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WO2012003259A1 - Système et procédé de production de rideaux de lumière non diffractants au moyen d'une pluralité de faisceaux optiques non diffractants minimalement interférants, se chevauchant spatialement - Google Patents

Système et procédé de production de rideaux de lumière non diffractants au moyen d'une pluralité de faisceaux optiques non diffractants minimalement interférants, se chevauchant spatialement Download PDF

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Publication number
WO2012003259A1
WO2012003259A1 PCT/US2011/042495 US2011042495W WO2012003259A1 WO 2012003259 A1 WO2012003259 A1 WO 2012003259A1 US 2011042495 W US2011042495 W US 2011042495W WO 2012003259 A1 WO2012003259 A1 WO 2012003259A1
Authority
WO
WIPO (PCT)
Prior art keywords
beams
nondiffracting
microscope
light
transforming
Prior art date
Application number
PCT/US2011/042495
Other languages
English (en)
Inventor
Hari Shroff
Andrew Gregory York
Yicong Wu
Original Assignee
The Government Of The U.S.A., Represented By The Secretary, Department Of Health And Human Services
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 The Government Of The U.S.A., Represented By The Secretary, Department Of Health And Human Services filed Critical The Government Of The U.S.A., Represented By The Secretary, Department Of Health And Human Services
Publication of WO2012003259A1 publication Critical patent/WO2012003259A1/fr

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Classifications

    • 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
    • G02B21/0004—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
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • 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
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
    • 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
    • G02B27/42—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect
    • G02B27/4294—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect in multispectral systems, e.g. UV and visible

Definitions

  • This application relates to a system of producing nondiffracting light sheets by a multiplicity of spatially overlapping optical beams, and in particular a selective plane illumination microscopy that utilizes such a system of producing nondiffracting light sheets.
  • SPIM Selective plane illumination microscopy
  • SPIM is a technique that uses a thin sheet of light for the illumination of a sample at a detection plane of a detection objective lens that detects a fluorescence signal generated by the sample when illuminated.
  • SPIM provides optical sectioning of the sample by illuminating only those fluorophores that are in the detection plane.
  • the system combines the advantages of wide-field methods (speed, flexibility, and dynamic range) with those of a confocal arrangement (optical sectioning).
  • SPIM also provides quantitative three-dimensional maps of the distribution of a fluorophore within the sample, for example, the expression pattern of GFP-labeled protein, with high spatiotemporal resolution and an excellent signal to noise ratio.
  • the standard SPIM technique produces nonuniform axial resolution, which is caused by the diffraction of the laser beam through the sample. As diffraction causes the laser beam to spread and thicken at distances far from its center (beam waist), optical sectioning degrades, thereby forcing a compromise between field of view and axial resolution.
  • a system may include an illumination source that transmits a beam of light.
  • a first optical arrangement transforms the beam of light into a plurality of beams with each of the plurality of beams having a different wavelength.
  • a second optical arrangement transforms the plurality of beams into a plurality of nondiffracting beams with each of the plurality of nondiffracting beams having different wavelengths such that the plurality of nondiffracting beams spatially overlap with at least another one of the plurality of nondiffracting beams with reduced interference.
  • a method for producing nondiffracting sheets of light may include:
  • each of the plurality of nondiffracting beams having different wavelengths such that the plurality of nondiffracting beams overlap with one another with reduced interference.
  • a microscope may include a laser source that transmits a pulsed laser beam, a diffraction grating for causing the pulsed laser beam to be split into a plurality of pulsed laser beams with each of the plurality of pulsed laser beams having different wavelengths.
  • a first optic arrangement magnifies the plurality of pulsed laser beams having different wavelengths from the diffraction grating and images the plurality of pulsed laser beams through an aperture for transforming the plurality of pulsed laser beams into a plurality of nondiffracting beams. Each of the plurality of nondiffracting beams has a different wavelength.
  • a second optic arrangement demagnifies the plurality of nondiffracting beams, and then focuses the plurality of nondiffracting beams onto a sample at the detection plane of the second optic arrangement, wherein the plurality of nondiffracting beams spatially overlap at least one other of the plurality of nondiffracting beams, but with reduced interference.
  • FIG. 1 is a simplified illustration of an embodiment of a selective plane illumination microscopy system
  • FIGS. 2 and 2B are simplified illustrations showing the plurality of nondiffracting beams relative to a detection plane of the microscopy system.
  • SPIM Selective plane illumination microscopy
  • interference is the addition (superposition) of two or more waves that produces a new wave pattern.
  • Interference usually refers to the interaction of waves that are correlated or coherent with each other, either because these waves come from the same source or because they have the same or nearly the same frequency.
  • the principle of superposition of waves states that the resultant displacement at a point is equal to the vector sum of the displacements of different waves at that point. For example, if a crest of a wave meets a crest of another wave at the same point then the crests interfere constructively and the resultant wave amplitude is increased. However, if a crest of a wave meets a trough of another wave then the waves interfere destructively, and the resultant wave amplitude is decreased.
  • both destructive and constructive interference produce unwanted concentric ringing that degrades the quality of the image detected by a Bessel-beam based SPIM microscope as discussed above.
  • constructive interference focuses energy into planes other than the detection plane of the microscope, thereby producing peaks in the rings, while destructive interference generates intensity nulls, which produces troughs in the rings.
  • both constructive and destructive interference above and below the detection plane of the microscope create difficulties in measuring the fluorescence signal generated by the illumination of a sample under detection by the microscope.
  • the term "interference" will refer to both constructive and destructive interference as described above.
  • embodiments of a system and method that produces nondiffracting sheets of light using a multiplicity of spatially overlapping optical beams as disclosed herein include particular properties and characteristics that address issues related to reducing interference.
  • the system and method as described herein overcomes these deficiencies by producing beams of light having different wavelengths that are transformed into nondiffracting beams that also have different wavelengths, which cause the nondiffracting beams to spatially overlap (causing a sheet) with minimal interference between the nondiffracting beams.
  • the interference due to any individual nondiffracting beam is reduced if the illumination source is a femtosecond laser beam, which produces multiphoton excitation of the sample and eliminates regions of high intensity that would otherwise exist due to the mechanism of linear absorption for generating fluorescence in a biological specimen under observation.
  • the microscope 10 may be a SPIM microscope that includes a laser device 12, such as a femtosecond laser device that generates a pulsed laser beam 36, which is reflected off a diffraction grating 14.
  • the plurality of beams 38 having different wavelengths imparted by the diffraction grating 14 is then imaged by a pair of relay lenses 18 and 20, such as lenses 18 and 20 having focal lengths of 300 mm and 100 mm, respectively, through an annular aperture 16 that transforms the plurality of beams 38 having different wavelengths into a plurality of nondiffracting beams 40 with different wavelengths.
  • the focal length of the relay lens 18 and 20 may be chosen so that the diameter of the pulsed laser beam 36 is demagnified (or magnified) to fill the annular aperture 16.
  • the annular aperture 16 may have an inner radius between 2.33 mm to 2.63 mm and an outer radius of 2.66 mm.
  • the value of the outer radius of the annular aperture 16 may be chosen so that, after magnification by the relay lenses 18, 20, 22, and 24, the back focal plane diameter of the first objective lens 26 is filled so as to produce the highest available numerical aperture.
  • the value of the inner radius of the annular aperture 16 determines the length of the nondiffacting beam 40 with smaller values producing a shorter nondiffracting beam 40 while larger values producing a longer nondiffracting beam 40
  • the range of values of the outer radius may be between 2.0-2.66 mm, and the inner radius may be any value less than the range of values for the outer radius.
  • other embodiments that transform the plurality of beams 38 into plurality of nondiffracting beams 40 may include an axicon, a spatial light modulator (SLM), or a binary phase mask.
  • nondiffracting beam means any beam of electromagnetic light that shows little or no diffraction over a significant propagation distance (i.e. the transverse intensity distributions of the beam do not vary over a significant propagation distance), including but not limited to Bessel-like beams (i.e., those beams whose wave amplitude is approximated by a Bessel function).
  • the plurality of nondiffracting beams 40 are then reimaged by another pair of relay lenses 22 and 24, for example lenses having focal lengths of 100 mm and 300 mm, respectively, onto the back detection plane of a first objective lens 26, for example an excitation objective lens.
  • the relay lenses 22 and 24 may have any suitable focal length with the only constraint being that the nondiffracting beams 40 must cover the back focal plane of the first objective lens 26
  • the first objective lens 26 may be a Nikon 0.8 NA 40x water immersion objective lens.
  • the annular aperture 16 may be replaced by an axicon plus additional lenses for increased power efficiency.
  • this arrangement transforms the energy into a series of nondiffracting beams 40 that overlap each other, but also reduces interference of the beams 40.
  • the resulting plurality of nondiffracting beams 40 may illuminate a biological sample 32 and induce a multiphoton fluorescence excitation in the sample 32 along the optical axis and in the vicinity of a detection plane 42 of the first objective lens 26.
  • the microscope 10 further includes a focusing optic arrangement having a second objective lens 28, for example a detection objective lens, positioned at a 90-degree angle relative to the sample 36 on the detection plane 42.
  • the second or detection objective lens 28 may be a Nikon 0.8 NA 40x water immersion objective lens.
  • the main constraint on the types of first and second objective lenses 26 and 28 is that these lenses must fit together at 90 degrees angle for a SPIM arrangement such that the 90 degree geometry imposes spatial constraints on the types of objective lenses that may be used.
  • a tube lens 34 is positioned along the optic axis of the second objective lens 28 for imaging the fluorescence excitation signal emitted by the sample 32 onto a widefield detector 30.
  • the tube lens 34 may be a 200 mm Nikon tube lens, however other focal lengths may be used to provide a given magnification.
  • the 200 mm tube lens in the example embodiment provides a magnification of 40x when combined with the first objective lens 26.
  • the widefield detector 30 may be a charge coupled detector (CCD), for example, an Andor iXon 888).
  • the optical overall arrangement of the microscope 10 described above creates a multiplicity of nondiffracting beams of light from an illumination source that overlap spatially with each other but with reduced interference, thereby producing sheets of light that resist diffraction along the optic axis. This arrangement results in better axial resolution of the image than is possible with conventional SPIM microscopes.
  • the concept of transforming a pulsed laser beam 36 generated from a laser source 12 that is split into a plurality of beams 38 having different wavelengths and then transformed into a plurality of nondiffracting beams 40 may be applied to other microscopy, such as fluorescence microscopy, where the application of nondiffracting sheets of light to illuminate a sample to generate the resulting excitation profile is desirable.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

La présente invention concerne un système et un procédé de production de rideaux de lumière non diffractants qui se chevauchent spatialement entre eux quand ils coupent le plan de détection d'un dispositif optique. Le système comporte une source d'éclairage servant à émettre un faisceau de lumière au moyen du dispositif optique qui comporte un réseau de diffraction utilisé pour diffracter le faisceau de lumière de façon à produire des faisceaux de lumière de différentes longueurs d'ondes, faisceaux que l'on fait passer au travers d'une fenêtre annulaire convertissant les faisceaux de lumière en faisceaux non diffractants de différentes longueurs d'ondes.
PCT/US2011/042495 2010-06-30 2011-06-30 Système et procédé de production de rideaux de lumière non diffractants au moyen d'une pluralité de faisceaux optiques non diffractants minimalement interférants, se chevauchant spatialement WO2012003259A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US36035210P 2010-06-30 2010-06-30
US61/360,352 2010-06-30

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WO2012003259A1 true WO2012003259A1 (fr) 2012-01-05

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Cited By (9)

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Publication number Priority date Publication date Assignee Title
WO2013150273A1 (fr) * 2012-04-03 2013-10-10 University Court Of The University Of St Andrews Imagerie haute résolution de volumes étendus
CN103792663A (zh) * 2014-01-17 2014-05-14 北京空间机电研究所 一种生成螺旋式贝塞尔光束的光学系统及生成方法
CN104677871A (zh) * 2015-02-27 2015-06-03 中国科学院自动化研究所 多光子激发光片照明显微成像系统
CN107003509A (zh) * 2015-05-22 2017-08-01 香港科技大学 产生用于多色荧光显微镜的无衍射光片的方法和系统
WO2018033581A1 (fr) * 2016-08-15 2018-02-22 Leica Microsystems Cms Gmbh Microscope à feuille de lumière
WO2018007469A3 (fr) * 2016-07-06 2018-03-01 Leica Microsystems Cms Gmbh Procédé d'analyse d'un échantillon et dispositif permettant la mise en œuvre dudit procédé
CN109656028A (zh) * 2019-01-11 2019-04-19 中国科学院广州生物医药与健康研究院 一种产生无衍射光的系统及方法
WO2020030410A1 (fr) * 2018-08-10 2020-02-13 Leica Microsystems Cms Gmbh Installation d'éclairage pour un microscope, microscope et procédé d'éclairage d'un volume d'échantillon dans un microscope
WO2020074855A1 (fr) * 2018-10-11 2020-04-16 University Court Of The University Of St Andrews Imagerie à feuille de lumière

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013150273A1 (fr) * 2012-04-03 2013-10-10 University Court Of The University Of St Andrews Imagerie haute résolution de volumes étendus
CN104204898A (zh) * 2012-04-03 2014-12-10 圣安德鲁斯大学董事会 扩展容积的高分辨率成像
US20150029325A1 (en) * 2012-04-03 2015-01-29 University Court Of The University Of St Andrews High resolution imaging of extended volumes
JP2015514235A (ja) * 2012-04-03 2015-05-18 ユニバーシティー コート オブ ザユニバーシティー オブ セイント アンドリューズＵｎｉｖｅｒｓｉｔｙ Ｃｏｕｒｔ Ｏｆ Ｔｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｓｔ Ａｎｄｒｅｗｓ 拡張ボリュームの高分解能イメージング
US10444520B2 (en) 2012-04-03 2019-10-15 University Court Of The University Of St Andrews High resolution imaging of extended volumes
CN103792663A (zh) * 2014-01-17 2014-05-14 北京空间机电研究所 一种生成螺旋式贝塞尔光束的光学系统及生成方法
CN103792663B (zh) * 2014-01-17 2015-11-18 北京空间机电研究所 一种生成螺旋式贝塞尔光束的光学系统及生成方法
CN104677871A (zh) * 2015-02-27 2015-06-03 中国科学院自动化研究所 多光子激发光片照明显微成像系统
CN107003509A (zh) * 2015-05-22 2017-08-01 香港科技大学 产生用于多色荧光显微镜的无衍射光片的方法和系统
EP3298449A4 (fr) * 2015-05-22 2019-02-06 The Hong Kong University of Science and Technology Procédés et systèmes permettant de générer des feuilles de lumière non diffractée pour une microscopie en fluorescence multicolore
LU93143B1 (de) * 2016-07-06 2018-03-05 Leica Microsystems Verfahren zum Untersuchen einer Probe sowie Vorrichtung zum Ausführen eines solchen Verfahrens
WO2018007469A3 (fr) * 2016-07-06 2018-03-01 Leica Microsystems Cms Gmbh Procédé d'analyse d'un échantillon et dispositif permettant la mise en œuvre dudit procédé
US10983321B2 (en) 2016-07-06 2021-04-20 Leica Microsystems Cms Gmbh Method for examining a sample, and device for carrying out such a method
US11835701B2 (en) 2016-07-06 2023-12-05 Leica Microsystems Cms Gmbh Method for examining a sample, and device for carrying out such a method
WO2018033581A1 (fr) * 2016-08-15 2018-02-22 Leica Microsystems Cms Gmbh Microscope à feuille de lumière
US10983327B2 (en) 2016-08-15 2021-04-20 Leica Microsystems Cms Gmbh Light sheet microscope
WO2020030410A1 (fr) * 2018-08-10 2020-02-13 Leica Microsystems Cms Gmbh Installation d'éclairage pour un microscope, microscope et procédé d'éclairage d'un volume d'échantillon dans un microscope
CN112567281A (zh) * 2018-08-10 2021-03-26 莱卡微系统Cms有限责任公司 用于显微镜的照明总成、显微镜和用于照明显微镜中的样本空间的方法
US12019229B2 (en) 2018-08-10 2024-06-25 Leica Microsystems Cms Gmbh Illumination arrangement for a microscope, microscope and method for illuminating a sample volume in a microscope
WO2020074855A1 (fr) * 2018-10-11 2020-04-16 University Court Of The University Of St Andrews Imagerie à feuille de lumière
CN109656028A (zh) * 2019-01-11 2019-04-19 中国科学院广州生物医药与健康研究院 一种产生无衍射光的系统及方法

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