EP3956710A1 - Microscope à fluorescence et procédé d'illustration d'un échantillon - Google Patents

Microscope à fluorescence et procédé d'illustration d'un échantillon

Info

Publication number
EP3956710A1
EP3956710A1 EP20723022.8A EP20723022A EP3956710A1 EP 3956710 A1 EP3956710 A1 EP 3956710A1 EP 20723022 A EP20723022 A EP 20723022A EP 3956710 A1 EP3956710 A1 EP 3956710A1
Authority
EP
European Patent Office
Prior art keywords
excitation light
light distribution
fluorescence
sample
light source
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.)
Pending
Application number
EP20723022.8A
Other languages
German (de)
English (en)
Inventor
Jonas FÖLLING
Lars Friedrich
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.)
Leica Microsystems CMS GmbH
Original Assignee
Leica Microsystems CMS GmbH
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 Leica Microsystems CMS GmbH filed Critical Leica Microsystems CMS GmbH
Publication of EP3956710A1 publication Critical patent/EP3956710A1/fr
Pending legal-status Critical Current

Links

Classifications

    • 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
    • G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling
    • 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
    • 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
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • 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
    • G02B21/0004—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
    • G02B21/008—Details of detection or image processing, including general computer control
    • G02B21/0084—Details of detection or image processing, including general computer control time-scale detection, e.g. strobed, ultra-fast, heterodyne detection
    • 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

Definitions

  • the so-called STED method is often used for high-resolution imaging of a sample, in which the sample is illuminated with a light distribution that is generated from a superposition of excitation light and de-excitation light.
  • STED stands for "Stimulated Emission Depletion”.
  • the excitation light is designed to stimulate the fluorescent light present in the sample to spontaneously emit fluorescent light.
  • the de-excitation light whose wavelength is different from the wavelength of the excitation light, is used to de-excite the the excitation light excited fluorophores by way of a stimulated emission of fluorescent light.
  • the solution proposed here therefore provides for the generation of two sample images which differ from one another with regard to the detected arrival times of the fluorescence photons which are used to generate the respective sample image distinguish.
  • the claimed fluorescence scanning microscope thus makes use of the fact that the dwell time of the fluorophores in the excited state, which is also referred to below as the life of the fluorophores, depends on whether the respective fluorophore is in the area of the zero point of the de-excitation light distribution or not . In the area of the zero point of the de-excitation light distribution, the lifetime is determined solely by the rate of spontaneous emission of the fluorophore. In contrast, in areas in which the de-excitation light distribution is not equal to zero, the rate of the stimulated emission is added to the rate of spontaneous emission.
  • the comparison of the two sample images directly enables both the direction and the distance to be determined in which the de-excitation light distribution is to be moved relative to the excitation light distribution in order to compensate for the offset and thus to achieve an optimal superimposition of the light distributions .
  • the direction and distance of the offset can be determined algorithmically so that only a small number of iterations are required to remove the offset, possibly even only a single iteration. In this way, the disadvantages explained at the beginning and resulting from an incorrect adjustment, such as the reduction in image brightness and the emphasis on undesired secondary maxima in the excitation light distribution, can be avoided.
  • the spatial offset between the excitation and de-excitation light distribution can in particular be determined using two-dimensional sample images. However, it is also possible to detect the offset in all three spatial directions. In this case, a three-dimensional stack of images is recorded instead of a two-dimensional sample image. In all cases, the sample image or the image stack, which represents the early fluorescence photons, contains information about the location of the maximum of the excitation light distribution, while the sample image or the image stack, which represents the late fluorescence photons, contains information about the location of the zero point of the de-excitation light distribution includes.
  • the offset can be determined in such a way that the two sample images or image stacks are related to one another, for example via a cross-correlation.
  • the de-excitation light source can be designed as a continuous wave laser light source or also as a pulsed or modulated laser light source.
  • the design as a continuous wave laser light source has the advantage that such a source is significantly cheaper than a pulsed source, in particular if the latter is to work with high laser powers as in conventional STED applications.
  • a spherical aberration in the de-excitation beam path has a significantly more disadvantageous effect than in the excitation beam path.
  • the sample image generated by the excitation beam alone as a reference, e.g. as a reference for the image brightness. Due to the brightness comparison of the two sample images, it is not necessary to use a quality measure that is laborious to determine and on the basis of which it must first be assessed how well the correction of the spherical aberration has succeeded.
  • the correction is, as it were, inherent in a reference, which is not the case, for example, if the quality of the correction is to be assessed on the basis of a single image.
  • FIG. 6 shows diagrams with examples to explain how pulsed excitation light and pulsed de-excitation light are to be coordinated with one another over time.
  • the fluorescence scanning microscope 100 comprises an excitation light source 102 which is designed to generate an excitation light distribution E of the type shown in FIG. 1, which excites fluorophores present in a sample 104 to spontaneously emit fluorescent light.
  • the wavelength of the excitation light distribution E generated by the excitation light source 102 is thus designed for the fluorophores used in the specific application.
  • the fluorescence scanning microscope 100 further comprises a de-excitation light source 106 which is designed to generate a de-excitation light distribution D of the type shown in FIG. 1, which excites the fluorophores excited by the excitation light distribution in the sample 104 by stimulated emission of fluorescent light.
  • the sample 104 illuminated with the superimposed light distribution emits fluorescent light L3, which is returned to the raster device 120 via the objective 126.
  • so-called descanned detection of the fluorescent light L3 is provided.
  • the fluorescent light L3 then successively passes the two wavelength-selective beam splitters 118, 116 and falls on the detector 110, which detects the fluorescent light L3 and outputs a corresponding output signal S to the processor 112.
  • the example according to FIG. 3 represents a particularly simple type of evaluation of the fluorescence photons.
  • An evaluation that is expanded in comparison is illustrated in the illustration according to FIG.
  • the processor 112 evaluates the fluorescence photons detected in the respective illumination target point by, for example, adapting the model function specified above according to relationship (1) to the detected temporal distribution of the fluorescence photons and determining two fit parameters aO, al from this. On the basis of the two fit parameters aO, a1, the processor 112 then generates two image points related to the same illumination target point and then, using a large number of such image points, the two sample images from which the spatial offset dx is determined.
  • FIG. 4 shows, by way of example, the course of the two fit parameters a0 and al along the axis x assuming a spatial offset dx between the maximum M of the excitation light distribution E and the zero point N of the de-excitation light distribution D.
  • the solid line shows the course of the fit parameter a1 and the dashed line the course of the fit parameter aO.
  • the course of a1 is similar to the fluorescence signal P2 in FIG. 3, which is represented by the late photons.
  • the fit parameter al is a measure of the amount of long-lived fluorophores and only these fluorophores can generate late fluorescence photons.
  • the profile of the fit parameter aO is similar to the fluorescence signal PI in FIG.
  • the excitation light source 102 outputs the trigger signal T both to the processor 112 and to the delay unit 228.
  • the delay unit 228 generates a trigger signal T ′ that is delayed with respect to the trigger signal T.
  • the delay caused by processor 112 can be set.
  • the delayed trigger signal T is used to synchronize the light pulses of the de-excitation light source 106 with those of the excitation light source 102.
  • the delay unit 228 in FIG. 5 can be configured, for example, in such a way that the light pulse emitted by the de-excitation light source 106 reaches the sample 104 by a time interval dt later than the light pulse that the excitation light source 102 emits.
  • This solution is illustrated in Figure 6b.
  • the fluorescence photons are emitted according to the excitation light distribution E.
  • the stimulated emission then takes place by emitting the de-excitation light pulse.
  • Part of the fluorescence is quenched, so that fluorophores preferably emit photons in the area of the zero point N of the de-excitation light distribution D.
  • the pulse length of the de-excitation light pulse can be set in such a way that it is in the range of the mean lifetime of the excited state of the fluorophores, for example in a range from 0.1 to 6.0 ns.
  • the stimulated emission is distributed over the time range of the pulse length of the de-excitation light pulse or over the part of the de-excitation light pulse that reaches the sample 104 after the excitation light pulse.
  • the fluorescence scanning microscope 100, 200 can also work in an operating mode in which the excitation takes place by means of a continuous wave laser light source and the de-excitation takes place by means of a pulsed laser light source.
  • the arrival time of the fluorescence photons at the detector 110 is related to the pulse times of the de-excitation light D.
  • the fluorescence photons that are detected shortly after the de-excitation pulse then contain information about the position of the zero point N of the de-excitation light distribution D.
  • Computer system can interact so that one of the methods described herein is carried out.
  • Another embodiment according to the invention comprises an apparatus or a system that is configured to transmit (for example electronically or optically) a computer program for carrying out one of the methods described herein to a receiver.
  • the receiver can be, for example, a computer, a mobile device, a storage device, or the like.
  • a programmable logic device eg, a field programmable gate array, FPGA
  • FPGA field programmable gate array
  • a field programmable gate arrangement can cooperate with a microprocessor to perform any of the methods described herein. In general, the methods are preferably performed by any hardware device.

Landscapes

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

Abstract

L'invention concerne un microscope à fluorescence comprenant une source de lumière d'excitation, laquelle est conçue de sorte à produire une répartition de la lumière d'excitation qui excite des fluorophores présents dans un échantillon à émettre spontanément des photons de fluorescence, une source de lumière de désexcitation qui est conçue de manière à produire une répartition de lumière de désexcitation qui désexcite les fluorophores excités dans l'échantillon par la répartition de lumière d'excitation par une émission stimulée de photons de fluorescence, une unité d'éclairage qui est conçue de sorte à concentrer la répartition de lumière d'excitation et la répartition de lumière de désexcitation afin qu'un maximum d'intensité de la répartition de lumière d'excitation et un minimum d'intensité de la répartition de lumière de désexcitation sont superposés dans l'espace en un point cible d'éclairage, un détecteur qui est conçu de sorte à détecter les photons de fluorescence émis de chaque point cible d'éclairage, en fonction de leurs temps d'arrivée, et un processeur. Le processeur est conçu de manière à évaluer les photons de fluorescence détectés dans chaque point cible d'éclairage en fonction de leurs temps d'arrivée, à générer sur la base de cette évaluation un premier point d'image et un second point d'image, lesquels représentent chaque point cible d'éclairage, à concentrer les premiers points d'image en une première image d'échantillon et à concentrer les seconds points d'image en une seconde image d'échantillon, et à déterminer sur la base des deux images d'échantillon un décalage spatial entre le maximum d'intensité de la répartition de lumière d'excitation et le minimum d'intensité de la répartition de lumière de désexcitation.
EP20723022.8A 2019-04-17 2020-04-17 Microscope à fluorescence et procédé d'illustration d'un échantillon Pending EP3956710A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102019110157.3A DE102019110157B4 (de) 2019-04-17 2019-04-17 Fluoreszenz-Rastermikroskop und Verfahren zur Abbildung einer Probe
PCT/EP2020/060836 WO2020212563A1 (fr) 2019-04-17 2020-04-17 Microscope à fluorescence et procédé d'illustration d'un échantillon

Publications (1)

Publication Number Publication Date
EP3956710A1 true EP3956710A1 (fr) 2022-02-23

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Application Number Title Priority Date Filing Date
EP20723022.8A Pending EP3956710A1 (fr) 2019-04-17 2020-04-17 Microscope à fluorescence et procédé d'illustration d'un échantillon

Country Status (5)

Country Link
US (1) US11650158B2 (fr)
EP (1) EP3956710A1 (fr)
CN (1) CN113711101A (fr)
DE (1) DE102019110157B4 (fr)
WO (1) WO2020212563A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102019110160B4 (de) * 2019-04-17 2023-07-27 Leica Microsystems Cms Gmbh Fluoreszenzmikroskop und Verfahren zur Abbildung einer Probe
DE102020134797B3 (de) 2020-12-23 2022-06-09 Abberior Instruments Gmbh Verfahren zum Abbilden einer interessierenden Struktur einer Probe und Mikroskop mit Array-Detektor zu dessen Durchführung
DE102022112384B4 (de) 2022-05-17 2024-02-29 Abberior Instruments Gmbh Verfahren, lichtmikroskop und computerprogramm zum einstellen einer zeitverzögerung zwischen lichtpulsen

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Also Published As

Publication number Publication date
WO2020212563A1 (fr) 2020-10-22
DE102019110157A1 (de) 2020-10-22
US20220196554A1 (en) 2022-06-23
DE102019110157B4 (de) 2021-06-17
US11650158B2 (en) 2023-05-16
CN113711101A (zh) 2021-11-26

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