WO2009141467A1 - Automated system and method for obtaining fully focused images with high-magnification microscopes - Google Patents

Automated system and method for obtaining fully focused images with high-magnification microscopes Download PDF

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Publication number
WO2009141467A1
WO2009141467A1 PCT/ES2009/000170 ES2009000170W WO2009141467A1 WO 2009141467 A1 WO2009141467 A1 WO 2009141467A1 ES 2009000170 W ES2009000170 W ES 2009000170W WO 2009141467 A1 WO2009141467 A1 WO 2009141467A1
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image
focused
images
focused images
automated system
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PCT/ES2009/000170
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Spanish (es)
French (fr)
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Joaquín MARTÍN CALLEJA
Moritz Kulawik
Francisco Javier Navas Pineda
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Universidad De Cádiz
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/241—Devices for focusing
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B7/00—Mountings, adjusting means, or light-tight connections, for optical elements
    • G02B7/28—Systems for automatic generation of focusing signals
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T5/00—Image enhancement or restoration
    • G06T5/50—Image enhancement or restoration by the use of more than one image, e.g. averaging, subtraction

Definitions

  • the use of a high magnification optical microscope for the realization of micrographs of three-dimensional objects presents enormous problems generated by the low depth of focus that can be obtained in the range of visible radiation, that is to say in the range of electromagnetic radiation that goes from 370 nanometers to 770 nanometers.
  • the depth of focus also called DOF of the English Depth Of Field
  • the focused observation of a sample that presents surface roughness with unevenness along the optical axis and within the observation field greater than 6 micrometers.
  • This DOF value varies with the square of the focal length, so that the use of objectives of greater magnifications (lower focal distances), entails a drastic decrease in the depth of focus or DOF, so that the greater the magnification, the greater will be the flatness requirements in the samples for their correct focused observation.
  • the confocal microscope is an optical microscope that uses a highly focused laser as a light source and incorporates two diaphragms: (a) a diaphragm or pinhole (in English terminology) of lighting that is located behind the light source, whose utility is to delimit The illumination of the object to a single point just at the point of focus and (b) a diaphragm or pinhole detection located in front of the photodetector whose utility is to restrict the acquisition of light only to that coming from the point of the object located just at the point of lighting corresponding to the focal point delimited by the lighting diaphragm.
  • the generation of an image of the object to be photographed implies traveling the entire surface of the same in the three directions of space through a micrometric displacement of the object to be photographed according to a sequence of planes at different distances from the objective, detecting in each plane only the points located in the Focal point.
  • the images thus obtained are very clear but monochromatic since the system uses a highly monochromatic laser as a source of illumination. This is a problem of great importance of confocai microscopy and in fact several techniques have been studied to obtain a solution that improves the quality of the images obtained.
  • One of the most used options is the use of fluorescent reagents (fluorochromes) to highlight areas of interest in the image.
  • Fluorochromes are substances that have the property of emitting light of a certain wavelength when they are illuminated with a radiation of a characteristic wavelength. Even so, the use of fluorochromes in microscopy presents several problems, such as (b1) the loss of fluorescence due to the great intensities of light used, so that the samples studied with this technique cannot be observed for long periods of time due to The disappearance of the fluorescence, a process called "fluorescence fading" and (b2) the Image obtained does not represent the actual chromaticity of the object but the chromaticity of the fluorochrome superimposed on that corresponding to that of the laser radiation. Therefore, the problem of obtaining polychromatic images using this technique is not solved, which limits its use. Despite this, it is not uncommon to find scientific articles in various fields of science, especially in the fields of biology, geology and materials science, in which the use of confocai microscopy is widespread. As an example, we can cite the following Bibliographical references:
  • Patent n 0 GB 2 385 481 A "Automated microscopy at a plurality of depth of focus through the thickness of a sample”. (Date: 08-20-2003).
  • This system allows obtaining multiple images of an object depending on the thickness of the object and the objective lens used to obtain images of all the parts of the object focused, but to document the object a very large set of images is necessary, and in many of them the area of interest or focused area can be very small and provide little information.
  • This system does not allow to obtain a fully automated image of the object focused in its entirety, since no algorithm is applied that is able to discern which is the focused area of each image obtained and subsequently join all these areas to obtain a single image of the object that will define us in its entirety.
  • Our system incorporates an algorithm that is capable of achieving this goal.
  • Patent No. WO 98/57211 "Optical system having an unlimited depth of focus”. (Date: 12-17-1998).
  • This patent describes a system composed of a system of lenses that generates a greater depth of field than conventional lenses, and that thanks to said set of lenses and a controlled and very rapid movement of the same generates in the observer a feeling of focused image at a greater depth of field than conventional lenses.
  • the restriction of this system is that the image that the observer sees is a sensation and is not captured by any system, so that this system is not comparable to the one presented in this document.
  • Patent No. US4661986 "Depth-of-focus imaging process method”. (Date: 04-28-1987).
  • This patent presents a system that allows images to be obtained at different object-objective lens distances and subsequently treats these images to obtain a more focused image of the object.
  • the treatment of these images is developed by means of a method called Burt's Pyramid, which is a method based on the correlation of regions, to which a Laplacian type operator is applied.
  • Burt's Pyramid is a method based on the correlation of regions, to which a Laplacian type operator is applied.
  • the limitation of this type of methods is that the analysis is carried out on regions of the samples, since their main utility lies in the field of movement studies, so they do not reach the level of resolution that the system presented in this patent.
  • the system that we expose in this document allows to obtain displaced images one of the following one less distance than the depth of focus of the objective lens used and then performs an analysis of all the captured images pixel by pixel, which allows to obtain a final image in where it is possible to visualize the object focused in all its extension.
  • the depth of focus with which it is possible to observe a three-dimensional object by means of a compound microscope is a property that depends on the magnification of the lens used as the objective, being practically independent of the characteristics of the eyepiece in the case of a direct observation or of the conversion lenses in the case of an observation by means of an image recording device, either digital or analog.
  • NA Opening Number
  • NA n * sen (u)
  • n is the refractive index of the medium in which the observation is made and u is the value of the flat half angle of the focal cone of the lens.
  • NA 1.25 for a 100X objective
  • the focusing characteristics of the objective lens can be known both in what refers to the minimum particle size that can be observed, usually referred to as spatial resolution, as in what refers to the value of the DOF
  • the resolution is defined as
  • Do is the minimum diameter of light in the focus or resolution
  • ⁇ is the number pi
  • n is the refractive index of the observation medium
  • ⁇ is the wavelength of the radiation used as a reference, whose value is 550 nanometers , since it is the one to which the human eye has the highest sensitivity and is also the one with the greatest intensity in the solar irradiation spectrum.
  • This value of D 0 is also used for the definition of the DOF, this being the distance forward and backward from the focal point of the objective lens and along the optical focus axis, between which the value of the focal point area the area in the focus is not greater than twice (figure 1).
  • This DOF distance can be calculated through the mathematical expression
  • Target of 40X * DOF of 0.83 micrometers.
  • Target of 6OX * DOF of 0.48 micrometers.
  • Objective of 8OX DOF of 0.32 micrometers.
  • Objective of 100X * DOF of 0.22 micrometers.
  • DOF depth of focus
  • the one that offers a better adjustment for said data is a decreasing exponential function of the second order, as it is possible to observe in Figure 2.
  • the exponential function of the second order is
  • the objective of the present invention is to obtain focused images in a range greater than the one defined by the depth of field by means of: (a) acquisition of a set of focused images, at different distances of the objective / object lens, with a variation of said objective / object distance not exceeding that defined by the DOF value of the objective used, (b) extraction, for each image, of the focused part by means of an adequate computer treatment of the data that make up the image and the application of a mathematical algorithm based on the maximization of the variance of each point with respect to those of its closest environment and (c) composition of a new complete image of the object as a sum of the focused parts extracted from each image.
  • the device used consists of several components described below (figure 3):
  • the microscope must have the ability to send the image observed by the objective (1a1) to an image capture device (1b). Since the object to be observed is three-dimensional and is supposed to be opaque, the microscope to be used must have some type of azimuthal illumination system, either intraocular (1a2) or extraocular (1a3), and in the latter case it may be with or without axial symmetry.
  • the procedure that will be described later for the generation of micrographs of wide focus works with digitized images, so that the element for its capture can be a digital image acquisition system coupled to the microscope (1a) or an image acquisition system non-digital (film, plate, etc.), in this case, it is necessary to carry out a subsequent digitalization of the images obtained.
  • This displacement capacity must be compatible with the resolutions derived from the indicated depth of field values, that is, to work with 100X lenses the system must be able to advance in steps no larger than 0.22 micrometers.
  • This system consists of: (2a) high precision movement element capable of complying with the aforementioned conditioner, (2b) necessary supports for the high precision movement element, (2c) electrical connection necessary with the computerized control subsystem (3) and (2d) necessary couplings so that the application of the movement from the high precision movement element (2a) to the object is optimal.
  • the computerized control and image processing subsystem (3) consists of a computer (3a) capable of processing the information contained in the acquired images, and an element composed of sentences organized according to a logical operating criterion (3b) that allows the processing of the images acquired to obtain micrographs of wide focus.
  • the equipment uses to obtain the desired result of a well focused image in a wide range of depths, a set of images that have been obtained at different distances object / objective lens.
  • the computerized subsystem of control and treatment of images (3) is based mainly on a mathematical algorithm that allows to obtain from each captured image the area of the same that is focused.
  • the algorithm is based on the determination of the variance of a perimeter set of points with respect to the analyzed point, the thickness of said perimeter can be defined by the user. This algorithm is completed with the logical routines necessary so that from the focused areas of each captured image it is possible to obtain a new image of the object throughout the entire length of study and fully focused.
  • the objective to be used is chosen based on the study to be developed, which will have a certain depth of field depending on its magnification, such and as stated above.
  • the value of the depth of field will establish the values of the displacement that the object will suffer between each of the images that will be acquired, and therefore the values in which the object / objective lens distance between each captured image will vary, which should be equal or less than the depth of field.
  • the computerized subsystem Control and image processing (3) will establish the number of displacements of the object and therefore the optimal number of images to be captured, using Ia expression obtained above that relates the DOF with the magnification and applying a factor, as previously stated, so that there is a certain overlap between an image and the next.
  • the image control and processing information subsystem (3) will begin the image taking process, controlling that the modification system of the object distance / objective lens (2) produces the displacement of the object until it is placed in a position suitable for taking images.
  • the element for capturing images (1 b) will acquire an image that will focus on a part of the object.
  • This acquisition can be carried out automatically through the computerized control and image processing subsystem (3) and using an automated image capture element, or manually if a device that performs its function in this way is used. In this case, the images must be transferred to the computerized system and digitalized if necessary.
  • the process of positioning the object in the image capture position and the acquisition of an image is repeated as many times as necessary to cover the entire depth of the object.
  • the displacement that this one will suffer in each step will be less than the depth of field that the objective lens used presents, thus ensuring that the image capture element (1 b) acquires as many as are necessary to cover the entire object.
  • each of the "n” "imacapas” is broken down into its primary components R, G, B, H, Sy L, generating the sub-layers of data nR, nG, nB, nH, nS and nL From These sub-layers are composed of a new layer of data obtained through the sum:
  • a calculation process is carried out consisting of the consecutive extraction and organized by rows and columns of sub-matrices of dimensions data (v, v), v being an odd number between 3 and 9, which will be set in advance, and will also be constant throughout the process.
  • the variance is calculated, defined as
  • the set of the values of the variance generated for each of the "anacapas” and organized by rows and columns forms a new matrix of data called “varcapa", obtaining a total of "n” "varcapas".
  • Each of the "varcapas” will have as dimensions ( ⁇ i-v ⁇ , ⁇ j-v ⁇ ) and will be a representation of the evolution of the similarity of the data that define an image at the length and width of the "imacapa" layer.
  • the image focused on the entire length of the object is formed with the original RGB-HSL values in the layer of best focus for each position.
  • another aspect of vital importance of the system and procedure presented here is that it does not modify the chromaticity of the object to be studied because it deals with numerical data of the colors that make up the image without interacting with the lighting system or detection in no time. In this way, this system acts as a microscope that was able to observe at a depth of field eligible by the user of the same, thus constituting a great advance in the field of optical microscopy in particular, and of microscopy in general.
  • Figure 1 Scheme in which the opening number and depth of field are defined from a geometric point of view.
  • Figure 3 General scheme of the automated system for obtaining fully focused images with high magnification microscopes, where the following components are observed: (1) Image capture subsystem. (1a) Optical microscope.

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Abstract

Automated system and method for obtaining fully focused images with high-magnification microscopes. The methodology and instrumentation for obtaining images with high-magnification microscopes, focused throughout the focal range of interest, independently of the objective used and maintaining the chromaticity of the observed object completely stable. A methodology is used that makes it possible to obtain partially focused images at different objective/object distances for subsequent extraction of the focused portion of each image and cumulative generation of the global focused image of the object. The invention is applicable to the automation of monocular, binocular or triocular optical microscopes with a view to obtaining perfectly focused optical images throughout the chromatic range.

Description

SISTEMA AUTOMATIZADO Y PROCEDIMIENTO PARA LA OBTENCIÓN DE IMÁGENES TOTALMENTE FOCALIZADASCON MICROSCOPIOSDE ELEVADA MAGNIFICACIÓN. AUTOMATED SYSTEM AND PROCEDURE FOR OBTAINING FULLY FOCUSED IMAGES WITH MICROSCOPES OF ELEVATED MAGNIFICATION.
Sectoresde Ia técnica G02B21/00M4A7F, G02B21/00M4A7Sectors of the technique G02B21 / 00M4A7F, G02B21 / 00M4A7
GeneralidadesGeneralities
La utilización de un microscopio óptico de elevada magnificación para Ia realización de micrografías de objetos tridimensionales presenta enormes problemas generados por Ia escasa profundidad de foco que es posible obtener en el rango de Ia radiación visible, es decir en el rango de Ia radiación electromagnética que va de los 370 nanómetros a los 770 nanómetros. En concreto y a modo de ejemplo, mediante un objetivo de 1OX (10 aumentos), Ia profundidad de foco (también llamada DOF del inglés Depth Of Field) tiene un valor aproximado de 6 micrómetros. Esto implica que no será factible Ia observación focalizada de una muestra que presente rugosidades superficiales con desniveles a Io largo del eje óptico y dentro del campo de observación superiores a 6 micrómetros. Este valor del DOF varía con el cuadrado de Ia distancia focal por Io que Ia utilización de objetivos de mayores magnificaciones (menores distancias focales), conlleva una drástica disminución de Ia profundidad de foco o DOF, de forma que cuanto mayor sea Ia magnificación tanto mayores serán los requerimientos de planitud en las muestras para su correcta observación focalizada.The use of a high magnification optical microscope for the realization of micrographs of three-dimensional objects presents enormous problems generated by the low depth of focus that can be obtained in the range of visible radiation, that is to say in the range of electromagnetic radiation that goes from 370 nanometers to 770 nanometers. Specifically and by way of example, by means of a 1OX (10x) objective, the depth of focus (also called DOF of the English Depth Of Field) has an approximate value of 6 micrometers. This implies that it will not be feasible the focused observation of a sample that presents surface roughness with unevenness along the optical axis and within the observation field greater than 6 micrometers. This DOF value varies with the square of the focal length, so that the use of objectives of greater magnifications (lower focal distances), entails a drastic decrease in the depth of focus or DOF, so that the greater the magnification, the greater will be the flatness requirements in the samples for their correct focused observation.
La metodología y Ia instrumentación que aquí se propone consigue salvar esta barrera ya que permite Ia obtención de imágenes enfocadas en todo el rango focal que nos interese, independientemente del objetivo utilizado y manteniendo totalmente estable Ia cromaticidad del objeto observado.The methodology and instrumentation proposed here manages to overcome this barrier since it allows obtaining images focused on the entire focal range that interests us, regardless of the objective used and keeping the chromaticity of the object observed completely stable.
Estadode Ia técnicaState of the technique
Existen algunos sistemas y diseños experimentales que abordan de forma parcial esta problemática tanto en el campo de Ia microfotog rafia (imágenes obtenidas a través de un microscopio), como en el de Ia macrofotog rafia (fotografía clásica pero utilizando lentes especiales para Ia fotografía de pequeños objetos). Algunos de estos sistemas son únicamente aplicables a Ia obtención de imágenes convencionales, no digitalizadas, que permiten Ia obtención de imágenes cromáticas de objetos con dimensiones centimétricas. En este caso se encuentra el sistema que utiliza como sistema de iluminación un proyector de luz especialmente adaptado para emitir un plano de luz perpendicular al eje óptico formado por el sistema de observación y el objeto a observar y situado justo a Ia distancia de focalización del objetivo de forma que sólo están iluminadas las partes del objeto que se encuentran a Ia distancia de focalización. Para conseguir obtener una imagen focalizada del objeto es preciso efectuar múltiples exposiciones sobre el mismo fotograma sincronizadas con el desplazamiento del objeto a fotografiar a Io largo del eje óptico definido por Ia cámara. Este sistema presenta severas complicaciones de uso por Ia necesidad de eliminar vibraciones o movimientos indeseados tanto de Ia cámara como del objeto así como de Ia iluminación secundaria de las partes del objeto fuera del plano de iluminación debidas a las reflexiones generadas por las zonas iluminadas y por Ia difracción de Ia luz generadas en los elementos limitantes del proyector usados para definir el plano de iluminación. Un ejemplo de este tipo de sistemas es posible encontrar en Ia patente " Method of and apparatus for the expansión of the range of the depth of focus beyond the limit given by conventional images" (patente n° US4,141 ,032, 20-02-1979), en el que las imágenes obtenidas de forma convencional son mostradas en un monitor una vez que se Ie han aplicado filtros para eliminar las zonas no focalizadas de las imágenes obtenidas en cada exposición al sistema de captura de imágenes.There are some experimental systems and designs that partially address this problem both in the field of raffia microfotog (images obtained through a microscope), and in that of macrophage raffia (classical photography but using special lenses for small photography objects). Some of these systems are only applicable to obtaining conventional, non-digitized images, which allow obtaining chromatic images of objects with centimeter dimensions. In this case there is the system that uses as a lighting system a light projector specially adapted to emit a plane of light perpendicular to the optical axis formed by the observation system and the object to be observed and located just at the target focusing distance so that only the parts of the object that are at the focusing distance are illuminated. To obtain a focused image of the object it is necessary to make multiple exposures on the same frame synchronized with the displacement of the object to be photographed along the optical axis defined by the camera. This system presents severe complications of use due to the need to eliminate unwanted vibrations or movements of both the camera and the object as well as the secondary illumination of the parts of the object outside the illumination plane due to the reflections generated by the illuminated areas and by The diffraction of the light generated in the limiting elements of the projector used to define the lighting plane. An example of this type of systems can be found in the "Method of and apparatus for the expansion of the range of the depth of focus beyond the limit given by conventional images" patent (US Pat. No. 4,141, 032, 20-02 -1979), in which the images obtained in a conventional manner are shown on a monitor once filters have been applied to eliminate the non-focused areas of the images obtained in each exposure to the image capture system.
En el caso de Ia fotografía digital y aplicado a Ia microfotografía uno de los sistemas más exitosos es el de los microscopios confocales. El microscopio confocal, es un microscopio óptico que utiliza como fuente de iluminación un láser altamente focalizado e incorpora dos diafragmas: (a) un diafragma o pinhole (en terminología inglesa) de iluminación que se encuentra localizado tras Ia fuente luminosa, cuya utilidad es delimitar Ia iluminación del objeto a un único punto justo en el punto de focalización y (b) un diafragma o pinhole de detección situando delante del fotodetector cuya utilidad es restringir Ia captación de luz únicamente a aquella proveniente del punto del objeto situado justo en el punto de iluminación correspondiente al punto focal delimitado por el diafragma de iluminación. Dado que tanto Ia iluminación como Ia observación están restringidas a un único punto, Ia generación de una imagen del objeto a fotografiar implica recorrer toda Ia superficie del mismo en las tres direcciones del espacio mediante un desplazamiento micrométrico del objeto a fotografiar según una secuencia de planos a diferentes distancias del objetivo, detectando en cada plano únicamente los puntos situados en el punto focal. Las imágenes así obtenidas son muy nítidas pero monocromáticas ya que el sistema utiliza como fuente de iluminación un láser altamente monocromático. Este es un problema de gran importancia de Ia microscopía confocai y de hecho se han estudiado diversas técnicas para obtener una solución que mejore Ia calidad de las imágenes obtenidas. Una de las opciones más utilizadas es el uso de reactivos fluorescentes (fluorocromos) para resaltar zonas de interés de Ia imagen. Los fluorocromos son sustancias que tienen Ia propiedad de emitir luz de una longitud de onda determinada cuando son iluminados con una radiación de una longitud de onda característica. Aún así, el uso de fluorocromos en microscopía presenta diversos problemas, como son (b1 ) Ia pérdida de fluorescencia debido a las grandes intensidades de luz empleadas, por Io que las muestras estudiadas con esta técnica no puede observadas durante largos periodo de tiempo debido a Ia desaparición de Ia fluorescencia, proceso denominado "fluorescence fading" y (b2) Ia Imagen obtenida no representa a Ia cromaticidad real del objeto sino a Ia cromaticidad del fluorocromo superpuesta a Ia correspondiente a Ia de Ia radiación láser. Por tanto, el problema de obtener imágenes policromáticas utilizando esta técnica no queda resuelto, Io que limita su uso. A pesar de ello, no es raro encontrar artículos científicos en diversos campos de Ia ciencia, sobre todo en los campos de Ia biología, Ia geología y Ia ciencia de los materiales, en los que el empleo de Ia microscopía confocai está muy extendido. Como ejemplo, podemos citar las siguientes referencias bibliográficas:In the case of digital photography and applied to the photomicrograph, one of the most successful systems is that of confocal microscopes. The confocal microscope is an optical microscope that uses a highly focused laser as a light source and incorporates two diaphragms: (a) a diaphragm or pinhole (in English terminology) of lighting that is located behind the light source, whose utility is to delimit The illumination of the object to a single point just at the point of focus and (b) a diaphragm or pinhole detection located in front of the photodetector whose utility is to restrict the acquisition of light only to that coming from the point of the object located just at the point of lighting corresponding to the focal point delimited by the lighting diaphragm. Since both the lighting and the observation are restricted to a single point, the generation of an image of the object to be photographed implies traveling the entire surface of the same in the three directions of space through a micrometric displacement of the object to be photographed according to a sequence of planes at different distances from the objective, detecting in each plane only the points located in the Focal point. The images thus obtained are very clear but monochromatic since the system uses a highly monochromatic laser as a source of illumination. This is a problem of great importance of confocai microscopy and in fact several techniques have been studied to obtain a solution that improves the quality of the images obtained. One of the most used options is the use of fluorescent reagents (fluorochromes) to highlight areas of interest in the image. Fluorochromes are substances that have the property of emitting light of a certain wavelength when they are illuminated with a radiation of a characteristic wavelength. Even so, the use of fluorochromes in microscopy presents several problems, such as (b1) the loss of fluorescence due to the great intensities of light used, so that the samples studied with this technique cannot be observed for long periods of time due to The disappearance of the fluorescence, a process called "fluorescence fading" and (b2) the Image obtained does not represent the actual chromaticity of the object but the chromaticity of the fluorochrome superimposed on that corresponding to that of the laser radiation. Therefore, the problem of obtaining polychromatic images using this technique is not solved, which limits its use. Despite this, it is not uncommon to find scientific articles in various fields of science, especially in the fields of biology, geology and materials science, in which the use of confocai microscopy is widespread. As an example, we can cite the following bibliographical references:
1. "Viscoelastíc properties of high pressure and heat induced tofu gels", Saowapark, S. et al, Food Chemístry, VoI. 107 (2008).1. "Viscoelastíc properties of high pressure and heat induced tofu gels", Saowapark, S. et al, Food Chemístry, VoI. 107 (2008).
2. "Láser scanning confocai arthroscopy of a fresh cadaveric knee joint", Jones, CW. Et al, Osteorarthritis and Cartilage, VoI. 15 (2007). 3. "Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia CeII Iine-K562", Sreekanth, D. et al, Phytomedicine, VoI. 14 (2007).2. "Laser scanning confocai arthroscopy of a fresh cadaveric knee joint", Jones, CW. Et al, Osteorarthritis and Cartilage, VoI. 15 (2007). 3. "Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia CeII Iine-K562", Sreekanth, D. et al, Phytomedicine, VoI. 14 (2007).
4. "A triphasic ceramic-coated porous hydroxyapatite for tissue engineering appllcation", Acta Biomaterialia, VoI. 4 (2008). Podemos observar en estas referencias el uso de microscopía confocal para Ia obtención de imágenes focalizadas. La actualidad de los trabajos citados Indica Ia necesidad de contar con un sistema que proporcione imágenes focalizadas más allá del límite impuesto por Ia focalización con lentes convexas y, por tanto, el sistema que aquí presentamos y que es objeto de Ia patente de invención será de gran utilidad en muchos campos científicos.4. "A triphasic ceramic-coated porous hydroxyapatite for tissue engineering appllcation", Acta Biomaterialia, VoI. 4 (2008). We can observe in these references the use of confocal microscopy to obtain focused images. The actuality of the works cited indicates the need to have a system that provides focused images beyond the limit imposed by focusing with convex lenses and, therefore, the system presented here and which is the subject of the invention patent will be Great utility in many scientific fields.
Además, de estas referencias que nos muestra que el problema que conseguimos resolver con el sistema y procedimiento que presentamos en esta patente es de gran importancia en Ia investigación actual, también podemos observar en Ia bibliografía de patentes de las últimas décadas Ia presencia de varios sistemas que intentan resolver este mismo problema, con Io que se redunda en Ia ¡dea de que el sistema que en este documento presentamos puede ser un avance de gran interés. Todos estos sistemas aportan unas mejoras con respecto a Ia microscopia óptica clásica, pero no resuelven en toda su extensión el problema, y presentan ciertas deficiencias que nuestro sistema supera. A continuación, se exponen algunos sistemas que podemos encontrar en Ia bibliografía, y se comparan con el que aquí se presenta:In addition, of these references that shows us that the problem that we managed to solve with the system and procedure that we present in this patent is of great importance in current research, we can also observe in the patent literature of the last decades the presence of several systems that try to solve this same problem, with what results in the idea that the system that we present in this document can be an advance of great interest. All these systems provide improvements with respect to classical optical microscopy, but they do not solve the problem in its entirety, and present certain deficiencies that our system overcomes. Next, some systems that we can find in the bibliography are exposed, and they are compared with the one presented here:
1. Patente n0 GB 2 385 481 A: "Automated microscopy at a plurality of depth of focus through the thickness of a sample". (Fecha: 20-08-2003). Este sistema permite obtener múltiples imágenes de un objeto en función del grosor de Ia misma y de Ia lente objetivo utilizada para obtener imágenes de todas las partes del objeto focalizadas, pero para documentar el objeto es necesario un conjunto de imágenes muy amplio, y en muchas de ellas Ia zona de interés o zona focalizada puede ser muy pequeña y aportar poca información. Este sistema no permite obtener de forma completamente automatizada una imagen del objeto focalizada en toda su extensión, pues no se aplica ningún algoritmo que sea capaz de discernir cual es Ia zona focalizada de cada imagen obtenida y con posterioridad unir todas estas zonas para obtener una única imagen del objeto que nos Io definirá en toda su extensión. Nuestro sistema incorpora un algoritmo que sí es capaz de lograr este objetivo. 2. Patente n° WO 98/57211 : "Optical system having an unlimited depth of focus". (Fecha: 17-12-1998).1. Patent n 0 GB 2 385 481 A: "Automated microscopy at a plurality of depth of focus through the thickness of a sample". (Date: 08-20-2003). This system allows obtaining multiple images of an object depending on the thickness of the object and the objective lens used to obtain images of all the parts of the object focused, but to document the object a very large set of images is necessary, and in many of them the area of interest or focused area can be very small and provide little information. This system does not allow to obtain a fully automated image of the object focused in its entirety, since no algorithm is applied that is able to discern which is the focused area of each image obtained and subsequently join all these areas to obtain a single image of the object that will define us in its entirety. Our system incorporates an algorithm that is capable of achieving this goal. 2. Patent No. WO 98/57211: "Optical system having an unlimited depth of focus". (Date: 12-17-1998).
En esta patente se describe un sistema compuesto por un sistema de lentes que genera una profundidad de campo mayor que las lentes convencionales, y que gracias a dicho conjunto de lentes y a un movimiento controlado y muy veloz de las mismas genera en el observador una sensación de imagen focalizada en una profundidad de campo mayor que las lentes convencionales. Obviamente, Ia restricción de este sistema es que Ia imagen que ve el observador es una sensación y no es capturada por ningún sistema, por Io que este sistema no es comparable al que en este documento se presenta.This patent describes a system composed of a system of lenses that generates a greater depth of field than conventional lenses, and that thanks to said set of lenses and a controlled and very rapid movement of the same generates in the observer a feeling of focused image at a greater depth of field than conventional lenses. Obviously, the restriction of this system is that the image that the observer sees is a sensation and is not captured by any system, so that this system is not comparable to the one presented in this document.
3. Patente n° US4661986: "Depth-of-focus imaging process method". (Fecha: 28-04- 1987). En esta patente se presenta un sistema que permite obtener imágenes a diferentes distancias objeto- lente objetivo y que posteriormente trata estas imágenes para obtener una imagen Io más focalizada del objeto. El tratamiento de estas imágenes se desarrolla por medio de un método denominado Pirámide de Burt, que es un método basado en Ia correlación de regiones, a las que se les aplica un operador tipo laplaciano. La limitación de este tipo de métodos es que se desarrolla el análisis sobre regiones de las muestras, pues su principal utilidad reside en el campo de estudios de movimientos, por Io que no llegan al nivel de resolución que el sistema que presentamos en esta patente. El sistema que exponemos en este documento permite obtener imágenes desplazadas una de Ia siguiente una distancia menor que Ia profundidad de foco de Ia lente objetivo utilizada y después realiza un análisis de todas las imágenes capturadas pixel a pixel, Io que permite obtener una imagen final en donde es posible visualizar el objeto focalizado en toda su extensión.3. Patent No. US4661986: "Depth-of-focus imaging process method". (Date: 04-28-1987). This patent presents a system that allows images to be obtained at different object-objective lens distances and subsequently treats these images to obtain a more focused image of the object. The treatment of these images is developed by means of a method called Burt's Pyramid, which is a method based on the correlation of regions, to which a Laplacian type operator is applied. The limitation of this type of methods is that the analysis is carried out on regions of the samples, since their main utility lies in the field of movement studies, so they do not reach the level of resolution that the system presented in this patent. The system that we expose in this document allows to obtain displaced images one of the following one less distance than the depth of focus of the objective lens used and then performs an analysis of all the captured images pixel by pixel, which allows to obtain a final image in where it is possible to visualize the object focused in all its extension.
Salvo los sistemas descritos anteriormente, no comparables al que aquí se presenta, no conocemos ningún otro sistema que permita Ia obtención de microfotog rafias bien focalizadas de objetos que presenten en su superficie una variación de niveles mayor que Ia correspondiente a Ia profundidad de foco del objetivo utilizado, manteniendo además totalmente estable Ia cromaticidad del objeto observado, condición ésta que, tal y como hemos explicado, no es fácil de obtener utilizando las técnicas descritas anteriormente, y que aporta un valor añadido al sistema que en esta patente se presenta.Except for the systems described above, not comparable to the one presented here, we do not know of any other system that allows the obtaining of well-focused microfotographs of objects that have a variation in levels greater than that corresponding to the depth of focus of the objective. used, also keeping the chromaticity of the object observed totally stable, this condition which, as we have explained, is not easy to obtain using the techniques described above, and that adds value to the system presented in this patent.
Descripción de Ia invención-Description of the invention-
Introducción La profundidad de foco con que es posible observar un objeto tridimensional mediante un microscopio compuesto es una propiedad que depende de Ia magnificación de Ia lente usada como objetivo, siendo prácticamente independiente de las características del ocular para el caso de una observación directa o de las lentes de conversión en el caso de una observación mediante un dispositivo de registro de imágenes, bien sea digital o analógico. Además, aunque existen en el mercado diversos tipos y calidades de objetivos de microscopio, prácticamente todos ellos presentan unas características ópticas fijas de forma que existe una correlación entre el valor de Ia magnificación del objetivo y Ia magnitud denominada Número de Apertura (NA), ésta última relacionada con el valor del ángulo del cono de focalización del objetivo (figura 1) y calculable a través de Ia expresiónIntroduction The depth of focus with which it is possible to observe a three-dimensional object by means of a compound microscope is a property that depends on the magnification of the lens used as the objective, being practically independent of the characteristics of the eyepiece in the case of a direct observation or of the conversion lenses in the case of an observation by means of an image recording device, either digital or analog. In addition, although there are several types and qualities of microscope lenses on the market, practically all of them have fixed optical characteristics so that there is a correlation between the magnification value of the objective and the magnitude called Opening Number (NA), this last related to the angle value of the objective focusing cone (figure 1) and calculable through the expression
NA=n*sen(u)NA = n * sen (u)
donde n es el índice de refracción del medio en el que se efectúa Ia observación y u es el valor del semiángulo plano del cono focal de Ia lente. Los valores que toma el Número de Apertura para las magnificaciones más habituales de los objetivos de microscopio son:where n is the refractive index of the medium in which the observation is made and u is the value of the flat half angle of the focal cone of the lens. The values taken by the Aperture Number for the most common magnifications of the microscope objectives are:
NA= 0.1 para un objetivo de 4X NA= 0.25 para un objetivo de 10X NA= 0.40 para un objetivo de 2OXNA = 0.1 for a 4X objective NA = 0.25 for a 10X objective NA = 0.40 for a 2OX objective
NA= 0.65 para un objetivo de 4OXNA = 0.65 for a 4OX objective
NA = 0.85 para un objetivo de 6OX,NA = 0.85 for a 6OX objective,
NA = 1.05 para un objetivo de 8OXNA = 1.05 for an 8OX objective
NA= 1.25 para un objetivo de 100X A partir del valor del Número de Apertura se pueden conocer las características de focalización de Ia lente objetivo tanto en Io que se refiere al tamaño mínimo de partícula que se puede observar, denominado habitualmente como resolución espacial, como en Io que se refiere al valor del DOF. Así, Ia resolución se define comoNA = 1.25 for a 100X objective From the value of the Aperture Number, the focusing characteristics of the objective lens can be known both in what refers to the minimum particle size that can be observed, usually referred to as spatial resolution, as in what refers to the value of the DOF Thus, the resolution is defined as
Dn=- *w* π NAD n = - * w * π NA
donde Do es el diámetro mínimo de luz en el foco o resolución, π es el número pi, n es el índice de refracción del medio de observación y λ es Ia longitud de onda de Ia radiación utilizada como referencia, cuyo valor es de 550 nanómetros, ya que es aquella a Ia que el ojo humano presenta Ia máxima sensibilidad y también es Ia de mayor intensidad en el espectro de irradiación solar. Este valor de D0 se utiliza también para Ia definición del DOF, siendo ésta Ia distancia hacia adelante y hacia atrás desde el punto focal de Ia lente objetivo y a Io largo del eje óptico de focalización, entre Ia cual el valor del área del punto focal no es mayor que dos veces el área en el foco (figura 1). Esta distancia DOF se puede calcular a través de Ia expresión matemáticawhere Do is the minimum diameter of light in the focus or resolution, π is the number pi, n is the refractive index of the observation medium and λ is the wavelength of the radiation used as a reference, whose value is 550 nanometers , since it is the one to which the human eye has the highest sensitivity and is also the one with the greatest intensity in the solar irradiation spectrum. This value of D 0 is also used for the definition of the DOF, this being the distance forward and backward from the focal point of the objective lens and along the optical focus axis, between which the value of the focal point area the area in the focus is not greater than twice (figure 1). This DOF distance can be calculated through the mathematical expression
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Figure imgf000009_0001
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Figure imgf000009_0001
donde todos los términos han sido previamente definidos. Utilizando esta expresión se pueden calcular los valores de profundidad de foco que presentan los objetivos más usuales: Objetivo de 4X=> DOF de 35 micrómetros.where all terms have been previously defined. Using this expression you can calculate the depth of focus values that have the most common objectives: 4X Objective => 35 micron DOF.
Objetivo de 10X=> DOF de 5.6 micrómetros.10X objective => 5.6 micron DOF.
Objetivo de 20X=* DOF de 2.19 micrómetros.20X objective = * DOF of 2.19 micrometers.
Objetivo de 40X=* DOF de 0.83 micrómetros.Target of 40X = * DOF of 0.83 micrometers.
Objetivo de 6OX=* DOF de 0.48 micrómetros. Objetivo de 8OX => DOF de 0.32 micrómetros.Target of 6OX = * DOF of 0.48 micrometers. Objective of 8OX => DOF of 0.32 micrometers.
Objetivo de 100X=* DOF de 0.22 micrómetros. En definitiva, esto significa que, por ejemplo, si se observa con un objetivo de 4OX, que presenta una profundidad de foco, DOF, de 0.83 micrómetros, un objeto tridimensional que tenga irregularidades superficiales mayores en tamaño que el valor indicado para Ia profundidad de foco, sólo podrá ser visible de forma bien focalizada una franja del objeto de espesor equivalente al valor del DOF, situada a cualquier altura del objeto, pero sólo de dicho espesor.Objective of 100X = * DOF of 0.22 micrometers. In short, this means that, for example, if it is observed with a 4OX objective, which has a depth of focus, DOF, of 0.83 micrometers, a three-dimensional object that has surface irregularities larger in size than the value indicated for the depth of focus, only a strip of the object of thickness equivalent to the value of the DOF, located at any height of the object, but only of said thickness may be visible in a well-focused manner.
Por otra parte, si se representan los datos de DOF frente a Ia magnificación es posible encontrar una relación entre ambas magnitudes, que nos permite definir una función genérica para cualquier lente objetivo, Io que nos posibilita poder obtener Ia distancia máxima de separación entre dos imágenes a capturar por el sistema.On the other hand, if the DOF data is represented against the magnification it is possible to find a relationship between both magnitudes, which allows us to define a generic function for any objective lens, which allows us to obtain the maximum separation distance between two images to capture by the system.
Hemos ajustado los datos teóricos a diversas funciones matemáticas encontrando queWe have adjusted the theoretical data to various mathematical functions finding that
Ia que ofrece un mejor ajuste para dichos datos es una función exponencial decreciente de segundo orden, tal y como es posible observar en Ia figura 2. La función exponencial de segundo orden esThe one that offers a better adjustment for said data is a decreasing exponential function of the second order, as it is possible to observe in Figure 2. The exponential function of the second order is
y=yo+Al e{~xlíl)+A2e{-χ2la] ,y = y o + Al e {~ xlíl) + A2e { - χ2la] ,
donde las variables x e y son Ia magnificación y DOF, respectivamente y las constantes toman los valores: yo=O;Al = 166,433; tl=2,3S5; A2 =4,530 ;t2=26,010 y R2=0,99995 que nos da una idea del buen ajuste entre los datos teóricos y Ia función definida.where the variables x and y are the magnification and DOF, respectively and the constants take the values: y o = O; Al = 166,433; tl = 2.3S5; A2 = 4,530; t2 = 26,010 and R 2 = 0,99995 that gives us an idea of the good fit between the theoretical data and the defined function.
En nuestro sistema las imágenes se espaciarán el valor de DOF que se obtiene multiplicando éste por un factor que oscila entre 0.7 y 0.9 para que exista un solapamiento entre una imagen y Ia siguiente. Así pues, hemos diseñado el sistema con Ia característica de que sea capaz de obtener imágenes espaciadas entre ellas, según el eje óptico de observación, una distancia menor que el valor de DOF de Ia lente objetivo utilizada. Descripción del procedimientoIn our system the images will be spaced the DOF value that is obtained by multiplying it by a factor that ranges between 0.7 and 0.9 so that there is an overlap between one image and the next. Thus, we have designed the system with the characteristic that it is capable of obtaining images spaced between them, according to the optical axis of observation, a distance smaller than the DOF value of the objective lens used. Procedure description
El objetivo de Ia presente invención es Ia obtención de imágenes focalizadas en un rango superior al meramente definido por Ia profundidad de campo mediante: (a) adquisición de un conjunto de imágenes focalizadas, a diferentes distancias lente objetivo/objeto, con una variación de dicha distancia objetivo/objeto no superior a la definida por el valor del DOF del objetivo utilizado, (b) extracción, para cada imagen, de Ia parte focalizada mediante un adecuado tratamiento informático de los datos que componen Ia imagen y Ia aplicación de un algoritmo matemático basado en Ia maximización de Ia varianza de cada punto respecto a los de su entorno más próximo y (c) composición de una nueva imagen completa del objeto como suma de las partes focalizadas extraídas de cada imagen.The objective of the present invention is to obtain focused images in a range greater than the one defined by the depth of field by means of: (a) acquisition of a set of focused images, at different distances of the objective / object lens, with a variation of said objective / object distance not exceeding that defined by the DOF value of the objective used, (b) extraction, for each image, of the focused part by means of an adequate computer treatment of the data that make up the image and the application of a mathematical algorithm based on the maximization of the variance of each point with respect to those of its closest environment and (c) composition of a new complete image of the object as a sum of the focused parts extracted from each image.
Para alcanzar esta meta, el dispositivo utilizado consta de diversos componentes que se describen a continuación (figura 3):To achieve this goal, the device used consists of several components described below (figure 3):
(1 ) Subsistema de captura de imágenes(1) Image capture subsystem
Se compone de:It consists of:
(1a) Microscopio óptico El microscopio debe poseer Ia capacidad de enviar Ia imagen observada por el objetivo (1a1) a un dispositivo de captura de imágenes (1b). Dado que el objeto a observar es tridimensional y se supone opaco, el microscopio a usar debe poseer algún tipo de sistema de iluminación azimutal, bien sea de tipo intraocular (1a2) o bien extraocular (1a3), pudiendo ser en este último caso con o sin simetría axial. (1a) Optical microscope The microscope must have the ability to send the image observed by the objective (1a1) to an image capture device (1b). Since the object to be observed is three-dimensional and is supposed to be opaque, the microscope to be used must have some type of azimuthal illumination system, either intraocular (1a2) or extraocular (1a3), and in the latter case it may be with or without axial symmetry.
(1b) Elemento para Ia captura de imágenes(1b) Element for image capture
El procedimiento que se describirá más adelante para Ia generación de micrografías de amplia focalización trabaja con imágenes digitalizadas, por Io que el elemento para su captura puede ser un sistema digital de adquisición de imágenes acoplado al microscopio (1a) o un sistema de adquisición de imágenes no digital (película, placa, etc), debiéndose en este caso, efectuar una posterior digital ización de las imágenes obtenidas.The procedure that will be described later for the generation of micrographs of wide focus works with digitized images, so that the element for its capture can be a digital image acquisition system coupled to the microscope (1a) or an image acquisition system non-digital (film, plate, etc.), in this case, it is necessary to carry out a subsequent digitalization of the images obtained.
(2) Subsistema de modificación de Ia distancia objeto/lente objetivo(2) Subsystem of modification of the object / objective lens distance
Se dispone de un sistema de traslación a Io largo del eje óptico del microscopio que permita posicionar el objeto a diferentes distancias de Ia lente objetivo. Esta capacidad de desplazamiento tiene que ser compatible con las resoluciones derivadas de los valores indicados de profundidad de campo, es decir que para trabajar con lentes de 100X el sistema debe ser capaz de avanzar en pasos no mayores de 0.22 micrómetros. Este sistema se compone de: (2a) elemento de movimiento de alta precisión capaz de cumplir con el condicionante anteriormente expuesto, (2b) soportes necesarios para el elemento de movimiento de alta precisión, (2c) conexionado eléctrico necesario con el subsistema informatizado de control (3) y (2d) acoplamientos necesarios para que Ia aplicación del movimiento desde el elemento de movimiento de alta precisión (2a) hasta el objeto sea óptimo.There is a translation system along the optical axis of the microscope that allows positioning the object at different distances from the objective lens. This displacement capacity must be compatible with the resolutions derived from the indicated depth of field values, that is, to work with 100X lenses the system must be able to advance in steps no larger than 0.22 micrometers. This system consists of: (2a) high precision movement element capable of complying with the aforementioned conditioner, (2b) necessary supports for the high precision movement element, (2c) electrical connection necessary with the computerized control subsystem (3) and (2d) necessary couplings so that the application of the movement from the high precision movement element (2a) to the object is optimal.
(3) Subsistema informatizado de control y tratamiento de imágenes(3) Computerized control and image processing subsystem
El subsistema informatizado de control y tratamiento de imágenes (3) consiste en un ordenador (3a) capaz de procesar Ia información contenida en las imágenes adquiridas, y un elemento compuesto por sentencias organizadas según un criterio lógico de operación (3b) que permite el tratamiento de las imágenes adquiridas para Ia obtención de micrografias de amplia focalización. El equipamiento utiliza para obtener el resultado buscado de una imagen bien focalizada en un amplio rango de profundidades, un conjunto de imágenes que hayan sido obtenidas a diferentes distancias objeto/lente objetivo. El subsistema informatizado de control y tratamiento de imágenes (3) se basa principalmente en un algoritmo matemático que permite obtener de cada imagen capturada Ia zona de Ia misma que está focalizada. El algoritmo está basado en Ia determinación de Ia varianza de un conjunto perimetral de puntos respecto al punto analizado, pudiendo ser definido por el usuario el espesor de dicho perímetro. Este algoritmo se completa con las rutinas lógicas necesarias para que a partir de las zonas focalizadas de cada imagen capturada sea posible obtener una nueva imagen del objeto en toda Ia extensión de estudio y completamente focalizada.The computerized control and image processing subsystem (3) consists of a computer (3a) capable of processing the information contained in the acquired images, and an element composed of sentences organized according to a logical operating criterion (3b) that allows the processing of the images acquired to obtain micrographs of wide focus. The equipment uses to obtain the desired result of a well focused image in a wide range of depths, a set of images that have been obtained at different distances object / objective lens. The computerized subsystem of control and treatment of images (3) is based mainly on a mathematical algorithm that allows to obtain from each captured image the area of the same that is focused. The algorithm is based on the determination of the variance of a perimeter set of points with respect to the analyzed point, the thickness of said perimeter can be defined by the user. This algorithm is completed with the logical routines necessary so that from the focused areas of each captured image it is possible to obtain a new image of the object throughout the entire length of study and fully focused.
De esta forma, es el elemento de tratamiento de imágenes el componente principal del sistema que aquí se presenta y que permite obtener imágenes focalizadas en un amplio rango de profundidades con las características descritas en este documento y que mejoran en gran medida a los sistemas que existen actualmente.In this way, it is the image processing element that is the main component of the system presented here and that allows to obtain focused images in a wide range of depths with the characteristics described in this document and that greatly improve the systems that exist currently.
Modo de trabajo de Ia invención.Work mode of the invention.
El modo de trabajo del sistema que aquí se presenta se puede resumir en los pasos que a continuación se detallan.The way of working of the system presented here can be summarized in the steps that are detailed below.
En primer lugar, una vez colocado el objeto a estudiar en el portamuestras del microscopio (1a), se elige el objetivo a utilizar en función del estudio que se desea desarrollar, que poseerá una determinada profundidad de campo en función de su magnificación, tal y como se ha expuesto anteriormente. El valor de Ia profundidad de campo establecerá los valores del desplazamiento que sufrirá el objeto entre cada una de las imágenes que se adquirirán, y por tanto los valores en que variará Ia distancia objeto/lente objetivo entre cada imagen capturada, que deberá ser igual o menor que Ia profundidad de campo. Tomándose un origen de coordenadas para Ia adquisición de imágenes en el objeto y en función de su morfología en Ia superficie de estudio (es decir, de Ia profundidad del objeto que es necesario observar), y del valor de profundidad de campo, el subsistema informatizado de control y tratamiento de imágenes (3) establecerá el número de desplazamientos del objeto y por tanto el número óptimo de imágenes que se deben capturar, utilizando Ia expresión obtenida anteriormente que relaciona Ia DOF con Ia magnificación y aplicando un factor, tal y como se expuso con anterioridad, para que se produzca un cierto solapamiento entre una imagen y Ia siguiente.In the first place, once the object to be studied has been placed in the microscope sample holder (1a), the objective to be used is chosen based on the study to be developed, which will have a certain depth of field depending on its magnification, such and as stated above. The value of the depth of field will establish the values of the displacement that the object will suffer between each of the images that will be acquired, and therefore the values in which the object / objective lens distance between each captured image will vary, which should be equal or less than the depth of field. Taking an origin of coordinates for the acquisition of images in the object and depending on its morphology on the study surface (that is, the depth of the object that is necessary to observe), and the value of depth of field, the computerized subsystem Control and image processing (3) will establish the number of displacements of the object and therefore the optimal number of images to be captured, using Ia expression obtained above that relates the DOF with the magnification and applying a factor, as previously stated, so that there is a certain overlap between an image and the next.
En segundo lugar, una vez determinados estos parámetros, el subsistema ¡nformatizado de control y tratamiento de imágenes (3) comenzará el proceso de toma de imágenes, controlando que el sistema de modificación de Ia distancia objeto/lente objetivo (2) produzca el desplazamiento del objeto hasta situarlo en una posición adecuada para Ia toma de imágenes. En esta situación, el elemento para Ia captura de imágenes (1 b), adquirirá una imagen que tendrá focalizada una parte del objeto. Esta adquisición puede desarrollarse de forma automática a través del subsistema informátizado de control y tratamiento de imágenes (3) y utilizando un elemento de captura de imágenes automatizado, o bien de forma manual si se utiliza un dispositivo que desarrolle su función de esta forma. En este caso, las imágenes deberán ser transferidas al sistema informátizado y dlgitalizadas en caso de ser necesario.Secondly, once these parameters have been determined, the image control and processing information subsystem (3) will begin the image taking process, controlling that the modification system of the object distance / objective lens (2) produces the displacement of the object until it is placed in a position suitable for taking images. In this situation, the element for capturing images (1 b) will acquire an image that will focus on a part of the object. This acquisition can be carried out automatically through the computerized control and image processing subsystem (3) and using an automated image capture element, or manually if a device that performs its function in this way is used. In this case, the images must be transferred to the computerized system and digitalized if necessary.
El proceso de posicionamiento del objeto en Ia posición de captura de imágenes y Ia adquisición de una imagen se repite tantas veces como sea necesario para poder abarcar toda Ia profundidad del objeto. El desplazamiento que sufrirá éste en cada paso será menor que Ia profundidad de campo que presente Ia lente objetivo utilizada, asegurándose de esta forma que el elemento para Ia captura de imágenes (1 b) adquiera tantas como sean necesarias para abarcar todo el objeto.The process of positioning the object in the image capture position and the acquisition of an image is repeated as many times as necessary to cover the entire depth of the object. The displacement that this one will suffer in each step will be less than the depth of field that the objective lens used presents, thus ensuring that the image capture element (1 b) acquires as many as are necessary to cover the entire object.
Por último, una vez adquiridas todas las imágenes necesarias, y transferidas, bien de forma automática o manual, al subsistema informátizado de control y tratamiento de imágenes (3), éste aplicará un procedimiento para obtener una sola imagen en Ia que todas las partes del objeto estén focalizadas. Este procedimiento es capaz de discernir las zonas focalizadas de las que no Io están dentro de una imagen cualquiera, es decir, se determinará en primer lugar cuales son las zonas del objeto focalizadas en cada una de las imágenes capturadas, y posteriormente se compondrá una imagen global del objeto formada por Ia superposición de las zonas focalizadas anteriormente determinadas. De esta forma podemos resumir el procedimiento que desarrolla el elemento de tratamiento de las imágenes (3b) para Ia generación de imágenes focalizadas a partir del conjunto de imágenes capturadas en los siguientes pasos:Finally, once all the necessary images have been acquired, and transferred, either automatically or manually, to the computerized control and image processing subsystem (3), it will apply a procedure to obtain a single image in which all parts of the Object are focused. This procedure is able to discern the focused areas from which they are not within any one image, that is, it will first determine which are the areas of the object focused on each of the captured images, and then an image will be composed global object formed by the superposition of the previously determined focused areas. In this way we can summarize the procedure developed by the image processing element (3b) for the generation of focused images from the set of images captured in the following steps:
1. Se parte de un conjunto de N capas, que denominamos "imacapas", de i*j elementos de señal RGB-HSL, que en conjunto forman una matriz tridimensional de datos. Cada "imacapa" se corresponde con los datos numéricos de señal RGBΗSLde Ia imagen proporcionada por el dispositivo de observación (microscopio) y que ha sido capturada mediante el elemento de captura de imágenes (1b), de forma que entre cada imagen existe un desplazamiento k entre el objeto y Ia lente objetivo. Este desplazamiento deberá ser menor que Ia profundidad de foco del objetivo utilizado para Ia observación y obtenido al multiplicar el valor de DOF por un coeficiente en el rango 0.7<Coef<0.9.1. It is based on a set of N layers, which we call "imacapas", of i * j RGB-HSL signal elements, which together form a three-dimensional array of data. Each "imacapa" corresponds to the RGBΗSL signal numerical data of the image provided by the observation device (microscope) and that has been captured by the image capture element (1b), so that between each image there is a shift k between the object and the objective lens. This displacement should be less than the depth of focus of the objective used for the observation and obtained by multiplying the DOF value by a coefficient in the range 0.7 <Coef <0.9.
2. A continuación, cada una de las "n" "imacapas" se descompone en sus componentes primarios R, G, B, H, Sy L, generando las subcapasde datos nR, nG, nB, nH, nS y nL A partir de estas subcapas se compone una nueva capa de datos obtenidos mediante Ia suma:2. Next, each of the "n" "imacapas" is broken down into its primary components R, G, B, H, Sy L, generating the sub-layers of data nR, nG, nB, nH, nS and nL From These sub-layers are composed of a new layer of data obtained through the sum:
uR*nR+uG*nG+uB*nB+uH*nH + ιιS*ttS+uL*nL,uR * nR + uG * nG + uB * nB + uH * nH + ιιS * ttS + uL * nL,
donde los elementos uR...uL son unos coeficientes que toman el valor 0 o 1 en función de cuales son las subcapas con las que se va a efectuar el análisis posterior. Cada una de las nuevas capas de datos formada se denomina "anacapa".where the elements uR ... uL are coefficients that take the value 0 or 1 depending on which are the sub-layers with which the subsequent analysis is to be carried out. Each of the new layers of data formed is called "anacapa".
3. Para cada una de las "n" "anacapas" se efectúa un proceso de cálculo que consiste en Ia extracción consecutiva y organizada por filas y columnas de submatrices de datos de dimensiones (v,v), siendo v un numero impar comprendido entre 3 y 9, el cual será fijado con anterioridad, y será además constante durante todo el proceso. Para el conjunto de datos existente en cada una de las submatrices indicadas se calcula Ia varianza, definida como
Figure imgf000016_0001
3. For each of the "n""anacapas" a calculation process is carried out consisting of the consecutive extraction and organized by rows and columns of sub-matrices of dimensions data (v, v), v being an odd number between 3 and 9, which will be set in advance, and will also be constant throughout the process. For the existing data set in each of the indicated sub-matrices, the variance is calculated, defined as
Figure imgf000016_0001
siendo x¡ cada uno de los datos de Ia submatriz, — 1).
Figure imgf000016_0002
being x¡ each of the submatrix data, - 1).
Figure imgf000016_0002
El conjunto de los valores de Ia varianza generados para cada una de las "anacapas" y organizado por filas y columnas conforma una nueva matriz de datos que se denomina "varcapa", obteniéndose un total de "n" "varcapas". Cada una de las "varcapas" tendrá como dimensiones ({i-v},{j-v}) y será una representación de Ia evolución de Ia similitud de los datos que definen una imagen a Io largo y ancho de capa "imacapa".The set of the values of the variance generated for each of the "anacapas" and organized by rows and columns forms a new matrix of data called "varcapa", obtaining a total of "n" "varcapas". Each of the "varcapas" will have as dimensions ({i-v}, {j-v}) and will be a representation of the evolution of the similarity of the data that define an image at the length and width of the "imacapa" layer.
4. Del conjunto total de "varcapas" se extrae el vector formado por los elementos (0,0) de cada "varcapa", siendo por Io tanto un vector de "n" datos, tantos como capas existan. Como cada uno de los datos del vector aquí considerado se corresponde con Ia varianza de una matriz de (v,v) elementos, se considera que el dato de Ia "imacapa" k que presenta el máximo valor de varianza se corresponde con el de Ia imagen focalizada. Este proceso se repite de forma consecutiva y organizada a todos los ({i-v},{j-v}) datos que conforman las diferentes "varcapas".4. From the total set of "varcapas" the vector formed by the elements (0,0) of each "varcapa" is extracted, being therefore a vector of "n" data, as many as layers exist. As each of the data of the vector considered here corresponds to the variance of a matrix of (v, v) elements, it is considered that the data of the "imacapa" k that presents the maximum value of variance corresponds to that of Ia focused image. This process is repeated consecutively and organized to all ({i-v}, {j-v}) data that make up the different "varcapas".
5. En el caso de que todos los valores del vector (i,j) de "varcapas" sean iguales o su variabilidad sea inferior a un determinado límite, entonces no es posible determinar con precisión cual es Ia capa de mejor focalización para dicha posición (i,j). En dicho caso, se escoge el dato de Ia "imacapa" que presente5. In the event that all the values of the vector (i, j) of "varcapas" are equal or their variability is less than a certain limit, then it is not possible to determine precisely which is the layer of best focus for said position (i, j). In this case, the data of the "imacapa" that is presented is chosen
Ia mejor focalización global, determinada ésta por Ia "anacapa" que presente Ia máxima varianza global.The best global targeting, determined by the "anacapa" that presents the maximum global variance.
6. Una vez determinada cual es Ia capa de mayor focalización para cada posición, se conforma Ia imagen enfocada en toda Ia extensión del objeto con los valores originales RGB-HSL en Ia capa de mejor focalización para cada posición. Por otra parte, otro aspecto de vital importancia del sistema y procedimiento que aquí se exponen es que no modifica Ia cromaticidad del objeto a estudiar debido a que trata con datos numéricos de los colores que componen Ia imagen sin interaccionar con el sistema de iluminación o de detección en ningún momento. De esta forma, este sistema actúa como un microscopio que fuese capaz de observar en una profundidad de campo elegible por el usuario del mismo, constituyendo así un gran avance en el campo de Ia microscopia óptica en particular, y de Ia microscopia en general.6. Once the layer of greatest focus for each position is determined, the image focused on the entire length of the object is formed with the original RGB-HSL values in the layer of best focus for each position. On the other hand, another aspect of vital importance of the system and procedure presented here is that it does not modify the chromaticity of the object to be studied because it deals with numerical data of the colors that make up the image without interacting with the lighting system or detection in no time. In this way, this system acts as a microscope that was able to observe at a depth of field eligible by the user of the same, thus constituting a great advance in the field of optical microscopy in particular, and of microscopy in general.
Descripción de las figuras.Description of the figures.
Figura 1. Esquema en el que se definen el número de apertura y Ia profundidad de campo desde un punto de vista geométrico.Figure 1. Scheme in which the opening number and depth of field are defined from a geometric point of view.
Figura 2. Representación de los valores teóricos de DOF frente a Ia magnificación y del ajuste de esos datos a una función exponencial decreciente de 2° orden.Figure 2. Representation of the theoretical values of DOF against magnification and the adjustment of these data to a decreasing exponential function of 2nd order.
Figura 3. Esquema general del sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, en donde se observan los siguientes componentes del mismo: (1 ) Subsistema de captura de imágenes. (1a) Microscopio óptico.Figure 3. General scheme of the automated system for obtaining fully focused images with high magnification microscopes, where the following components are observed: (1) Image capture subsystem. (1a) Optical microscope.
(1a1) Lentes objetivo. (1a2) Sistema de iluminación.(1a1) Objective lenses. (1a2) Lighting system.
(1b) Elemento para Ia captura de imágenes. (2)Subsistema de modificación de Ia distancia objeto/lente objetivo. (2a) Sistema de movimiento de alta precisión. (2b) Soportes del sistema de movimiento. (2c) Conexionado del sistema de movimiento.(1b) Element for image capture. (2) Subsystem of modification of the object / objective lens distance. (2a) High precision movement system. (2b) Movement system supports. (2c) Movement system connection.
(2d) Acoplamientos.(2d) Couplings.
(3) Subsistema informatizado de control y tratamiento de imágenes. (3a) Ordenador. (3b) Software de control y tratamiento de imágenes. (3) Computerized control and image processing subsystem. (3a) Computer. (3b) Control and image processing software.

Claims

REIVlNDtCACK*eS REIVlNDtCACK * eS
1. Sistema automatizado para la obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, basado en Ia captura secuencial de imágenes digitalizadas parcialmente foclizadas, a través de Ia óptica del microscopio y a diferentes alturas de observación, con un desplazamiento entre ellas inferior a Ia profundidad de foco de Ia lente objetivo, a partir de las cuales se extrae Ia parte que está focalizada de cada una de las imágenes capturadas y parcialmente focalizadas mediante Ia aplicación de un algoritmo que contempla Ia varianza matemática en los valores cromáticos de cada píxel de cada imagen parcialmente focalizada, generando Ia imagen global focalizada por apilamiento de las partes focalizadas.1. Automated system for obtaining fully focused images with high magnification microscopes, based on the sequential capture of digitized images partially focused, through the microscope optics and at different observation heights, with a displacement between them less than the depth of focus of the objective lens, from which the part that is focused of each one of the captured and partially focused images is extracted by means of the application of an algorithm that contemplates the mathematical variance in the chromatic values of each pixel of each image partially focused, generating the global image focused by stacking the focused parts.
2. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque Ia instrumentación utilizada comprende: a) Un subsistema para Ia captura de imágenes parcialmente focalizadas. b) Un subsistema de modificación de Ia distancia objeto/lente objetivo. c) Un subsistema informatizado de control y tratamiento de imágenes parcialmente focalizadas.2. Automated system for obtaining fully focused images with microscopes of high magnification, according to claim 1, characterized in that the instrumentation used comprises: a) A subsystem for capturing partially focused images. b) A subsystem of modification of the object / objective lens distance. c) A computerized subsystem of control and treatment of partially focused images.
3. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el subsistema de captura de imágenes parcialmente focalizadas comprende un microscopio óptico donde se situará el objeto a estudiar, y un elemento para Ia captura de imágenes parcialmente focalizadas.3. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the partially focused image capture subsystem comprises an optical microscope where the object to be studied will be located, and an element for the capture of partially focused images.
4. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el microscopio óptico dispone de un sistema de iluminación azimutal, bien sea de tipo intraocular o bien extraocular con o sin simetría axial. 4. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the optical microscope has an azimuthal illumination system, either intraocular or extraocular with or without axial symmetry.
5. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el microscopio óptico deberá poseer Ia capacidad de enviar Ia imagen parcialmente focalizada observada por el objetivo a un dispositivo de captura de imágenes.5. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the optical microscope must have the ability to send the partially focused image observed by the objective to an image capture device.
6. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el subsistema de captura de imágenes comprende un elemento para Ia captura de imágenes de forma digital o en su defecto comprenderá un sistema de digitalización de imágenes.6. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the image capture subsystem comprises an element for capturing images digitally or else will comprise a digitalization system for images.
7. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el subsistema de modificación de Ia distancia objeto/lente objetivo permite posicionar el objeto a diferentes distancias de Ia lente objetivo.7. Automated system for obtaining fully focused images with microscopes of high magnification, according to claim 1, characterized in that the subsystem of modification of the object / objective lens distance allows positioning the object at different distances from the objective lens.
8. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el subsistema de modificación de Ia distancia objeto/lente objetivo comprende un elemento de movimiento de alta precisión y los soportes y acoplamientos necesarios para interactuar con el objeto de forma óptima.8. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the subsystem for modifying the object / objective lens distance comprises a high precision movement element and the supports and couplings necessary to interact with the object optimally.
9. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el elemento de movimiento de alta precisión se acopla con el objeto de forma que es capaz de producir el movimiento de éste a través del eje de observación de Ia lente objetivo. 9. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the high-precision movement element is coupled with the object so that it is capable of producing its movement through the axis of observation of the objective lens.
10. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1, caracterizado porque el elemento de movimiento de alta precisión es capaz de producir movimientos menores a Ia profundidad de campo de las lentes objetivo comerciales más comunes.10. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the high precision movement element is capable of producing smaller movements at the depth of field of the most common commercial objective lenses.
11. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque posee los acoplamientos y soportes necesarios para que el elemento de movimiento de alta precisión desarrolle su función según las reivindicaciones 8 y 9.11. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that it has the necessary couplings and supports for the high precision movement element to perform its function according to claims 8 and 9.
12. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque para determinar cual es el desplazamiento óptimo entre dos imágenes parcialmente focalizadas consecutivas utiliza el siguiente procedimiento:12. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that to determine which is the optimal displacement between two consecutive partially focused images uses the following procedure:
(a) determina cual es Ia profundidad de foco de Ia lente objetivo utilizada en Ia observación mediante Ia fórmula matemática DOF= 166.433*EXP[-(a) determine which is the depth of focus of the objective lens used in the observation by means of the mathematical formula DOF = 166.433 * EXP [-
X/2.385] +4.530*EXP[-(X2)/26.070].X / 2,385] + 4,530 * EXP [- (X 2 ) /26,070].
(b) Multiplica el valor de DOF obtenido según el apartado 12.a por un factor en el rango 0.7<Coef<0.9, siendo el resultado el valor de desplazamiento que debe aplicarse al objeto en Ia dirección del eje óptico de observación para Ia captura de Ia imagen parcialmente focalizada. (b) Multiply the DOF value obtained according to section 12.a by a factor in the range 0.7 <Coef <0.9, the result being the displacement value that should be applied to the object in the direction of the optical observation axis for the capture of the partially focused image.
13. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el subsistema informatizado de control y tratamiento de imágenes consiste en un ordenador capaz de procesar las entradas y salidas de información y un software de control y tratamiento de imágenes que, mediante su lógica estructurada, maneja Ia información requerida para: a) Controlar el movimiento que debe producir el elemento de movimiento de alta precisión para situar el objeto en Ia posición deseada para Ia adquisición de cada una de las imágenes parcialmente focalizadas. b) Controlar Ia adquisición de cada una de las imágenes parcialmente focalizadas, una vez posicionado el objeto en el caso de usar un sistema de captura de imágenes automatizado. c) Guardar las imágenes parcialmente focalizadas una vez capturadas, y d) Aplicar el algoritmo para, a partir de todas las imágenes parcialmente focalizadas adquiridas del objeto, obtener una imagen del mismo completamente focalizada en toda su extensión y en toda su profundidad.13. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the computerized control and image processing subsystem consists of a computer capable of processing information inputs and outputs and control software and image processing that, through its structured logic, handles the information required to: a) Control the movement that the high precision motion element must produce to place the object in the desired position for the acquisition of each of the images partially focused. b) Control the acquisition of each of the partially focused images, once the object is positioned in the case of using an automated image capture system. c) Save partially focused images once captured, and d) Apply the algorithm to, from all partially focused images acquired of the object, obtain a fully focused image of it in its entirety and in all its depth.
14. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el software de control y tratamiento de imágenes se compone de un algoritmo matemático capaz de determinar las zonas focalizadas de las imágenes parcialmente focalizadas capturadas y de obtener una imagen totalmente focalizada a partir de todas las imágenes parcialmente focalizadas capturadas, utilizando únicamente Ia zona focalizada de las mismas.14. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the image control and treatment software is composed of a mathematical algorithm capable of determining the focused areas of the partially focused images captured and to obtain a fully focused image from all partially focused images captured, using only the focused area thereof.
15. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el software de control y tratamiento de imágenes se compone de un algoritmo matemático capaz de discernir cual es Ia imagen con mejor focalización en cada pixel que componen las imágenes capturadas. 15. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the image control and treatment software is composed of a mathematical algorithm capable of discerning which is the image with the best focus on each pixel that make up the captured images.
16. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el algoritmo usado para Ia extracción de Ia parte focalizada de cada imagen parcialmente focalizada se basa en:16. Automated system for obtaining fully focused images with microscopes of high magnification, according to claim 1, characterized in that the algorithm used for the extraction of the focused part of each partially focused image is based on:
(a) Descompone cada imagen digital parcialmente focalizada en sus componentes RGB (Red, Green y Blue).(a) Decomposes each digital image partially focused on its RGB components (Red, Green and Blue).
(b) Calcula las componentes HSL (Hue, Saturation y Luminance) de cada imagen a partir de los valores RGB indicados en el apartado 16.a. (c) Obtiene una nueva imagen como suma de las componentes R, G, B, H, Sy(b) Calculate the HSL components (Hue, Saturation and Luminance) of each image from the RGB values indicated in section 16.a. (c) Obtains a new image as a sum of the components R, G, B, H, Sy
L obtenidos según se indica en los apartados 16.a y 16. b, multiplicando cada componente por un factor de peso estadístico comprendido en el rango 0-1.L obtained as indicated in sections 16.a and 16. b, multiplying each component by a statistical weight factor in the range 0-1.
(d) Para Ia imagen obtenida según el apartado 16. c, se determinan los píxeles que cumplen el criterio de focalización, expresando éste en función del valor estadístico de Ia varianza de cada uno de los píxeles de Ia imagen y los de su entorno perimetral, respecto a un determinado límite.(d) For the image obtained according to section 16. c, the pixels that meet the focusing criterion are determined, expressing this according to the statistical value of the variance of each of the pixels of the image and those of its perimeter environment , regarding a certain limit.
(e) Extrae de cada imagen parcialmente focalizada los píxeles que han superado el criterio de focalización expresado en el punto 16. d, que por sustitución de los mismos en una imagen negra de referencia, genera Ia imagen final prefocalizada.(e) Extracts from each partially focused image the pixels that have exceeded the focusing criteria expressed in point 16. d, which by substituting them in a black reference image, generates the pre-focused final image.
(f) Determina, de acuerdo con Ia reivindicación 15, cual de las imágenes parcialmente focalizada presenta la máxima focalización mediante Ia maximización del valor de Ia varianza global de cada imagen, calculando dicho valor mediante Ia aplicación de alguno de los siguientes criterios: i. el criterio expuesto en el punto 16. d referido al punto central de Ia imagen y usando como entorno perimetral todos los píxeles de Ia imagen. ii. La suma de los valores de varianza obtenida para cada uno de los píxeles de Ia imagen según Io expuesto en el punto 16. d y usando como entorno perimetral los 8 píxeles más próximos al píxel considerado, iii. La suma de los valores de varianza obtenida para cada uno de los píxeles de Ia imagen según Io expuesto en el punto 16.d y usando como entorno perimetral un número de píxeles a determinar por el usuario.(f) Determine, according to claim 15, which of the partially focused images has the maximum focus by maximizing the value of the overall variance of each image, calculating said value by applying one of the following criteria: i. the criteria set forth in point 16. d referred to the central point of the image and using as a perimeter environment all the pixels of the image. ii. The sum of the variance values obtained for each of the pixels of the image according to what is stated in point 16. d and using the 8 pixels closest to the pixel considered as a perimeter environment, iii. The sum of the variance values obtained for each of the pixels of the image according to what is stated in point 16.d and using as a perimeter environment a number of pixels to be determined by the user.
(g) Rellena los píxeles que un estuviesen cubiertos en Ia imagen final prefocalizada obtenida según se expresa en el apartado 16.e, con los píxeles equivalentes de Ia imagen parcialmente focalizada de mejor focalización determinada según el criterio expuesto en el punto 16. f.(g) Fill in the pixels that were covered in the final prefocalized image obtained as expressed in section 16.e, with the equivalent pixels of the partially focused image with better focus determined according to the criteria set forth in point 16. f.
17. Sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación, según reivindicación 1 , caracterizado porque el algoritmo capaz de conformar una imagen a partir de los píxeles que presenten mejor focalización de cada una de las imágenes parcialmente focalizadas, según Io descrito en Ia reivindicación 16, permite modificar por el usuario las variables de contorno entre las que se incluyen: (a) El peso estadístico de cada una de las componentes R, G, B, H, S y L de las imágenes parcialmente focalizadas.17. Automated system for obtaining fully focused images with high magnification microscopes, according to claim 1, characterized in that the algorithm capable of shaping an image from the pixels that present better focus of each of the partially focused images, according to Io described in claim 16, allows the user to modify the contour variables among which are included: (a) The statistical weight of each of the components R, G, B, H, S and L of the partially focused images.
(b) La amplitud del número de capas perimetrales que se emplea para determinar Ia varianza característica de cada píxel.(b) The amplitude of the number of perimeter layers used to determine the characteristic variance of each pixel.
(c) El valor umbral que determina cuando Ia varianza es tal que el píxel debe ser considerado como píxel focalizado.(c) The threshold value that determines when the variance is such that the pixel must be considered as a focused pixel.
18. Un procedimiento para Ia obtención de imágenes totalmente focalizadas con microscopios de elevada magnificación que, haciendo uso de Ia instrumentación descrita en las reivindicaciones 1 a 17, se caracteriza por: a) Llevar a cabo, con precisión submicrométrica, el movimiento del objeto a estudiar a Io largo del eje óptico de observación, necesario para colocarlo en Ia posición de captura de imágenes parcialmente focalizadas según Ia profundidad de campo de Ia lente objetivo, b) Capturar imágenes parcialmente focalizadas del objeto a estudiar en las posiciones adecuadas de forma automatizada o bien de forma manual. c) Obtener una imagen completamente focalizada del objeto a estudiar en toda su extensión y en toda su profundidad a partir de las imágenes parcialmente focalizadas capturadas. 18. A procedure for obtaining fully focused images with high magnification microscopes which, using the instrumentation described in claims 1 to 17, is characterized by: a) Carrying out, with submicron precision, the movement of the object to study along the optical axis of observation, necessary to place it in the partially focused image capture position according to the depth of field of the objective lens, b) Capture partially focused images of the object to be studied in the appropriate positions automatically or Well manually. c) Obtain a fully focused image of the object to be studied in its entirety and in all its depth from the partially focused images captured.
19. Uso del sistema automatizado para Ia obtención de imágenes totalmente focalizadas con microscopios de alta magnificación, descrito en las reivindicaciones 1 a 17, para obtener imágenes totalmente focalizadas con lentes objetivo de diversas y altas magnificaciones. 19. Use of the automated system for obtaining fully focused images with high magnification microscopes, described in claims 1 to 17, to obtain fully focused images with objective lenses of various and high magnifications.
PCT/ES2009/000170 2008-05-23 2009-03-26 Automated system and method for obtaining fully focused images with high-magnification microscopes WO2009141467A1 (en)

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US4661986A (en) * 1983-06-27 1987-04-28 Rca Corporation Depth-of-focus imaging process method
US20020181762A1 (en) * 2001-05-30 2002-12-05 Mitutoyo Corporation Systems and methods for constructing an image having an extended depth of field
EP1336888A1 (en) * 2002-02-13 2003-08-20 Fairfield Imaging Ltd. Microscopy imaging system and data acquisition method
US20060038144A1 (en) * 2004-08-23 2006-02-23 Maddison John R Method and apparatus for providing optimal images of a microscope specimen

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US4661986A (en) * 1983-06-27 1987-04-28 Rca Corporation Depth-of-focus imaging process method
US20020181762A1 (en) * 2001-05-30 2002-12-05 Mitutoyo Corporation Systems and methods for constructing an image having an extended depth of field
EP1336888A1 (en) * 2002-02-13 2003-08-20 Fairfield Imaging Ltd. Microscopy imaging system and data acquisition method
US20060038144A1 (en) * 2004-08-23 2006-02-23 Maddison John R Method and apparatus for providing optimal images of a microscope specimen

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