnica s¡n embargo tiene restringida su apiicabilidad ai caso de muestras transparentes y que pueden ser observadas mediante iluminación coherente. s recientemente, se ha propuesto la implementación de la microscopía integral [1 1 j- [17] (también llamada microscopía plenóptica o microscopía "iightíieid"). El problema de esta técnica es su pobre resolución transversal. Prior art The problem of obtaining three-dimensional (3D) images of microscopic samples is currently solved, in general, by taking numerous captures after an axial scanning process (as in the case of the "Ught-sheet microscope" [1] - | 3], or the microscope by structured illumination [4]) or 3D scanning (as in the case of the confocal microscope [5] - [6] or, for application in perfííomeíría, the confocal perfiiómetrc [7]). An alternative to these techniques, but which does not require the realization of this sample, is digital hoiographic microscopy [8] -f10]. However, this technique has restricted its applicability in the case of transparent samples that can be observed by coherent illumination. s recently, the implementation of integral microscopy [1 1 j- [17] (also called plenoptic microscopy or "iightieid" microscopy) has been proposed. The problem with this technique is its poor transversal resolution.
La microscopía integral tiene como principal característica su capacidad para registrar la información 3D de muestras gruesas sin necesidad de realizar más de una única toma o disparo. Estos sistemas se basan en ¡a captura simultánea de toda una serie de perspectivas (verticales y horizontales) de la muestra, lo cual se consigue situando un "array" o disposición ordenada de micro-lentes en frente del sensor (CCD o CMOS). En la Fig. 1 se muestra un esquema de un microscopio integral (ÍMic) convencional, es decir en su configuración clasica, donde la muestra Re se encuentra dispuesta en el plano objeto Pe, y el microscopio comprende un objetivo Oc con un correspondiente diafragma de apertura De y, fuera del objetivo Oc, se dispone una lente de tubo Le de manera que su foco objeto coincide con el diafragma de apertura De. En el foco imagen de ia lente de íubo Le se dispone una disposición ordenada da micro-lentes Me, en cuyo foco imagen se dispone e! sensor Se. Integral microscopy has as its main characteristic its ability to record 3D information from thick samples without the need to perform more than one single Shooting or shooting These systems are based on the simultaneous capture of a whole series of perspectives (vertical and horizontal) of the sample, which is achieved by placing an "array" or orderly arrangement of micro-lenses in front of the sensor (CCD or CMOS). In Fig. 1 a diagram of a conventional integral microscope (ÍMic) is shown, that is to say in its classical configuration, where the sample Re is arranged in the object plane Pe, and the microscope comprises an Oc objective with a corresponding diaphragm of aperture From and, outside the Oc lens, a Le tube lens is arranged so that its object focus coincides with the aperture diaphragm From. In the image image of the lens of the lens, an orderly arrangement of micro-lenses is provided Me , in whose image focus is available! sensor Se.
En el esquema de la Fig. 1 , eí sensor Se recoge un conjunto de micro-imágenes que contienen una versión muesíreada de! mapa de radiancias (el mapa de radiancias contiene información sobre ia energía, posición y orientación de los rayos luminosos emitidos por ia muestra). Aunque el mapa de radiancias tiene 4 dimensiones (2 espaciales (r. y) y dos angulares ( , φ)), en este documento solamente se muestra, para simplificar, un corte bidimenslona! (χ, θ) del mismo. En la Fig. 2 se muestra una versión esquemática del mapa de radiancias capturado con el esquema de la Fig. 1 . En este esquema, cada columna representa los píxeies registrados por cada micro-imagen, mientras que si se extraen y agrupan ia información de la filas se pueden generar diferentes vistas de la muestra desde posiciones equidistantes en el diafragma de apertura. A partir de esta información es posible calcular, usando los algoritmos adecuados, tanto una reconstrucción en profundidad (enfocando en diferentes planos) como calcular un mapa de profundidades. La mayor limitación de esta técnica en el momento actual es que la resolución de las imágenes reconstruidas está determinada por el tamaño de las micro-lenies que componen el "array". Estas micro-lentes no pueden tener tamaños inferiores a ios 100 μπι, ya que ello incrementaría sensiblemente ios efectos difractivos durante ia captura del mapa de radiancias. Por ello, la resolución de los iMic en el momento actual esta limitada a, aproximadamente, 1/3 de la resolución nativa de! microscopio. Este hecho constituye el mayor cuello de botella que impide la utilización de la microscopía integral con muestras microscópicas. Otro aspecto mejorable es e! tamaño del dispositivo. Tingase en cuenta que típicamente las lentes de íubo tienen por focai fr ~ 200 mm, lo que implica un camino óptico desde ia muestra Re a! sensor Se superior a 400 mm. In the scheme of Fig. 1, the sensor A set of micro-images containing a muesíreada version of! radiation map (the radiation map contains information on the energy, position and orientation of the light rays emitted by the sample). Although the radiation map has 4 dimensions (2 spatial (r. Y) and two angular (, φ)), this document only shows, to simplify, a two-dimensional cut! (χ, θ) of it. A schematic version of the radiation map captured with the scheme of Fig. 1 is shown in Fig. 2. In this scheme, each column represents the pixels recorded by each micro-image, while if the information from the rows is extracted and grouped, different views of the sample can be generated from equidistant positions in the aperture diaphragm. From this information it is possible to calculate, using the appropriate algorithms, both a reconstruction in depth (focusing on different planes) and calculating a depth map. The major limitation of this technique at the present time is that the resolution of the reconstructed images is determined by the size of the micro-lenies that make up the "array." These micro-lenses cannot have sizes smaller than ios 100 μπι, since this would significantly increase the diffractive effects during the capture of the radiation map. Therefore, the resolution of the iMic at the present time is limited to approximately 1/3 of the native resolution of! microscope. This fact constitutes the largest bottleneck that prevents the use of integral microscopy with microscopic samples. Another improvable aspect is e! device size Keep in mind that typically the eyeglass lenses have by focai f r ~ 200 mm, which implies an optical path from the sample Re a! sensor Be greater than 400 mm.
Resulta necesario ofrecer una aiternativa ai estado de ia técnica que cubra las lagunas halladas en eí mismo, proporcionando un microscopio integrai configurado de manera que permita registrar la información 3D de muestras microscópicas sin necesidad de realizar más de una única toma o disparo y con una resolución que no esté limitada por el tamaño de las micro-lentes. Referencias: It is necessary to offer a state-of-the-art technique that covers the gaps found in it, providing an integrated microscope configured to allow the 3D information of microscopic samples to be recorded without the need to make more than a single shot or shot and with a resolution that is not limited by the size of the micro-lenses. References:
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[16] N . Cohén, S. Yang, A. Andalman. M. Broxton, L. Grosenick, . Deisseroth, .  [16] N. Cohen, S. Yang, A. Andalman. M. Broxton, L. Grosenick,. Deisseroth,.
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[17] A. Llavador, E. Sánchez-Ortiga, J.C. Barreiro, G. Saavedra, and M. Martinez- CorraU Resolution enhancement in integral microscopy by physicai ínterpolation," Biomed Opt Express 6, 2854-2863 (2015).  [17] A. Llavador, E. Sánchez-Ortiga, J.C. Barreiro, G. Saavedra, and M. Martinez-CorraU Resolution enhancement in integral microscopy by physicai interpolation, "Biomed Opt Express 6, 2854-2863 (2015).
Explicación de la invención Explanation of the invention.
Con tal fin, la presente invención concierne a un microscopio integral que comprende, de manera en si conocida: To this end, the present invention concerns an integral microscope comprising, in a manner known per se:
- un sistema óptico que incluye: - an optical system that includes:
- un objetivo con un diafragma de apertura, configurados y dispuestos para recibir luz proveniente de una muestra, según un camino óptico, al ser dicha muestra iluminada con luz incoherente; y - an objective with an aperture diaphragm, configured and arranged to receive light from a sample, according to an optical path, as said sample is illuminated with incoherent light; Y
- una disposición ordenada (o "array") de micro-lentes, dispuestas para que ia luz proveniente de la muestra que entra en dicho objetivo incida en cada una de las micro-lentes de dicha disposición ordenada de micro-lentes; y - an ordered arrangement (or "array") of micro-lenses, arranged so that the light coming from the sample entering said target affects each of the micro-lenses of said ordered arrangement of micro-lenses; Y
- unos medios de adquisición de imágenes configurados y dispuestos para recibir la luz que atraviesa a cada una de las micro-lentes con el fin de adquirir imágenes de ia muestra desde diferentes perspectivas, de manera simultánea. A las imágenes obtenidas de esta forma se las denomina, en la presente invención, imágenes elementales, A diferencia de los microscopios integrales del estado de la técnica, donde ia disposición ordenada de micro-lentes se dispone en el plano del foco imagen de la lente de tubo insertada tras el diafragma de apertura, en el propuesto por el primer aspecto de ia invención, de manera característica, la disposición ordenada de micro- lentes se encuentra dispuesta en el plano pupilar definido por el diafragma de apertura, es decir en el piano de Fourier. - means for acquiring images configured and arranged to receive the light that passes through each of the micro-lenses in order to acquire images of the sample from different perspectives, simultaneously. The images obtained in this way are called, in the present invention, elementary images, Unlike the integral microscopes of the state of the art, where the ordered arrangement of micro-lenses is arranged in the plane of the lens image focus of tube inserted after the opening diaphragm, in that proposed by the first aspect of the invention, characteristically, the arranged arrangement of micro-lenses is arranged in the pupillary plane defined by the opening diaphragm, that is to say in the piano of Fourier.
Para llegar a ia concepción de ia presente invención, se ha tenido en cuenta que aplicando algebra de matrices ABCD al mapa de radiancias capturado por un iMic en su configuración clásica, es decir al mostrado esquemáticamente en la Fig. 2, puede demostrarse que esta información no es más que la traspuesta de ia que se podría obtener situando un "array" de micro-lentes sobre el plano de la apertura (o plano de Fourier) y realizando de ese modo la captura. In order to arrive at the conception of the present invention, it has been taken into account that applying algebra of ABCD matrices to the radiation map captured by an iMic in its classical configuration, that is to say shown schematically in Fig. 2, it can be demonstrated that this information it is nothing more than the transposition of the ia that could be obtained by placing an "array" of micro-lenses on the plane of the aperture (or Fourier plane) and thereby performing the capture.
En otras palabras, la información capturada por un iMic clásico trabajando con un "array "de N micro-lentes y P pixeles por micro-lente, es ia misma (aunque ordenada de diferente forma) que la capturada según el microscopio del primer aspecto de la presente invención, es decir situando P lentes sobre el plano pupilar, con N psxeles por lente. La diferencia es que en el plano de Fourier se puede insertar directamente un "array" de micro-cámaras fotográficas (cada micro-cámara captura una imagen elemental diferente), lo cual da lugar a un Incremento muy significativo de la resolución (en este caso el número total de pixeles es el resultado de multiplicar e! numero de píxeles de una micro-cámara por el n mero de cámaras). Una ventaja adicional es que esta nueva configuración, propuesta por la presente invención, captura directamente las vistas de la muestra. Es decir que al realizarse ia adquisición de imágenes elementales en el plano de Fourier, la resolución obtenida es sensiblemente mayor a la conseguida con los microscopios integrales convencionales, el tiempo de cálculo es menor, y ademas eí tamafio dei microscopio es mucho menor, permitiendo la fabricación de microscopios 3D portátiles (con un volumen equivalente al de una cámara de fotos compacta). In other words, the information captured by a classic iMic working with an "array" of N micro-lenses and P pixels per micro-lens, is the same (although ordered in a different way) than the one captured according to the microscope of the first aspect of the present invention, that is, placing P lenses on the pupillary plane, with N pspixels per lens. The difference is that in the Fourier plane you can directly insert an "array" of micro-cameras (each micro-camera captures a different elementary image), which results in a very significant increase in resolution (in this case the total number of pixels is the result of multiplying the number of pixels of a micro-camera by the number of cameras). An additional advantage is that this new configuration, proposed by the present invention, directly captures the views of the sample. That is, when the acquisition of elementary images in the Fourier plane is performed, the resolution obtained is significantly greater than that achieved with conventional integral microscopes, the calculation time is shorter, and also the size of the microscope is much smaller, allowing the manufacture of portable 3D microscopes (with a volume equivalent to that of a compact photo camera).
De acuerdo con un ejemplo de realización, eí microscopio dei primer aspecto está configurado para que ¡as imágenes elementales adquiridas por los citados medios de adquisición de imágenes contengan una versión mueslreada del mapa de radiancias de la muestra. According to an exemplary embodiment, the microscope of the first aspect is configured so that the elementary images acquired by said image acquisition means contain a sample version of the sample's radiation map.
De manera preferida, cada uno de dichos sensores de imágenes comprende N elementos fotosensibles, o psxeles activos, de manera que la resolución obtenida con el microscopio es igual a N x P, siendo P el número de micro-lentes. Preferably, each of said image sensors comprises N photosensitive elements, or active pspixels, so that the resolution obtained with the microscope is equal to N x P, where P is the number of micro-lenses.
En general, ¡os medios de adquisición de imágenes comprenden una disposición ordenada (o "array") de sensores de imagen, cada uno de eílos dispuesto, según dicho camino óptico, tras una de ¡as micro-lentes de la disposición ordenada de micro-lentes. De manera alternativa, para otro ejemplo de realización, los medios de adquisición de imágenes comprenden un único sensor de imagen (CCD, CMOS, etc.) que comprende M elementos fotosensibles, o psxeles activos, agrupados en una pluralidad de regiones contiguas, cada una de las cuales comprende N píxeies y está dispuesta, según dicho camino óptico, tras una de ias micro-lentes de la disposición ordenada de rnicro~¡eníes. In general, the image acquisition means comprise an ordered arrangement (or "array") of image sensors, each of them arranged, according to said optical path, after one of the micro-lenses of the ordered micro arrangement -glasses. Alternatively, for another embodiment, the image acquisition means comprise a single image sensor (CCD, CMOS, etc.) comprising M photosensitive elements, or active psxels, grouped into a plurality of contiguous regions, each of which it comprises N pixels and is arranged, according to said optical path, after one of the micro-lenses of the ordered arrangement of rnicro.
De acuerdo con un ejemplo de realización, cada uno de dichos sensores de imagen forma parte de una respectiva micro-cámara sin lente. According to an exemplary embodiment, each of said image sensors is part of a respective micro-camera without a lens.
De manera alternativa, y preferida, para otro ejemplo de realización dicha disposición de sensores de imagen y dicha disposición de micro-lentes están agrupados formando una disposición de micro-cámaras, donde cada una de las micro-cámaras comprende un sensor de imagen y un micro-objetivo con una micro-lente. Alternatively, and preferably, for another embodiment, said image sensor arrangement and said micro-lens arrangement are grouped together forming a micro-camera arrangement, where each of the micro-cameras comprises an image sensor and a micro-objective with a micro-lens.
Ventajosamente, la citada disposición de micro-cámaras comprende un soporte en el que se encuentran fijadas las micro-cámaras debidamente alineadas, estando dicho soporte configurado, dimensíonado y dispuesío tras el diafragma de apertura, de manera que la disposición de micro-ientes quede insertada en ia apertura del diafragma de apertura que define ei piano pupílar. Aunque ei microscopio propuesto por el primer aspecto de ia invención puede funcionar bajo luz ambiental, ésta no es la aplicación más habitual ni la que ofrece mejores resultados, por io que, de acuerdo con un ejemplo de realización más habitual y eficiente, el microscopio del primer aspecto de la invención comprende unos medios de iluminación que incluyen al menos una fuente de luz incoherente, configurada y dispuesta para iluminar a ía muestra. Advantageously, said micro-camera arrangement comprises a support in which the micro-cameras are properly aligned, said being configured, sized and arranged support behind the opening diaphragm, so that the micro-tooth arrangement is inserted into the opening of the opening diaphragm that defines the pupil piano. Although the microscope proposed by the first aspect of the invention can operate under ambient light, this is not the most common application or the one that offers the best results, so that, according to a more usual and efficient embodiment, the microscope of the The first aspect of the invention comprises lighting means that include at least one incoherent light source, configured and arranged to illuminate the sample.
De modo similar el microscopio de ¡a presente invención también puede utilizarse para adquirir imágenes de muestras externas ai mismo, pero tal uso tampoco es el más habitual ni ofrece unos resultados tan buenos como el relativo a adquirir imágenes de una muestra interna ai microscopio. Similarly, the microscope of the present invention can also be used to acquire images of external samples to itself, but such use is also not the most common nor does it offer results as good as that related to acquiring images of an internal sample under the microscope.
En cualquier caso, para un ejemplo de realización preferido, el microscopio del primer aspecto de la invención comprende un soporte para la muestra. Según un ejemplo de realización, ei microscopio de ia invención comprende una estructura de soporte en ia que se encuentran montados, según el orden definido por el anteriormente citado camino óptico: los medios de iluminación, ei soporte para la muestra, ei sistema óptico y los medios de adquisición de imágenes. Para una variante de dicho ejemplo de realización, el sistema Óptico y los medios de adquisición de imágenes se encuentran montados en la estructura de soporte de manera desplazabie y guiada en aproximación/alejamiento respecto al soporte para ia muestra, para enfocar a ia muestra y/o distintas partes de ia misma. De acuerdo con un ejemplo de realización, ei microscopio de la invención comprende una carcasa que aloja en su interior a la citada estructura de soporte con todos ios elementos montados en ia misma, o que constituye dicha estructura de soporte. In any case, for a preferred embodiment, the microscope of the first aspect of the invention comprises a sample holder. According to an exemplary embodiment, the microscope of the invention comprises a support structure in which they are mounted, in the order defined by the aforementioned optical path: the lighting means, the sample holder, the optical system and the means of image acquisition. For a variant of said exemplary embodiment, the Optical system and the image acquisition means are mounted on the support structure in a displaced and guided manner in approximation / distance with respect to the support for the sample, to focus on the sample and / or different parts of it. According to an exemplary embodiment, the microscope of the invention comprises a housing that houses said support structure with all the elements mounted therein, or that constitutes said support structure.
El microscopio del primer aspecto de la invención comprende, para un ejemplo de realización, un sistema electrónico en conexión con por io menos ios medios de adquisición de imágenes para controlarlos para adquirir imágenes simultáneamente en un único disparo. Ventajosamente, el citado sistema electrónico también esta conectado con los medios de iluminación para controlarlos para que emitan luz incoherente al menos durante eí citado único disparo de ios medios de adquisición de imágenes. The microscope of the first aspect of the invention comprises, for one embodiment, an electronic system in connection with at least the means of image acquisition to control them to acquire images simultaneously in a single shot. Advantageously, said electronic system is also connected to the lighting means to control them so that they emit incoherent light at least during said single shot of the image acquisition means.
Según un ejemplo de realización del microscopio dei primer aspecto de la invención, eí sistema electrónico comprende unos medios de procesamiento internos (es decsr, locales) adaptados para procesar las imágenes adquiridas para realizar al menos una de !as siguientes tareas, a partir de ía información tridimensional de la muestra contenida en las imágenes adquiridas; According to an embodiment of the microscope of the first aspect of the invention, the electronic system comprises internal processing means (ie, local) adapted to process the acquired images to perform at least one of the following tasks, based on the three-dimensional information of the sample contained in the acquired images;
- calcular y mostrar en ai menos una pantalla una reconstrucción en profundidad de la muestra, con imágenes enfocadas a diferentes profundidades; y - calculate and show at least one screen a reconstruction in depth of the sample, with images focused at different depths; Y
- realizar de un mapa de profundidades o matriz tridimensional, en la que se indica la posición tridimensional de cada punto de la muestra. - make a map of depths or three-dimensional matrix, which indicates the three-dimensional position of each point of the sample.
Alternativamente, o de manera complementaria, eí sistema electrónico comprende unos medios de comunicación, vía cable o inalámbricos (por ejemplo Bluetooth®), para el envío de las imágenes adquiridas a un sistema de procesamiento externo (es decir, remoto) adaptado para procesar las imágenes adquiridas para realizar al menos una de las siguientes tareas, a partir de ia información tridimensional de ia muestra contenida en las Imágenes: Alternatively, or in a complementary manner, the electronic system comprises means of communication, via cable or wireless (for example Bluetooth®), for sending the acquired images to an external (ie, remote) processing system adapted to process the images acquired to perform at least one of the following tasks, based on the three-dimensional information of the sample contained in the Images:
- calcular y mostrar en al menos una pantalla, del microscopio o externa ai mismo, una reconstrucción en profundidad de la muestra, con imágenes enfocadas a diferentes profundidades; y - realizar de un mapa de profundidades o matriz tridimensional, en la que se índica ia posición tridimensional de cada punto de la muestra - calculate and display on at least one screen, from the microscope or external to itself, an in-depth reconstruction of the sample, with images focused at different depths; and - make a map of depths or three-dimensional matrix, in which the three-dimensional position of each point of the sample is indicated
La presente invención también concierne, en un segundo aspecto, a un sistema de microscopía integral, que comprende: - el microscopio según el primer aspecto de ia invención; y The present invention also concerns, in a second aspect, an integral microscopy system, comprising: - the microscope according to the first aspect of the invention; Y
- un sistema de procesamiento (por ejemplo imptementado en un ordenador, "lablef , o I:Smartphone!:) adaptado para procesar ias imágenes adquiridas por ei microscopio para realizar al menos una de las siguientes tareas, a partir de la información tridimensional de ia muestra contenida en las imágenes: - a processing system (for example installed on a computer, "lablef, or I: Smartphone ! ) : adapted to process the images acquired by the microscope to perform at least one of the following tasks, based on the three-dimensional information of the ia Sample contained in the images:
- calcular y mostrar en al menos una pantalla del sistema una reconstrucción en profundidad de ¡a muestra, con imágenes enfocadas a diferentes profundidades; y - calculate and display at least one screen of the system a deep reconstruction of the sample, with images focused at different depths; Y
- realizar de un mapa de profundidades o matriz tridimensional, en ia que se indica la posición tridimensional de cada punto de la muestra. De acuerdo con un ejemplo de realización del sistema propuesto por el segundo aspecto de la invención, éste comprende como mínimo una impresora 3D comunicada con el sistema de procesamiento para al menos recibir del mismo información de perfilometría reiativa a dicho mapa de profundidades o matriz tridimensional, y adaptada para imprimir una maqueta tridimensional de ¡a muestra a partir de la información recibida. - make a map of depths or three-dimensional matrix, in which the three-dimensional position of each point of the sample is indicated. According to an embodiment of the system proposed by the second aspect of the invention, it comprises at least one 3D printer communicated with the processing system to at least receive from the same profiling geometry information related to said depth map or three-dimensional matrix, and adapted to print a three-dimensional model of the sample based on the information received.
Un tercer aspecto de ia presente invención concierne a un uso del microscopio según el pnmer aspecto para aplicaciones de hio-medieina, incluyendo el cribado de muestras biológicas (potencialmente ligadas a aigún patógeno) in situ, analizando el relieve de ia muestra o de un seccionado en profundidad de ia misma, Esto puede ser muy útil, por ejemplo, para la detección in situ de posibles contagios. Evidentemente, el uso en laboratorio para las mismas aplicaciones también es posible. A third aspect of the present invention concerns a use of the microscope according to the pnmer aspect for hyo-mediein applications, including the screening of biological samples (potentially linked to some pathogen) in situ, analyzing the relief of the sample or a section In depth of the same, This can be very useful, for example, for the detection in situ of possible infections. Obviously, laboratory use for the same applications is also possible.
Un cuarto aspecto de la invención concierne a un uso del microscopio según el primer aspecto para aplicaciones de perfilometría, tales como- control de calidad de piezas o componentes (tal como componentes de microelectrónica y semiconductores, testado de micro-lentes y de lentes infraoculares), y ciencia forense. En relación a este cuarto aspecto, las aplicaciones de perfilometría de muestras microscópicas opacas en ambientes alejados de un laboratorio son realmente ventajosas. Esto puede ser muy útil, por ejemplo, para ia comprobación inmediata de las posibles imperfecciones de una pieza industriar aturalmente, el uso del microscopio deS primer aspecto de la invención en laboratorios también está contemplado, con e! mismo rigor que fuera del laboratorio Los datos obtenidos de la medición perfiíométrica (mapa de profundidades) se pueden utilizar para construir maquetas 3D con una impresora 3D. A fourth aspect of the invention concerns a use of the microscope according to the first aspect for profilometry applications, such as quality control of parts or components (such as microelectronic and semiconductor components, testing of micro-lenses and infraocular lenses) , and forensic science. In relation to this fourth aspect, the applications of profilometry of opaque microscopic samples in environments far from a laboratory are really advantageous. This can be very useful, for example, for the immediate verification of possible imperfections of An industrial part, the use of the microscope of the first aspect of the invention in laboratories is also contemplated, with e! same rigor as outside the laboratory The data obtained from the perfiometric measurement (depth map) can be used to build 3D models with a 3D printer.
Brg^descrjflcj^ dibujos Las anteriores y otras ventajas y características se comprenderán más plenamente a partir de la siguiente descripción detallada de unos ejemplos de realización con referencia a los dibujos adjuntos, que deben tomarse a título ilustrativo y no limitativo, en los que. La Figura 1 muestra un esquema de un microscopio integral convencional (¡Míe) en su configuración clásica. Brg ^ describjflcj ^ drawings The foregoing and other advantages and features will be more fully understood from the following detailed description of some embodiments with reference to the attached drawings, which should be taken by way of illustration and not limitation, in which. Figure 1 shows a diagram of a conventional integral microscope (¡Mì) in its classical configuration.
La Figura 2 se muestra una versión esquemática del mapa de radiancias capturado mediante ei microscopio de ía Figura 1 . Figure 2 shows a schematic version of the radiation map captured by the microscope of Figure 1.
La Figura 3a muestra, de manera esquemática, ai microscopio propuesto por ei primer aspecto de la invención, para un ejemplo de realización, Figure 3a shows, schematically, the microscope proposed by the first aspect of the invention, for an exemplary embodiment,
La Figura 3b muestra una versión esquemática del mapa de radiancias capturado mediante ei microscopio del primer aspecto de la presente invención , según el esquema de la Figura 3; Figure 3b shows a schematic version of the radiation map captured by the microscope of the first aspect of the present invention, according to the scheme of Figure 3;
La Figura 4 muestra, en su vista (a) unas micro-imágenes de una muestra obtenidas con el microscopio iMic de la Figura 1 , y la vista (b) una vista aumentada de unas pocas de dichas micro -imágenes; Figure 4 shows, in its view (a) micro-images of a sample obtained with the iMic microscope of Figure 1, and view (b) an enlarged view of a few of said micro-images;
La Figura 5 muestra sendas vistas con diferente perspectiva calculadas para el iMIc a partir de las micro-imágenes de la Figura 4; La Figura 8 muestra sendas reconstrucciones de ia muestra a dos profundidades, para el Mic de ia Figura 1 , a partir de las micro -imágenes de la Figura 4; Figure 5 shows paths with different perspectives calculated for the iMIc from the micro-images of Figure 4; Figure 8 shows two reconstructions of the sample at two depths, for the Mic of Figure 1, from the micro-images of Figure 4;
La Figura 7 muestra dos vistas con diferente perspectiva capturadas directamente con el microscopio propuesto por el primer aspecto de la invención, denominado también FiMic, según ei esquema de la Figura 3a; Figure 7 shows two views with different perspective captured directly with the microscope proposed by the first aspect of the invention, also called FiMic, according to the scheme of Figure 3a;
La Figura 8 muestra sendas reconstrucciones de la muestra a dos profundidades, para ei FiMic, es decir para microscopio propuesto por el primer aspecto de la invención, según el esquema de ia Figura 3a; Figure 8 shows two reconstructions of the sample at two depths, for the FiMic, that is to say for a microscope proposed by the first aspect of the invention, according to the scheme of Figure 3a;
La Figura 9 muestra otro ejemplo de realización del microscopio propuesto por el primer aspecto de la presente invención, según un prototipo ilustrado de manera esquemática; y Figure 9 shows another embodiment of the microscope proposed by the first aspect of the present invention, according to a prototype illustrated schematically; Y
La Figura 10 muestra diferentes distribuciones de micro-cámaras en un soporte de las mismas, incluidas en el microscopio propuesto por ei primer aspecto de la invención, según diferentes realizaciones. Descripción detallada de unos ejemplos de realización Figure 10 shows different distributions of micro-cameras on a support thereof, included in the microscope proposed by the first aspect of the invention, according to different embodiments. Detailed description of some embodiments
Ei microscopio propuesto por el primer aspecto de ia presente invención proporciona una nueva configuración o diseño en la que la captura del mapa de radiancias no se produce en el plano imagen del microscopio, s¡no en el piano papilar o de Fourier. Este nuevo diseño, ai que ios presentes inventores han denominado Microscopio Integral de Fourier (Fí íc) presenta en su diserlo final, según realizaciones preferidas, una estructura simple y compacta. The microscope proposed by the first aspect of the present invention provides a new configuration or design in which the capture of the radiation map does not occur in the microscope image plane, but not in the papillary or Fourier piano. This new design, which the present inventors have called Fourier Integral Microscope (Fí íc) presents in its final design, according to preferred embodiments, a simple and compact structure.
En la Figura 3a muestra, de manera esquemática, ai microscopio propuesto por el primer aspecto de ia invención, para un ejemplo de realización , para el cual éste comprende; In Figure 3a shows, schematically, the microscope proposed by the first aspect of the invention, for an exemplary embodiment, for which it comprises;
- un objetivo O con un diafragma de apertura D, configurados y dispuestos para recibir luz proveniente de una muestra R (situada en ei plano objeto Pe), según un camino Ó tieO; al ser dicha muestra R iluminada con luz incoherente; y - una disposición de micro-cámaras C, donde cada una de las micro-cámaras C comprende un sensor de imagen 3 y un micro-objetivo 1 con una micro-lente 2, estando ¡as micro-lentes 2 agrupadas según una disposición ordenada o "array" de rnicro-íentes 2 dispuesta en el plano pupilar definido por el diafragma de apertura D. - an objective O with an aperture diaphragm D, configured and arranged to receive light from a sample R (located in the object plane Pe), according to a path OR; when said sample R is illuminated with incoherent light; Y - an arrangement of micro-cameras C, where each of the micro-cameras C comprises an image sensor 3 and a micro-lens 1 with a micro-lens 2, the micro-lenses 2 being grouped according to an ordered arrangement or "array" of rnicro-íentes 2 arranged in the pupillary plane defined by the aperture diaphragm D.
En la Figura 3b se muestra ei mapa de radiancias obtenido con el esquema de la Figura 3a. Nótese que la información capturada ahora es esencialmente Sa misma que en ei caso del ÍMic convencional (ver Figura 2), pero con una arquitectura mucho más compacta La segunda, y no menos importante, ventaja de este segundo diseño, es que las perspectivas de la muestra (columnas del nuevo mapa de radiancias) se capturan directamente y con una resolución que ya no está limitada por el tamaño de las micro-lentes, sino por e! de los pixeles del sensor de imagen. Este diseño permite reconstruir imágenes de muestras 3D con resoluciones mucho mejores que las alcanzadas hasta el momento en microscopía integral Figure 3b shows the radiation map obtained with the scheme of Figure 3a. Note that the information captured now is essentially Sa same as in the case of the conventional ÍMic (see Figure 2), but with a much more compact architecture The second, and not least, advantage of this second design, is that the perspectives of the Sample (columns of the new radiation map) are captured directly and with a resolution that is no longer limited by the size of the micro-lenses, but by e! of the image sensor pixels. This design allows to reconstruct images of 3D samples with much better resolutions than those achieved so far in integral microscopy
En las Figuras 4 a 8 se ilustran los resultados obtenidos con el i ie, es decir con eí microscopio integra! convencional de la Figura 1. con el fin de compararlos con los obtenidos con el microscopio propuesto por la presente invención, habiéndose ilustrado estos últimos en ¡as Figuras 7 y 8, The results obtained with the i ie are illustrated in Figures 4 to 8, that is, with the integrated microscope! conventional of Figure 1. in order to compare them with those obtained with the microscope proposed by the present invention, the latter having been illustrated in Figures 7 and 8,
La imágenes mostradas han sido obtenidas en laboratorio por los presentes inventores, con un montaje experimental tipo iMic como el ilustrado en ia Figura 1 y con otro del tipo Fi íc como e! ilustrado en la Figura 3a. En ambos casos, se ba utilizado el mismo objetivo de microscopio (20X/Q.40). En el caso i íc se na utilizado un "array" compuesto por micro-lentes de diámetro 0=0.1 10 mm. En el caso del Fi ic se han implementado ópticamente micro-cámaras que estaban separadas una distancia Λ-2.0 mm. En ambos casos se ha utilizado como objeto una muestra de tejido tintada con un colorante fluorescente. The images shown have been obtained in the laboratory by the present inventors, with an experimental assembly type iMic as illustrated in Figure 1 and with another of the Fi Fi type such as e! illustrated in Figure 3a. In both cases, the same microscope objective (20X / Q.40) was used. In the case where an "array" consisting of micro-lenses of diameter 0 = 0.1 10 mm has been used. In the case of Fi ic, micro-cameras that were separated by a distance Λ-2.0 mm have been optically implemented. In both cases, a sample of tissue stained with a fluorescent dye has been used as the object.
En el caso de la geometría iMic, el resultado proporcionado por el microscopio es el conjunto de micro-imágenes (de 0, 1 10 mm de diámetro) ilustrado en la Figura 4. In the case of the iMic geometry, the result provided by the microscope is the set of micro-images (0.1 mm in diameter) illustrated in Figure 4.
A partir de las micro-imágenes de la Figura 4 es posible calcular un conjunto de vistas en perspectiva de la muestra microscópica. Ei procedimiento de cálculo es bien conocido y se detalla, por ejemplo, en Biomed. Opí Express 6, 2854-2863 (2015). En ¡a Figura 5 se muestran dos de estas perspectivas calculadas según dicho procedimiento de cálculo. A partir de las vistas en perspectiva es pasible calcular (el algoritmo también está descrito en Biomed. Opt. Express 6, 2854-2863 (2015)) las diferentes fccalizaciones en profundidad. En la Figura 8 se muestran reconstrucciones de la muestra a dos profundidades, para el ÍMÍc de la Figura 1 , a partir de las micro- imágenes de la Figura 4. La distancia entre el plano más lejano y el más próximo en las reconstrucciones de la Figura 6 es de 18 mieras. En ias Figuras 7 y 8 se muestran las imágenes obtenidas con el FíMíc, es decir con el microscopio de la presente invención, según el esquema de la Figura 3a. En este caso las imágenes obtenidas ya constituyen directamente ias vistas de la muestra 3D, sin necesidad de aplicar ningún cálculo. Aunque la muestra utilizada es la misma que en el caso de! ¡Míe, ia región observada es diferente. En particular, en la Figura 7 se muestran dos vistas en perspectiva de la muestra y en la Figura 8 se muestran reconstrucciones correspondientes a dos profundidades. En este caso, ia distancia entre el plano más lejano y el más próximo en ias reconstrucciones de la Figura 8 también es de 18 mieras. Puede comprobarse que ia resolución de las vistas obtenidas con el FiMic es cuatro veces superior a las obtenidas con el sMic, y en general se puede afirmar que es casi tres veces superior a la obtenida en cualquier experimento de M\a publicado anteriormente. Además, el campo visual y la relación señal/ruido son también sensiblemente superiores From the micro-images of Figure 4 it is possible to calculate a set of perspective views of the microscopic sample. The calculation procedure is fine known and detailed, for example, in Biomed. Opí Express 6, 2854-2863 (2015). Figure 5 shows two of these perspectives calculated according to said calculation procedure. From the perspective views it is possible to calculate (the algorithm is also described in Biomed. Opt. Express 6, 2854-2863 (2015)) the different depth settings. Figure 8 shows reconstructions of the sample at two depths, for the IMIc of Figure 1, from the micro-images of Figure 4. The distance between the farthest and the closest plane in the reconstructions of the Figure 6 is 18 microns. Figures 7 and 8 show the images obtained with the FíMíc, that is, with the microscope of the present invention, according to the scheme of Figure 3a. In this case the images obtained already directly constitute the views of the 3D sample, without the need to apply any calculation. Although the sample used is the same as in the case of! Me, the region observed is different. In particular, in Figure 7 two perspective views of the sample are shown and in Figure 8 reconstructions corresponding to two depths are shown. In this case, the distance between the furthest and the closest plane in the reconstructions of Figure 8 is also 18 microns. It can be verified that the resolution of the views obtained with the FiMic is four times higher than those obtained with the sMic, and in general it can be affirmed that it is almost three times higher than that obtained in any previously published M \ a experiment. In addition, the visual field and the signal-to-noise ratio are also significantly higher.
En la Figura 9 se ilustra otro ejemplo de realización de! microscopio propuesto por el primer aspecto de la presente invención, según un prototipo para el cual el microscopio incluye una estructura de soporte formada por dos partes S1 , S2 despiazahies entre sí según una dirección vertical (según ia posición ilustrada en la Figura 9) , de manera guiada en aproximación/alejamiento a través de una barra G, sobre la que se encuentra montada de manera guiada la parte S 1 a través de un elemento despíazador 8 (accionable manualmente o de manera automática) Another example of embodiment of! Is illustrated in Figure 9. Microscope proposed by the first aspect of the present invention, according to a prototype for which the microscope includes a support structure formed by two parts S1, S2 splitting each other according to a vertical direction (according to the position illustrated in Figure 9), of guided approach / distance way through a bar G, on which the part S 1 is mounted in a guided way through a stripper element 8 (manually or automatically operated)
En la parte S I de ia estructura de soporte se encuentran montados un objetivo de microscopio 4 que incluye un diafragma de apertura D: y una disposición o "array" de micro-cámaras C, soportadas debidamente distribuidas en un soporte Cs, donde cada una de las micro-cámaras C incluye un sensor de imagen 3 de N pixeles activos (CCD, CMOS, etc.) y un objetivo 1 con una micro-lente 2, estando montado el soporte S1 sobre el diafragma D (Según la posición ilustrada en la Figura 9) de manera que las mierc-!entes 2 quedan dispuestas en el piano pupilar del mismo. In the SI part of the support structure a microscope objective 4 is mounted which includes an aperture diaphragm D : and an array or array of C micro-cameras, supported properly distributed on a Cs support, where each of the C micro-cameras includes an image sensor 3 of N active pixels (CCD, CMOS, etc.) and a lens 1 with a micro-lens 2 , the support S1 being mounted on the diaphragm D (According to the position illustrated in Figure 9) so that the two are arranged in the pupillary piano thereof.
Asimismo, en la parte S2 de ia estructura de soporte se encuentran montados una fuente de luz incoherente 5 {ta! como un LED}, un soporte 7 para ia muestra R (el cual es preferiblemente móvil según unos ejes X-Y perpendiculares ai eje 2), asi corno un conector USB 9 (aunque podría ser de otro tipo) para ia conexión vía cable de las imágenes adquiridas a un dispositivo USB conectado ai mismo o, de manera inalámbrica (por ejemplo, Biuetooth®) a través de un dispositivo de comunicaciones conectado ai mismo a un sistema de procesamiento externo, ímpiementado por ejemplo en un ordenador, "tahleí" o "srnartphone". En la parte 52 también se encuentra montada una batería recargable 8 para ia alimentación de un sistema electrónico local (no ilustrado) del microscopio. In addition, an incoherent light source 5 {ta! as an LED}, a support 7 for the sample R (which is preferably mobile along XY axes perpendicular to axis 2), as well as a USB connector 9 (although it could be of another type) for the connection via cable of the images acquired from a USB device connected to itself or, wirelessly (for example, Biuetooth®) through a communications device connected to itself to an external processing system, implemented for example on a computer, "tahleí" or "srnartphone ". In part 52 a rechargeable battery 8 is also mounted for the power of a local electronic system (not shown) of the microscope.
Ventajosamente, para un ejemplo de realización no ilustrado, todo el conjunto ilustrado en ia Figura 9 se encuentra alojado en el interior de una carcasa, con el fin de proporcionar un dispositivo compacto, similar a una cámara fotográfica compacta, para su uso fuera del laboratorio. Advantageously, for an exemplary embodiment, the entire assembly illustrated in Figure 9 is housed inside a housing, in order to provide a compact device, similar to a compact camera, for use outside the laboratory. .
Finalmente, en la Figura 10 se muestran diferentes distribuciones de micro-cámaras C en un soporte Cs de las mismas, incluidas en el microscopio propuesto por el primer aspecto de ia invención, según diferentes realizaciones. Las distribuciones están adaptadas a ia forma y tamaño dei diafragma D en ei que deben insertarse las micro- lentes 2 de ia micro-cámaras C, Se muestran distribuciones hexagonales con 7 micro- cámaras (vista (a)} o 19 micro-cámaras (vista (b)), así como distribuciones rectangulares con 12 micro-cámaras (vista (c)) y 24 micro-cámaras (vista (d)). Finally, in Figure 10 different distributions of micro-cameras C are shown on a support Cs thereof, included in the microscope proposed by the first aspect of the invention, according to different embodiments. The distributions are adapted to the shape and size of the diaphragm D in which the micro-lenses 2 of the micro-cameras C must be inserted. Hexagonal distributions with 7 micro-cameras (view (a)) or 19 micro-cameras ( view (b)), as well as rectangular distributions with 12 micro-cameras (view (c)) and 24 micro-cameras (view (d)).
Un experto en ia materia podría introducir cambios y modificaciones en ios ejemplos de realización descritos sin salirse del alcance de la invención según está definido en las reivindicaciones adjuntas. A person skilled in the art could introduce changes and modifications in the described embodiments described without departing from the scope of the invention as defined in the appended claims.

Claims

1- Microscopio integral que comprende:  1- Integral microscope comprising:
- un sistema óptico que incluye:  - an optical system that includes:
- un objetivo (O) con un diafragma de apertura (D), configurados y dispuestos para recibir luz proveniente de una muestra {R), segün un camino óptico, ai ser dicha muestra (R) iluminada con luz incoherente; y - una disposición ordenada de micro-lentes (2), dispuestas para que la luz proveniente de la muestra (R) que entra en dicho objetivo (O) incida en cada una de las micro-lentes (2} de dicha disposición ordenada de micro-lentes; y - a target (O) with an aperture diaphragm (D), configured and arranged to receive light from a sample {R), according to an optical path, if said sample (R) is illuminated with incoherent light; and - an ordered arrangement of micro-lenses (2), arranged so that the light coming from the sample (R) entering said objective (O) affects each of the micro-lenses (2) of said ordered arrangement of micro-lenses; and
- unos medios de adquisición de imágenes configurados y dispuestos para recibir la luz que atraviesa a cada una de las micro-lentes (2) con el fin de adquirir imágenes de la muestra ( ) desde diferentes perspectivas, de manera simultánea; estando ei microscopio caracterizado porque dicha disposición ordenada de micro- lentes (2) se encuentra dispuesta en ei plano pupiiar definido por dicho diafragma de apertura (D). - means for acquiring images configured and arranged to receive the light that passes through each of the micro-lenses (2) in order to acquire images of the sample () from different perspectives, simultaneously; the microscope being characterized in that said ordered arrangement of micro-lenses (2) is arranged in the pupil plane defined by said aperture diaphragm (D).
2.- Microscopio según la reivindicación 1 , que está configurado para que ¡as micro- imágenes adquiridas por dichos medios de adquisición de imágenes contengan una versión maestreada del mapa de radiancias de la muestra. 2. Microscope according to claim 1, which is configured so that the micro-images acquired by said image acquisition means contain a mastered version of the sample radiation map.
3 - Microscopio según ia reivindicación 1 ó 2, en el que dichos medios de adquisición de imágenes comprenden una disposición ordenada de sensores de imagen (3), cada uno de ellos dispuesto, según dicho camino óptico, tras una de las micro-íentes (2) de la disposición ordenada de micro-lentes. 3 - Microscope according to claim 1 or 2, wherein said image acquisition means comprise an ordered arrangement of image sensors (3), each arranged, according to said optical path, after one of the micro-elements ( 2) of the orderly arrangement of micro-lenses.
4,- Microscopio según la reivindicación 3, en el que cada uno de dichos sensores de imagen (3) forma parte de una respectiva micro-cámara sin ¡ente. 4, - Microscope according to claim 3, wherein each of said image sensors (3) is part of a respective micro-camera without an entity.
5 - Microscopio según la reivindicación 1 , 2 ó 3, en el que dicha disposición de sensores de imagen (3) y dicha disposición de micro-lentes (2) están agrupados formando una disposición de micro-cámaras (C), donde cada una de las micro- cámaras (C) comprende un sensor de imagen (3) y un micro-objetivo (1 ) con una micro-lente (2). 5 - Microscope according to claim 1, 2 or 3, wherein said image sensor arrangement (3) and said micro-lens arrangement (2) are grouped together forming a micro-camera arrangement (C), wherein each of the micro-cameras (C) comprises an image sensor (3) and a micro-lens (1) with a micro-lens (2).
8 - Microscopio según ía reivindicación 4 ó 5, en ei que dicha disposición de micro- cámaras (C) comprende un soporte (Cs) en eí que se encuentran fijadas ias micro- cámaras (C) debidamente alineadas, estando dicho soporte (Cs) configurado, dimensionado y dispuesto tras el diafragma de apertura (D), de manara que la disposición de micro-ientes (2) quede insertada en la apertura del diafragma de apertura (D) que define ei piano pupilar. 8 - Microscope according to claim 4 or 5, in which said micro-camera arrangement (C) comprises a support (Cs) in it that the micro-cameras (C) are properly aligned, said support (Cs) being configured, sized and arranged after the opening diaphragm (D), so that the micro-tooth arrangement (2) is inserted into the opening of the opening diaphragm (D) that defines the pupil piano.
7 - Microscopio según ía reivindicación 4, 5 ó 6, en el que cada uno de dichos sensores de imágenes (3) comprende N elementos fotosensibles, o pixeles activos. 7 - Microscope according to claim 4, 5 or 6, wherein each of said image sensors (3) comprises N photosensitive elements, or active pixels.
8.- Microscopio según una cualquiera de las reivindicaciones anteriores, que comprende unos medios de iluminación que incluyen al menos una fuente de luz incoherente (5), configurada y dispuesta para iluminar a dicha muestra (R). 8. Microscope according to any one of the preceding claims, comprising lighting means that include at least one incoherent light source (5), configured and arranged to illuminate said sample (R).
9 - Microscopio según una cualquiera de las reivindicaciones anteriores, que comprende un soporte (7) para dicha muestra (R) 9 - Microscope according to any one of the preceding claims, comprising a support (7) for said sample (R)
10. - Microscopio según la reivindicación 9 cuando depende de la 8, que comprende una estructura de soporte (SI , S2) en ¡a que se encuentran montados, según el orden definido por dicho camino óptico: dicha fuente de luz incoherente (5), dicho soporte (7) para ia muestra (R), ei sistema Óptico y los medios de adquisición de imágenes. 10. - Microscope according to claim 9 when it depends on the 8, which comprises a support structure (SI, S2) in which they are mounted, according to the order defined by said optical path: said incoherent light source (5) , said support (7) for the sample (R), the Optical system and the image acquisition means.
1 1 . - Microscopio según la reivindicación 10, en el que el sistema óptico y los medios de adquisición de imágenes se encuentran montados en la estructura de soporte (S 1 ,eleven . - Microscope according to claim 10, wherein the optical system and the image acquisition means are mounted on the support structure (S 1,
52) de manera desplazable y guiada en aproximación/alejamiento respecto al soporte (7) para la muestra (R). 52) in a scrollable and guided approach / distance from the support (7) for the sample (R).
12.- Microscopio según la reivindicación 10 u 1 1 , que comprende una carcasa que aloja en su interior a dicha estructura de soporte £S1 , S2) con todos ios elementos montados en la misma, o que constituye dicha estructura de soporte (S1 , S2). 12. Microscope according to claim 10 or 1, comprising a housing that houses said support structure £ S1, S2) with all the elements mounted therein, or that constitutes said support structure (S1, S2).
1 3.- Microscopio según una cualquiera de las reivindicaciones anteriores, que comprende un sistema electrónico en conexión con ai menos Sos medios de adquisición de imágenes para controlarlos para adquirir imágenes simultáneamente en un único disparo. 1. Microscope according to any one of the preceding claims, comprising an electronic system in connection with at least Sos image acquisition means to control them to acquire images simultaneously in a single shot.
14:- Microscopio según la reivindicación 13 cuando depende de la 8, caracterizado porque dicho sistema electrónico también está conectado con los medios de iluminación para controlarlos para que emitan luz incoherente ai menos durante dicho único disparo de los medios de adquisición de imágenes. 14 : - Microscope according to claim 13 when it depends on the 8, characterized in that said electronic system is also connected with the lighting means to control them so that they emit incoherent light at least during said single shot of the image acquisition means.
15.- Microscopio según la reivindicación 13 ó 14, en ei que el sistema electrónico comprende unos medios de procesamiento internos adaptados para procesar las imágenes adquiridas para realizar al menos una de las siguientes tareas, a partir de la información tridimensional de la muestra contenida en las imágenes: - calcular y mostrar en ai menos una pantalla una reconstrucción en profundidad de la muestra, con imágenes enfocadas a diferentes profundidades; y 15. Microscope according to claim 13 or 14, wherein the electronic system comprises internal processing means adapted to process the acquired images to perform at least one of the following tasks, based on the three-dimensional information of the sample contained in the images: - calculate and show at least one screen a reconstruction in depth of the sample, with images focused at different depths; Y
- realizar de un mapa de profundidades o matriz tridimensional, en la que se indica la posición tridimensional de cada punto de ía muestra. - make a map of depths or three-dimensional matrix, which indicates the three-dimensional position of each point of the sample.
16.- Microscopio según la reivindicación 13, 14 ó 1 5, en ei que ei sistema electrónico comprende unos medios de comunicación, vía cable o inalámbricos, para el envió de las imágenes adquiridas a un sistema de procesamiento externo adaptado para procesar las imágenes adquiridas para realizar al menos una de las siguientes tareas, a partir de la información tridimensional de ¡a muestra contenida en las imágenes: 16. Microscope according to claim 13, 14 or 1, in which the electronic system comprises communication means, via cable or wireless, for sending the acquired images to an external processing system adapted to process the acquired images to perform at least one of the following tasks, based on the three-dimensional information of the sample contained in the images:
- calcular y mostrar en al menos una pantalla, del microscopio o externa al mismo, una reconstrucción en profundidad de la muestra, con imágenes enfocadas a diferentes profundidades; y - realizar de un mapa de profundidades o matriz tridimensional, en la que se indica ía posición tridimensional de cada punto de la muestra. - calculate and display on at least one screen, from the microscope or external to it, an in-depth reconstruction of the sample, with images focused at different depths; Y - make a map of depths or three-dimensional matrix, which indicates the three-dimensional position of each point of the sample.
17. - Sistema de microscopía integral, caracterizado porque comprende: 17. - Integral microscopy system, characterized in that it comprises:
- el microscopio según ¡a reivindicación 16; y - the microscope according to claim 16; Y
- un sistema de procesamiento adaptado para procesar las imágenes adquiridas por el microscopio para realizar ai menos una de las siguientes tareas, a partir de la información tridimensional de la muestra contenida en las imágenes: - a processing system adapted to process the images acquired by the microscope to perform at least one of the following tasks, based on the three-dimensional information of the sample contained in the images:
■ calcular y mostrar en ai menos una pantalla de! sistema una reconstrucción en profundidad de la muestra, con imágenes enfocadas a diferentes profundidades; y ■ calculate and display at least one screen of! system a deep reconstruction of the sample, with images focused at different depths; Y
- realizar de un mapa de profundidades o matriz tridimensional, en la que se indica la posición íridímensionaS de cada punto de ía muestra. - make a map of depths or three-dimensional matrix, in which the iridescent position of each point of the sample is indicated.
18. ;- Sistema según la reivindicación 17, caracterizado porque comprende al menos una impresora 3D comunicada con el sistema de procesamiento para al menos recibir dei mismo información relativa a dicho mapa de profundidades o matriz tridímensionai, y adaptada para imprimir una maqueta tridimensional de la muestra a partir de la información recibida. 18.; - System according to claim 17, characterized in that it comprises at least one 3D printer communicated with the processing system to at least receive the same information relating to said depth map or three-dimensional matrix, and adapted to print a three-dimensional model of the Sample from the information received.
19.- Uso del microscopio según una cualquiera de las reivindicaciones 1 a 16, para aplicaciones de bio-medicina, incluyendo el cribado de muestras biológicas in situ, analizando e! relieve de la muestra o de un seccionado en profundidad de la misma. 19. Use of the microscope according to any one of claims 1 to 16, for bio-medicine applications, including the screening of biological samples in situ, analyzing e! relief of the sample or an in-depth section of it.
20■■ Uso dei microscopio según una cualquiera de las reivindicaciones 1 a 18, para aplicaciones de perfiíomatría. Use of the microscope according to any one of claims 1 to 18, for perfiomatometry applications.
21,~ Uso según la reivindicación 20, en a! que dichas aplicaciones de perfílometria incluyen ai menos una de las siguientes aplicaciones: control de calidad de piezas o componentes, y ciencia forense. 21, Use according to claim 20, in a! that such profilometry applications include at least one of the following applications: quality control of parts or components, and forensic science.
PCT/ES2016/000135 2015-12-30 2016-12-20 Integral microscope, uses of same and integral microscopy system WO2017114985A1 (en)

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