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EP-4741906-A1 - ACCESSORY FOR OBTAINING A FOCAL IMAGE STACK, MICROSCOPE COMPRISING SAME AND METHOD FOR OBTAINING A FOCAL IMAGE

EP4741906A1EP 4741906 A1EP4741906 A1EP 4741906A1EP-4741906-A1

Abstract

The present invention relates to an accessory comprising a first converging lens coupling (3); a second converging lens coupling (6), inverted with respect to the first coupling (3); and a varifocal lens (7) displaceable along the optical axis between the first and second coupling (3, 6) on a mechanical means (8), said accessory (100) being couplable to a host microscope (200) such that the first object focal plane, F 1 , coincides with the image focal plane, F T ′ , of the tube lens (203) of the host microscope (200), the varifocal lens (7) being adjustable by a distance z 2 with respect to the first image focal plane, F 1 ′ . The invention also relates to a microscope comprising the accessory and a method for obtaining a focal image.

Inventors

  • MARTÍNEZ CORRAL, Manuel
  • SAAVEDRA TORTOSA, GENARO
  • TOLOSA RUIZ, Ángel
  • SÁNCHEZ ORTIGA, Emilio
  • INCARDONA, Nicolo

Assignees

  • Universitat de València

Dates

Publication Date
20260513
Application Date
20240619

Claims (16)

  1. - An accessory for obtaining a focal image stack couplable to a host microscope forming a coupled optical system, wherein said host microscope (200) comprises, consecutively arranged centered with respect to an optical axis (204): a support means (201) of a sample-object, an objective (202) comprising at least one lens (221) with a focal length f ob and an aperture diaphragm (222), defining an object focal plane F ob comprising an object focus, a plane Q 0 , located at a distance z 0 with respect to the object focal plane F ob , and an image focal plane F ob ′ said image focal plane, F ob ′ comprising an image focus; wherein said planes are perpendicular to the optical axis (204) of the host microscope (200); and a converging tube lens (203), located at a distance d with respect to the image focal plane, F ob ′ said tube lens (203) defining an image focal plane F T ′ , said image focal plane, F T ′ comprising an image focus, at a distance f T with respect to the axial position of the tube lens (203); wherein the accessory (100) is characterized in that it comprises a first converging lens coupling (3) comprising at least one lens, wherein said first converging lens coupling (3) has a focal length f AC 1 and an optical power P AC 1 , defining a first object focal plane F 1 , a plane Q 1 , located at a distance z 1 with respect to the plane F 1 , and a first image focal plane, F 1 ′ ; a second converging lens coupling (6) comprising at least one lens, wherein said second converging lens coupling (6) is a lens coupling inverted with respect to said first converging lens coupling (3), where the second converging lens coupling (6) has a focal length f AC 2 , and an optical power P AC 2 , defining a second object focal plane, F 2 that coincides with the first image focal plane F 1 ′ , and a second image focal plane F 2 ′ ; and a varifocal lens (7), of variable power P L , displaceable along the optical axis between the first and second converging lens coupling (3, 6) on a mechanical means (8) of axial displacement, the accessory (100) being couplable to a host microscope (200) such that the first object focal plane, F 1 , coincides with the image focal plane, F T ′ , of the tube lens (203) of the host microscope (200); and said varifocal lens (7) being adjustable by a distance z 2 with respect to the first image focal plane, F 1 ′ , z 2 being: z 2 = f AC 1 f T 2 f T − d .
  2. The accessory for obtaining a focal image stack according to claim 1, wherein the accessory (100) comprises a photosensitive recording medium (9) configured to record, sequentially, the focal image stack generated in the second image focal plane, F 2 ′ .
  3. The accessory for obtaining a focal image stack according to claim 2, wherein said photosensitive recording medium (9) further comprises a communication medium configured to transmit the stack of recorded images to an image processing means.
  4. The accessory for obtaining a focal image stack according to any one of claims 1 to 3, wherein the first converging lens coupling (3) comprises a first converging lens (1) of optical power P 1 , and a second converging lens (2), spaced at a distance e 3 with respect to the first converging lens (1), of optical power P 2 , the power of the coupling being P AC 1 = P 1 + P 2 - e 3 P 1 P 2 .
  5. The accessory for obtaining a focal image stack according to claim 4, wherein the first and/or second converging lens (1, 2) comprises an achromatic doublet.
  6. The accessory for obtaining a focal image stack according to any one of claims 1 to 3, wherein the first coupling (3) comprises an M12 lens (10) and a housing (12).
  7. The accessory for obtaining a focal image stack according to any one of claims 1 to 6, wherein the second coupling (6) comprises a first converging lens (4) of optical power P 4 , and a second converging lens (5), spaced at a distance, e 6 , with respect to the first converging lens (4) of the second converging coupling (6), of optical power P 5 , the power of the coupling being P AC 2 = P 4 + P 5 - e 6 P 4 P 5
  8. The accessory for obtaining a focal image stack according to any one of claims 1 to 6, wherein the second coupling (6) comprises an M12 lens (11) and a housing (13).
  9. The accessory for obtaining a focal image stack according to any one of claims 1 to 8, wherein the optical power, P AC 1 , of the first converging lens coupling (3) is equal to the optical power, P AC 2 , of the second converging lens coupling (6).
  10. The accessory for obtaining a focal image stack according to any one of claims 1 to 9, wherein the mechanical axial displacement means (8) is manually actuated.
  11. The accessory for obtaining a focal image stack according to any one of claims 1 to 9, wherein the mechanical axial displacement means (8) is electronically actuated.
  12. Optical microscope comprising an optimal system arranged centered with respect to an optical axis, wherein said optical microscope is characterized in that it comprises an accessory (100) according to any one of claims 1 to 11, coupled to the optical system of a host microscope (200).
  13. Method for obtaining a focal image, the method comprising: - coupling an accessory (100) according to any one of claims 1 to 11 on a host microscope (200), such that F 1 , coincides with the image focus, F T ′ , of the tube lens (203) comprised in the host microscope (200) whereto it is coupled; - adjusting the varifocal lens (7) of said accessory (100) at a distance z 2 , z 2 being: z 2 = f AC 1 f T 2 f T − d ; - adjusting the optical power P L of the varifocal lens (7) such that: the object focal plane, F ob , the object focal plane F 1 , corresponding to the first coupling (3), and the image focal plane F 2 ′ , corresponding to the second coupling (6), of the accessory (100) form conjugate planes, either the plane, Q 0 , the plane Q 1 , corresponding to the first coupling (3), and the image focal plane F 2 ′ , corresponding to the second coupling (6), of the accessory (100) form conjugate planes; and - recording the focal image.
  14. Method for obtaining a focal image, according to claim 13, wherein the method comprises, once the focal image has been recorded, modifying the optical power P L , of the varifocal lens (7) and recording a second focal image.
  15. Method for obtaining a focal image, according to any one of claims 13 to 14, the recorded focal image is generated in the focal plane image of the accessory, F 2 ′ .
  16. Method for obtaining a focal image, according to any one of claims 14 to 15, wherein the method further comprises transmitting the recorded image stack to an external image processing means

Description

Technical field of the invention The present invention falls within the field of optics. Specifically, it relates to an accessory for obtaining a focal image stack couplable to a host microscope. Background of the invention An optical microscope is an instrument designed to provide two-dimensional (2D) images of essentially flat microscopic samples. An optical microscope comprises an optical system formed by different elements centered with respect to an axis of symmetry, called optical axis: The stage plate is a support means where the sample is placed. It is a medium that is mechanically displaceable in the direction of the optical axis, that is, axially.The objective is formed by at least one lens and an aperture diaphragm. Said objective has a numerical aperture, NA, and an object focal length, fob, that is, distance between the lens, or lens system, of the microscope objective and the object focus, Fob a point that, after the rays pass through it, are refracted parallel to the optical axis.The tube lens is a lens, or lens systems, configured to collect the light emerging from the objective, and provide an actual 2D image of the sample flat on its image focal plane, that is, the plane perpendicular to the optical axis comprising the image focus, FT′. The image focal length, fT, depends on the manufacturer of the tube lens, or lens system, used. Traditionally, the objectives used in a microscope define a focal length, fT, that ranges between 150 - 200 mm.The photosensitive recording medium is a pixelated medium, such as a CCD sensor or a CMOS sensor, with a pixel size δp, configured to record the image formed in the image focal plane, FT′, of the tube lens. The focal length of the objective, fob, allows defining an object focal plane, a plane perpendicular to the optical axis that contains the object focus, Fob. By placing a sample at the object focal plane, the objective provides, at infinity, an image of the sample. Additionally, the focal length of the objective, fob, is also the distance between the optical center, the lens, or lens system, of the microscope objective and its image focus, Fob′, that point at which, after being refracted, the rays that reach the objective in a direction parallel to the optical axis coincide. Similar to the object focal plane, an image focal plane of the objective, Fob′, may be defined as the plane perpendicular to the optical axis that coincides with the image focus. Typically, the aperture diaphragm is located in the image focal plane of the objective Fob′, and the lenses that compose it are designed to minimize the effect of aberrations. In this way, in the present invention the object focus of a lens is designated indistinctly, F, and the object focal plane, which is that which is perpendicular to the optical axis and contains said object focus, F. This way of designating will also be applied to the image focus, F', and the image focal plane. An optical microscope is represented by its lateral magnification, Mh, defined as the ratio between the size of the image and the size of the object, calculated as: Mh=fT/fob The spatial resolution is also defined, which is given by the resolution limit: rlim=maxλ2NA+δpMh,2δpMh Where λ is the average wavelength of the light emitted by the sample, δp is the pixel size of the photosensitive recording medium, NA is the numerical aperture of the objective and Mh is the lateral magnification of the microscope. The resolution limit is the minimum distance between two points located in the object plane, below which it is not possible to distinguish their images. Finally, the depth of field (DoF) is defined, which indicates the length of the axial interval, around the plane that contains the focus Fob, wherein the object can be placed without the image losing resolution, which can be calculated as: DoF=λNA2+δpNAMh Microscopes are prepared to focus to a single plane, specifically the object focal plane (Fob) of the objective. As indicated above; to perform the focusing tasks the stage plate is the displaced element, so that modification of the focused planes in the microscope is achieved. For a 2D sample, the stage plate is axially displaced until the sample is located at the object focal plane, Fob, of the objective. In that case, the sample image falls on the photosensitive recording medium or sensor. In the case of 3D samples, only one section of the sample, that corresponding to the section above Fob, provides a focused image. The displacement of the stage plate allows focusing different sections, transversal to the optical axis, of the sample. To capture an image stack of cross sections of 3D samples, an axial mechanical scanning process is therefore necessary wherein, in each step, the stage plate is mechanically displaced. However, it is necessary to wait a while until the system stabilizes to finally capture the image with the pixelated sensor. However, this whole process may prove too slow for the case that the sample is in mo