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WO2016156092A1 - Procédé pour localiser au moins un émetteur au moyen d'un microscope de localisation - Google Patents

Procédé pour localiser au moins un émetteur au moyen d'un microscope de localisation Download PDF

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Publication number
WO2016156092A1
WO2016156092A1 PCT/EP2016/056166 EP2016056166W WO2016156092A1 WO 2016156092 A1 WO2016156092 A1 WO 2016156092A1 EP 2016056166 W EP2016056166 W EP 2016056166W WO 2016156092 A1 WO2016156092 A1 WO 2016156092A1
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WO
WIPO (PCT)
Prior art keywords
emitter
optical detector
emission radiation
lenses
optical
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2016/056166
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German (de)
English (en)
Inventor
Alexander Egner
Claudia Geisler
Haugen MITTELSTÄDT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Laser Laboratorium Goettingen eV
Original Assignee
Laser Laboratorium Goettingen eV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Laser Laboratorium Goettingen eV filed Critical Laser Laboratorium Goettingen eV
Publication of WO2016156092A1 publication Critical patent/WO2016156092A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B11/00—Measuring arrangements characterised by the use of optical techniques
    • G01B11/002—Measuring arrangements characterised by the use of optical techniques for measuring two or more coordinates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B9/00—Measuring instruments characterised by the use of optical techniques
    • G01B9/04—Measuring microscopes
    • 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
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/082—Condensers for incident illumination only
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/362—Mechanical details, e.g. mountings for the camera or image sensor, housings

Definitions

  • the invention relates to a method for locating at least one emitter of electromagnetic emission radiation by means of a localization microscope and to an apparatus for carrying out such a method.
  • Such methods are today known in various configurations in the prior art. Emitters can be observed in imaging and / or tracking processes that are smaller than the Abbe's
  • fluorophores for example, carbon nanotubes, quantum dots or fluorophores.
  • the fluorophores become electromagnetic
  • the electromagnetic emission radiation emitted by the emitter is collected and applied to an optical detector via suitable objectives and optical arrangements.
  • Dot images correspond to the lateral position of the emitter in the
  • Fluorophore prevails in the active state, it is by the electromagnetic Excitation radiation excitable. Only in this case, he can be excited zürn lights.
  • Activation radiation can be brought from a passive state to an active state.
  • fluorophores are known which can be brought from an active to a passive state. The following is from
  • PAM localization microscopy
  • STORM stochastic optical reconstruction microscopy
  • FPALM fluorescence photoactivation localization microscopy
  • PALMIRA ground state depletion microscopy followed by individual molecule return ( GSDIM) or "direct STORM” (dSTORM).
  • GSDIM ground state depletion microscopy followed by individual molecule return
  • dSTORM direct STORM
  • the methods have a high lateral localization accuracy of less than 20-30 nanometers. In the axial direction, however, the observable volume is limited to a layer of about 1 pm. Such methods are described, for example, in WO 2009/146016 A1. Attempts have been made to achieve good localization accuracy even in the axial direction. So far, however, the information on the positions in this direction must be determined indirectly. For this purpose, techniques such as two-plane detection, astigmatism or double helix are used, whereby the point-spread-function (PSF) is modified to determine the position along the optical axis from the z-dependent change of this function. However, this does not lead to an isotropic resolution in all spatial directions In the interference method, opposing objectives (4Pi) achieve better axial resolution, but all these methods are limited to layer thicknesses of about 1 pm.
  • PSF point-spread-function
  • the invention is therefore based on the object, a method for
  • the invention achieves the stated object by a method for locating at least one emitter of electromagnetic emission radiation by means of a localization microscope, the method having the following steps:
  • the measured values correspond to the distribution of the on the optical
  • Directed detector whose optical axes in linearly independent directions from each other. This corresponds to observation of the radiating emitter from two different, linearly independent directions.
  • the measured values of the electromagnetic emission radiation form an image of the emitter on the optical detector for each objective through which the electromagnetic emission radiation is conducted.
  • image is understood merely as a summary of the measured values which are caused by the electromagnetic emission radiation of one or more emitters which has been conducted through one of the at least two objectives.
  • the electromagnetic emission radiation of several emitters usually forms several "images" in this understanding, even if it was passed through a single one of the at least two objectives. in that, for at least one of the plurality of objectives, the plurality of emitters are closer together than the Abbe's dissolution criterion
  • the electromagnetic emission radiation of multiple emitters form a common image. This case can occur, for example, if two emitters are arranged offset along the optical axis of the respective objective. The emitters then shine for that
  • Lens to be very close to each other, although the actual distance may exceed the resolution criterion corresponding dimension. This can usually be resolved by the "images" of the at least one further objective.
  • Parameters that determine the position and viewing direction of the respective objective can be determined, for example, by solving a linear Gieichungssystems three-dimensional coordinates of each emitting emitter. By observing the volume in which the emitter is located, from two different, linearly independent viewing directions, one obtains information directly on the position of the emitter in two
  • Each lens directs the emission radiation to at least one optical detector.
  • the various optical detectors may be part of a single detector array, which may be in the form of a CCD chip of a digital camera, for example. In this case, for example, the different lenses would forward each one of them
  • Electromagnetic emission radiation passed through at least three, preferably at least four lenses on the at least one optical detector, wherein the optical axes of the lenses extend in pairs linearly independent directions.
  • the optimal positioning of, for example, four lenses is the tetrahedral arrangement. Between each two of the lenses then the tetrahedral angle of 09.5 ° is included. In this case, the localization accuracy is almost isotropic when the
  • the location accuracy with which the position of an emitter can be determined depends on the number of photons transmitted by the lenses which are emitted by the respective emitter.
  • the dyes used as fluorophore have only a limited duration of activity, so that the number of photons that can be emitted is limited. Since the individual fluorophores mostly by stochastic
  • a fluorophore that has been activated once can not be reactivated to increase, for example, the "exposure time" for that fluorophore and thus improve the locating accuracy of the emitted photons with the objectives, so that it is advantageous to use lenses with the largest possible numerical aperture and thus the largest possible aperture angle, for an isotropic image field in which the
  • the emitter is a fluorophore which emits electromagnetic excitation radiation to emit the
  • the Fiuorophor in an active by:
  • Electromagnetic excitation radiation excitable state and in a passive state in which excitation is not possible.
  • it can be brought from the active state to the passive state or from the passive state to the active state by electromagnetic radiation.
  • the at least one Fiuorophor is brought into a stimulable state before being excited by an electromagnetic activation radiation. This is often the case in particular with those already described
  • the measured values of the electromagnetic emission radiation emitted by one or more emitters each form an image of the emitters or emitters passing through one of the at least two
  • Lenses was passed to the at least one optical detector.
  • the images are weighted differently when determining the position of the at least one emitter.
  • the images are weighted less heavily the wider they are on the optical detector and / or the less emission radiation has been detected by the optical detector for the particular image.
  • the wider the image on the detector the more "blurred" the image of the emitter is, for example, because the emitter is relatively far out of the focal plane or the focal volume of the lens. Since the localization accuracy depends on the amount of collected photons, it decreases as the less emission radiation from the optical detector for an image is detected.
  • the different weighting can also be iterative. Thus, it is possible, for example, first to determine the position of the emitter in the aforementioned manner with or without additional weighting and to determine from the positions thus determined which emitters are furthest outside the focal planes of the individual objectives.
  • one or more iteratively can renew
  • Position determinations are performed in which the images of these emitters are weighted according to the determined in the previous step positions.
  • the position of the emitter in the imaging plane belonging to the respective objective is determined from the measured values recorded by the optical detector and / or the images formed therefrom.
  • Brightness distribution by a function such as a Gaussian function, or by several, for example, two or three Gaussian functions, attach and determine in this way how many emitters are at least likely responsible. In this way, a probable
  • electromagnetic emission radiation of an emitter which is passed through in each case one of the at least two lenses on the optical detector, determines an orientation of the emitter in space. Assuming that the emitter is a radiating dipole, the amount of radiated is
  • electromagnetic radiation is not distributed isotropically over all spatial directions.
  • the radiation characteristic of the dipole is known, so that from the
  • Subsets of incident on the optical detector emission radiation for each different lenses on the orientation of the dipole and thus the orientation of the emitter can be closed in space.
  • the invention also achieves the stated object by an apparatus for carrying out one of the methods described here, comprising at least two lenses with optical axes which are linearly independent of one another
  • the device also has an optical detector, an electrical controller for evaluating the detector measured values and at least one
  • the device has at least two, preferably at least three, more preferably at least four objectives. These should be positioned as close as technically feasible to the tetrahedral arrangement, which offers a nearly isotropic spatial resolution. This is difficult with commercially available lenses, possibly even impossible.
  • a device manufactured by the inventors has four lenses, one of which is arranged vertically below the sample holder, and at the same time serves as a supply for the excitation radiation and optionally the activation radiation.
  • This objective is advantageously oriented vertically upwards and designed as an air immersion objective with a numerical aperture of 0.75.
  • Above the sample holder there are three identical lenses, which are advantageously designed as water immersive lenses with a numerical aperture of, for example, 0.8.
  • the optical axes of the upper lenses intersect the optical axis of the lower one
  • Lens in the aforementioned embodiment at an angle of 125.5 °.
  • the deviation from the optimum tetrahedral angle of 109.5 ° has the consequence that more information is collected via the positioning in the x and y directions, ie a direction perpendicular to the optical axis of the lower objective, than via the perpendicularly extending z-axis.
  • the focal volume, in which the highest resolution and localization accuracy is present, is formed by the four focal planes of the four lenses, each having a thickness of the focal depth.
  • lenses with a smaller numerical aperture they can be arranged at an angle closer to the optimum
  • Tetrahedral angle of 09.5 ° Due to the lower numerical aperture, however, not so many photons emitted by the respective fluorophore are captured and directed to the optical detector, so that the
  • lenses with the largest possible aperture angle are used. This corresponds to half the opening angle and is preferably at least 17.5 °.
  • At least one of the lenses is designed as an immersion objective, preferably as a water immersion objective.
  • the sample holder in this case has an immersion liquid,
  • the vessel advantageously has a depth which allows the vessel to be filled with immersion liquid so high that the entrance lenses of the immersion objectives are completely submerged therein.
  • immersion liquid water or an aqueous medium is advantageously used.
  • the proportion of water in the aqueous medium used is advantageously 80%, particularly preferably 89%.
  • Immersion liquids are preferably liquids, as they are in the state of Technique of super-resolution microscopy can be used in particular with regard to the fluorescence properties of the markers and / or the structure of the cell. These are known to the person skilled in the art, so that a further description is omitted here.
  • the sample holder has a cover glass, which is arranged such that at least a part of a emitted from the emitter
  • Emission radiation on the way to one of the lenses passes through the coverslip, and which has a refractive index that the
  • Immersion liquid in particular at a wavelength of
  • Cover glass does not affect the beam path in the upper lenses, in particular due to the transition at the interface between the coverslip and the surrounding water.
  • the cover glass consists of a fluoropolymer and has a refractive index of 1.34. This minimizes influences of the cover glass on the optical image.
  • cover glass with a refractive index deviating from the refractive index of the immersion liquid it is preferable to use a suitable cover slip.
  • the device has an environmental structure surrounding the sample, in which at least one lens is embedded.
  • These are advantageously lenses that simultaneously meet the sine and the Herrschel conditions.
  • Such lenses are known in the art.
  • Both the lateral and the axial magnification corresponds to the ratio of the refractive indices in the sample and in the image space. This results in a "swapping out" of the image, which results in the individual lenses being able to be arranged at a greater distance from one another, which means that the installation space required for the objectives no longer becomes a limiting factor, so that lenses with a larger numerical aperture are also used can be.
  • a particularly advantageous embodiment of the environmental structure is tetrahedral with four lenses embedded therein, which are arranged in the surfaces of the tetrahedron.
  • the environmental structure may also be part of the sample holder.
  • At least one of the lenses used has a holder which allows translation in different, advantageously all, spatial directions and permits tilting or rotation of the objective. Only a rotation around the optical axis is not necessary.
  • the different optical detectors are particularly preferably part of a single optical chip, for example a CCD chip, which is part of a digital camera. If the embodiment described above is used with three upper and one lower objective, it is sufficient for one, preferably the lower one
  • Lenses allow a movement only along its optical axis.
  • the sample is attached to a sample holder separately on a sample table on which it is placed, in all three if possible
  • the entire device is preferably mounted on a vibration-damped table, for example a Heiliagerston.
  • a vibration-damped table for example a Luftiagerston.
  • the position was measured 100 times and the emitter position was determined with two, three or four lenses. From the distribution of these emitter positions calculated in this way, the standard deviation and the FWHM can be used as a measure of the localization accuracy, including the lower lens and one, two or three of the upper lenses used lenses have a half-width of 9 nm in the x-direction, 8 nm in the y-direction and 20 nm in the z-direction resulting in standard deviations of 3.8 nm, 3.4 nm and 8.5 nm used three lenses, two of which are located above the sample holder, results in a half-width of 11 nm in the x-direction, 9 nm in the y-direction and 15 nm in the z-direction, resulting in standard deviations of 4.7 nm, 3 When using all four objectives, a half-width of 11 nm in the x-direction, 8 nm in the y-direction
  • Embodiment of the present invention explained in more detail. It shows the schematic representation of a section of a device according to a first embodiment of the present invention, the schematic section through a part of a device according to the embodiment of the present invention, the schematic section through a sample holder of such a device and the schematic plan view of the lens assembly Figure 1 and a schematic plan view of an objective assembly with a modified sample holder.
  • Figure 1 shows the detail of a device according to a first
  • Embodiment of the present invention Centrally there is a sample holder 2, which is designed as a water-fillable vessel. Below the sample holder 2 is a first lens 4, which is formed in the present embodiment as Heilimmersions solicitiv with a numerical aperture of 0.75.
  • the first objective 4 serves to guide at least part of the electromagnetic emission radiation emitted by the fluorophore to an optical detector (not shown) and, on the other hand, to conduct electromagnetic radiation
  • FIG. 2 shows a section through the embodiment shown in FIG. One recognizes the sample holder 2, the first objective 4 and one of the second objectives 8. In the central region of the sample holder 2 there is an elevation 10 on which the sample 6 is arranged and which ensures that the sample 6 is at the optimum distance from one another first input lens 12 of the first lens 4 can be arranged.
  • the sample holder 2 is up to one
  • Water level 14 which is shown in Figure 2 by a dashed line, filled with water. This water level 14 must be so high that a second input lens 16 is completely covered with water.
  • Figure 3 shows an enlarged sectional view of the sample holder 2 with the central elevation 10, on which the sample 6 is to be arranged, and the
  • the three second lenses 8 can be seen, which are arranged around the sample 6.
  • wavefronts 18 are shown, which correspond to the electromagnetic emission radiation. Due to the design of the lenses 8, the space is the limiting factor and the lenses 8 are very close to each other.
  • the three second objectives 8 are arranged around the sample 6, a sample holder 2 having an environmental structure 20 being arranged around the sample 6.
  • the first objective 4 is not shown in this illustration. It "looks" from below from the plane of the drawing on the sample 6.
  • lenses 22 are embedded, which have a lateral and an axial magnification, which correspond to the ratio of the refractive indices in the sample 2 lying outside the surrounding structure 20 image space 24.
  • the perfect real intermediate images 28 are observed by the second objectives 8. As a result, they can be arranged at a greater distance from the sample 6 and the installation space of the Lenses 8 is no longer the limiting factor.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

La présente invention concerne un procédé pour localiser au moins un émetteur d'un rayonnement électromagnétique d'émission au moyen d'un microscope de localisation, le procédé comprenant les étapes consistant : a) à faire passer le rayonnement électromagnétique d'émission à travers au moins deux objectifs (4, 8) pour atteindre au moins un détecteur optique, les axes optiques des au moins deux objectifs (4, 8) s'étendant dans des directions linéairement indépendantes ; b) à détecter le rayonnement électromagnétique d'émission par l'au moins un détecteur optique, l'au moins un détecteur optique déterminant des valeurs de mesure ; et c) à déterminer la position de l'au moins un émetteur à partir des valeurs de mesure déterminées par le détecteur optique.
PCT/EP2016/056166 2015-03-27 2016-03-21 Procédé pour localiser au moins un émetteur au moyen d'un microscope de localisation Ceased WO2016156092A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102015004104.5 2015-03-27
DE102015004104.5A DE102015004104B4 (de) 2015-03-27 2015-03-27 Verfahren zum Lokalisieren wenigstens eines Emitters míttels eines Lokalisationsmikroskops

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WO2016156092A1 true WO2016156092A1 (fr) 2016-10-06

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DE102016119262B4 (de) * 2016-10-10 2018-06-07 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Verfahren zum räumlich hochauflösenden Bestimmen des Orts eines vereinzelten, mit Anregungslicht zur Emission von Lumineszenzlicht anregbaren Moleküls in einer Probe
DE102017126128A1 (de) * 2017-11-08 2019-05-09 Endress+Hauser SE+Co. KG System und Verfahren zur ortsaufgelösten Bestimmung von zumindest einer physikalischen oder chemischen Prozessgröße
DE102020134495B4 (de) 2020-12-21 2024-02-15 Abberior Instruments Gmbh Verfahren und Mikroskop zur Aufnahme von Trajektorien einzelner Partikel in einer Probe
DE102022120952B4 (de) 2022-08-18 2024-03-14 Abberior Instruments Gmbh Verfahren und vorrichtung zum simultanen verfolgen zweier emitter

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DE102015004104A1 (de) 2016-09-29

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