WO2005029151A1 - Rastermikroskop mit evaneszenter beleuchtung - Google Patents
Rastermikroskop mit evaneszenter beleuchtung Download PDFInfo
- Publication number
- WO2005029151A1 WO2005029151A1 PCT/EP2004/052296 EP2004052296W WO2005029151A1 WO 2005029151 A1 WO2005029151 A1 WO 2005029151A1 EP 2004052296 W EP2004052296 W EP 2004052296W WO 2005029151 A1 WO2005029151 A1 WO 2005029151A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- scanning microscope
- microscope according
- illuminating light
- light
- sample
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/32—Micromanipulators structurally combined with 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
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
-
- 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
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/02—Objectives
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
-
- 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
Definitions
- the invention relates to a scanning microscope.
- a sample In scanning microscopy, a sample is illuminated with a light beam in order to observe the detection light emitted by the sample, as reflection or fluorescent light.
- Illuminating light beams are moved in a sample plane with the aid of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another, so that one mirror deflects in the x direction and the other in the y direction.
- the mirrors are tilted, for example, with the help of galvanometer control elements.
- the power of the detection light coming from the object is measured depending on the position of the scanning beam.
- the control elements are usually equipped with sensors for determining the current mirror position. Especially in confocal scanning microscopy, an object is scanned in three dimensions with the focus of a light beam.
- a confocal scanning microscope generally comprises a light source, focusing optics, with which the light from the source is focused on a pinhole - the so-called excitation diaphragm - a beam splitter, one Beam deflection device for beam control, microscope optics, a detection diaphragm and the detectors for detecting the detection or fluorescent light to be focused behind which the detectors are located
- This detection arrangement is called a descan arrangement. Detection light that does not originate directly from the focus region takes a different light path and does not pass through the detection aperture, so that point information is obtained that is obtained by sequential scanning of the object leads to a three-dimensional image with the focus of the illuminating light beam.
- US 2002/0097489 A1 discloses a microscope with evanescent illumination of a sample annt
- the microscope contains an ice light source, the light of which is coupled via a slit diaphragm through the microscope objective into the specimen slide for evanescent illumination.
- the illuminating light propagates in the slide by total internal reflection, whereby the illumination of the sample only in the area from which Microscope of this type is known under the term TIRFM (Total Internal Reflection Fluorescent Microscope)
- the task is performed by a scanning microscope with a light source that evanescently illuminates a sample arranged on a slide, with a
- Point detector which starts from a raster point of the sample Detection light received, and solved with a beam deflection device arranged in the beam path of the detection light for moving the position of the raster point in the sample.
- the invention has the advantage that scanning of the sample in two dimensions or in three dimensions, as well as a greatly increased resolution in the z direction, is possible.
- the scanning of the sample in the lateral direction (xy direction) is effected with the aid of the beam deflection device arranged in the beam path of the detection light.
- the relative distance between the sample and the objective can be adjusted to scan the sample in the axial direction (z direction).
- the sample can be arranged on a height-adjustable table or an objective adjustable in the z direction can be used.
- the illuminating light can preferably be coupled into the cover glass of the sample through the objective of the scanning microscope.
- the illuminating light is coupled into the slide by the condenser of the scanning microscope.
- the coupling occurs neither through the lens nor through the condenser but directly, for example via a prism, into the slide.
- the illuminating light preferably runs through the outer edge region of the objective pupil in order to ensure that the critical angle of the total reflection in the cover glass is reached.
- the illuminating light is preferably formed into an illuminating light beam which preferably has a focus in the plane of the objective pupil.
- the illuminating light beam can remain stationary during the examination of a sample.
- the illuminating light beam is with the help of another
- the objective preferably has a numerical aperture which is larger than 1.3 and is particularly advantageously between 1.35 and 1.42.
- a color-selective segment diaphragm is arranged in the beam path of the illuminating light, preferably in the plane of the objective pupil.
- the color-selective segment panel has different optical properties in the outer edge area than in the inner area.
- the color-selective segment diaphragm is preferably transparent in the outer edge region for light of the wavelength of the illuminating light, while in the inner region it is only transparent for light above the wavelength of the illuminating light. This embodiment variant is particularly preferred for fluorescence applications in which the wavelength of the detection light is naturally above the wavelength of the illuminating light.
- the color-selective segment aperture in the interior is only transparent to light below the wavelength of the illuminating light.
- This variant is particularly suitable for multi-photon excitation of the sample.
- the illuminating light is preferably pulsed infrared light.
- the illuminating light has several wavelengths.
- several different sample dyes can be optically excited at the same time.
- the point detector preferably contains a detection pinhole in a plane corresponding to the focal plane of the objective.
- the spatial position of the grid points from which the point detector can receive detection light is determined by the position of the detection pinhole and by the position of the beam deflection device.
- the point detector contains a multiband detector or a spectrometer. This makes it possible to obtain spectral point information from the sample. This variant is particularly advantageous in combination with multi-color lighting.
- the scanning microscope according to the invention can additionally be designed as a confocal scanning microscope, wherein at the same time both a confocal examination of the sample can take place through the interior of the color-selective segment aperture, while at the same time TIRF illumination is made possible through the exterior of the color-selective segment aperture.
- imaging optics preferably a Bertrandt lens, are provided in the beam path of the illuminating light.
- the light path of the detection light comprises a plurality of detection channels, wherein a bandpass filter can be provided in each of the detection channels.
- Fig. 4 shows another scanning microscope according to the invention.
- the 1 shows a scanning microscope according to the invention with a light source 1 which is designed as an argon ion laser 3.
- the light source 1 generates an illuminating light beam 5, which is reflected by a beam splitter 7 to the objective 9.
- the illuminating light beam 5 runs through the outer edge region of the objective pupil 11 (indicated by the double arrow 24) and is coupled into the cover glass 13 of the sample 15 for evanescent illumination.
- In the beam path of the Illumination light beam bundle 5 contains imaging optics 19 designed as a Bertrandt lens 17, which produces a focus in the plane of the objective pupil 11.
- a further beam deflection device 21 is located in the beam path of the illuminating light beam 5, which contains a gimbal-mounted scanning mirror, not shown.
- the color-selective segment diaphragm 23 shown in FIG. 2 is arranged in the plane of the objective pupil 11.
- the color-selective segment diaphragm 23 has an outer edge region 25 which is transparent to the illuminating light.
- the color-selective segment diaphragm 23 has an inner region 27 which is transparent to light above the wavelength of the illuminating light.
- the detection light 51 originating from the sample passes through the lens and the interior of the color-selective segment diaphragm to the beam splitter 7, passes through it and reaches the point detector 33 via the beam deflection device 29, which contains a gimbal-mounted scanning mirror 31.
- the point detector 33 contains a pinhole aperture 35, whose spatial position together with the position of the gimbal-mounted scanning mirror 31 determines the position of the raster point in the sample from which the point detector 33 receives detection light 51.
- the point detector 33 contains a multiband detector 36 which can simultaneously receive detection light 51 in a plurality of adjustable wavelength bands.
- the illuminating light beam of the argon ion laser 3 contains illuminating light of several wavelengths, which enables multicolor excitation of the sample.
- FIG. 3 shows a further scanning microscope according to the invention, in which a confocal examination of a sample is made possible simultaneously with the TIRF examination of a sample.
- This scanning microscope contains a further light source 37, which is designed as a pulsed titanium sapphire laser 39 and which emits a further illuminating light beam 41.
- the further Illumination light beam bundle 41 passes through a second beam splitter 43 and via the beam deflection device 29, as well as through the beam splitter 7 and a third beam splitter 45 to the objective 9 and illuminates directly through the inner region 27 of the segment diaphragm 23 through the sample 15.
- In the sample 15 is independent of the TIRF illumination with the illuminating light beam 5 by the other
- Illumination light beam 41 causes two-photon excitation of the sample.
- the further detection light 53 resulting from the two-photon excitation of the sample is detected with the aid of a non-descan detector 47, which is designed as a CCD element 49.
- This further detection light 53 reaches the non-descan detector 47 via the inner area of the objective, by reflection at the third beam splitter 45.
- another color-selective segment diaphragm is used in the objective pupil, which is transparent in the outer edge area for the illuminating light beam 5 of the light source 1 is and which is designed reflective for this light in the interior. This ensures that no illuminating light is radiated directly onto the sample.
- the beam splitters 7, 45 and 43 are designed such that neither the light from the illuminating light beam 5 nor from the titanium sapphire laser 39 reaches the point detector 33 or the non-descan detector 47.
- the light source 1 consists of a titanium sapphire laser 55, which emits an illuminating light beam 5, which is guided as TIRF illumination through the outer edge region 25 of a color-selective segment diaphragm 23.
- the evanescent illumination induces 15 multi-photon excitation in the sample.
- the resulting fluorescent light passes through the entire segment aperture 23 via the third beam splitter 45 to the non-descan detector 47, which is designed as a CCD element 49.
- a three-dimensional image of the sample is recorded by confocal illumination with a light source 37, which consists of an argon ion laser 57, and detection with a point detector 33, which is designed as a multiband detector 36.
- a light source 37 which consists of an argon ion laser 57
- a point detector 33 which is designed as a multiband detector 36.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/573,435 US7480046B2 (en) | 2003-09-25 | 2004-09-23 | Scanning microscope with evanescent wave illumination |
EP04787202A EP1664888B1 (de) | 2003-09-25 | 2004-09-23 | Rastermikroskop mit evaneszenter beleuchtung |
DE502004008546T DE502004008546D1 (de) | 2003-09-25 | 2004-09-23 | Rastermikroskop mit evaneszenter beleuchtung |
JP2006527425A JP2007506955A (ja) | 2003-09-25 | 2004-09-23 | エバネッセント波照明を備えた走査顕微鏡 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10344410A DE10344410A1 (de) | 2003-09-25 | 2003-09-25 | Rastermikroskop mit evaneszenter Beleuchtung |
DE10344410.6 | 2003-09-25 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005029151A1 true WO2005029151A1 (de) | 2005-03-31 |
Family
ID=34353078
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2004/052296 WO2005029151A1 (de) | 2003-09-25 | 2004-09-23 | Rastermikroskop mit evaneszenter beleuchtung |
Country Status (7)
Country | Link |
---|---|
US (1) | US7480046B2 (de) |
EP (1) | EP1664888B1 (de) |
JP (1) | JP2007506955A (de) |
CN (1) | CN1856725A (de) |
AT (1) | ATE415641T1 (de) |
DE (2) | DE10344410A1 (de) |
WO (1) | WO2005029151A1 (de) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1698929A1 (de) * | 2005-03-01 | 2006-09-06 | Leica Microsystems CMS GmbH | Objektiv und Mikroskop |
US7486441B2 (en) | 2005-03-01 | 2009-02-03 | Leica Microsystems Cms Gmbh | Objective and microscope |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8040597B2 (en) * | 2006-05-16 | 2011-10-18 | Olympus Corporation | Illuminating device |
JP2009198903A (ja) * | 2008-02-22 | 2009-09-03 | Olympus Corp | 光学機器 |
US8456725B2 (en) * | 2008-03-26 | 2013-06-04 | Yale University | Optical system that selectively provides either of a collimated light beam or a convergent light beam |
US9012872B1 (en) | 2010-01-15 | 2015-04-21 | Iowa State University Research Foundation, Inc. | Auto-calibrated scanning-angle prism-type total internal reflection microscopy for nanometer-precision axial position determination and optional variable-illumination-depth pseudo total internal reflection microscopy |
DE102010034122B4 (de) | 2010-08-12 | 2020-03-26 | Carl Zeiss Microscopy Gmbh | Mikroskop und Objektiv, insbesondere für die TIRF-Mikroskopie |
DE102011017078B4 (de) * | 2011-04-15 | 2019-01-31 | Leica Microsystems Cms Gmbh | Weitfeld-Mikroskop-Beleuchtungssystem, Verwendung desselben und Weitfeld-Beleuchtungsverfahren |
DE102012102983A1 (de) * | 2012-04-05 | 2013-10-10 | Carl Zeiss Microscopy Gmbh | Verfahren und Vorrichtung zum Bestimmen eines kritischen Winkels eines Anregungslichtstrahls |
DE102012023024B4 (de) | 2012-11-07 | 2023-05-04 | Carl Zeiss Microscopy Gmbh | Lichtmikroskop und Mikroskopieverfahren |
US9696264B2 (en) * | 2013-04-03 | 2017-07-04 | Kla-Tencor Corporation | Apparatus and methods for determining defect depths in vertical stack memory |
DE102013222562B4 (de) | 2013-11-06 | 2023-01-26 | Leica Microsystems Cms Gmbh | Mikroskop und Verfahren sowie Verwendung eines Mikroskops für evaneszente Beleuchtung und punktförmige Rasterbeleuchtung |
US10061111B2 (en) | 2014-01-17 | 2018-08-28 | The Trustees Of Columbia University In The City Of New York | Systems and methods for three dimensional imaging |
WO2015109323A2 (en) * | 2014-01-17 | 2015-07-23 | The Trustees Of Columbia University In The City Of New York | Systems and methods for three-dimensional imaging |
WO2015157769A1 (en) * | 2014-04-11 | 2015-10-15 | The Regents Of The University Of Colorado, A Body Corporate | Scanning imaging for encoded psf identification and light field imaging |
DE102014110341A1 (de) | 2014-07-22 | 2016-01-28 | Leica Microsystems Cms Gmbh | Verfahren und Vorrichtung zum mikroskopischen Untersuchen einer Probe |
LU92505B1 (de) | 2014-07-22 | 2016-01-25 | Leica Microsystems | Verfahren und vorrichtung zum mikroskopischen untersuchen einer probe |
CN104536132A (zh) * | 2014-12-09 | 2015-04-22 | 中国科学院上海技术物理研究所 | 一种多孔径近距大视区光学系统及扫描镜的设计方法 |
KR102580771B1 (ko) * | 2015-05-04 | 2023-09-19 | 매직 립, 인코포레이티드 | 가상 및 증강 현실을 위한 분리된 동공 광학 시스템들 및 이를 사용하여 이미지들을 디스플레이하기 위한 방법들 |
EP3482238A1 (de) | 2016-07-10 | 2019-05-15 | The Trustees of Columbia University in the City of New York | Dreidimensionale bildgebung mit gewobbelter, konfokal ausgerichteter planarer anregung mit einem bildrelais |
CN108614351B (zh) * | 2016-12-12 | 2020-07-10 | 凝辉(天津)科技有限责任公司 | 一种具有可旋转式物镜的显微镜 |
US10712545B2 (en) * | 2017-03-07 | 2020-07-14 | The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Systems and methods for conducting contact-free thickness and refractive-index measurements of intraocular lenses using a self-calibrating dual confocal microscopy system |
CN109597195B (zh) * | 2019-01-30 | 2021-02-23 | 清华大学 | 基于晶格光照的超分辨三维光场显微成像系统及方法 |
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DE19902234A1 (de) * | 1998-08-21 | 2000-02-24 | Witec Wissenschaftliche Instr | Kombinationsmikroskop |
US6255642B1 (en) * | 1999-06-23 | 2001-07-03 | Massachusetts Institute Of Technology | Standing wave total internal reflection imaging |
US20030058530A1 (en) * | 2001-09-25 | 2003-03-27 | Yoshihiro Kawano | Microscope switchable between observation modes |
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JP3093145B2 (ja) * | 1995-12-13 | 2000-10-03 | 科学技術振興事業団 | 光照射切り替え方法 |
JP2001356272A (ja) * | 2000-06-12 | 2001-12-26 | Olympus Optical Co Ltd | 画像取得方法及び走査型光学顕微鏡 |
JP4686015B2 (ja) * | 2000-09-21 | 2011-05-18 | オリンパス株式会社 | 照明装置 |
US6597499B2 (en) * | 2001-01-25 | 2003-07-22 | Olympus Optical Co., Ltd. | Total internal reflection fluorescence microscope having a conventional white-light source |
JP4827335B2 (ja) * | 2001-08-13 | 2011-11-30 | オリンパス株式会社 | 走査型レーザ顕微鏡 |
DE10143481A1 (de) * | 2001-09-05 | 2003-03-20 | Europ Lab Molekularbiolog | Mikroskop |
JP2003131141A (ja) * | 2001-10-24 | 2003-05-08 | Japan Science & Technology Corp | 回転式輪帯全反射照明機構 |
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2003
- 2003-09-25 DE DE10344410A patent/DE10344410A1/de not_active Withdrawn
-
2004
- 2004-09-23 JP JP2006527425A patent/JP2007506955A/ja active Pending
- 2004-09-23 AT AT04787202T patent/ATE415641T1/de not_active IP Right Cessation
- 2004-09-23 EP EP04787202A patent/EP1664888B1/de not_active Not-in-force
- 2004-09-23 CN CNA2004800278045A patent/CN1856725A/zh active Pending
- 2004-09-23 WO PCT/EP2004/052296 patent/WO2005029151A1/de active Application Filing
- 2004-09-23 US US10/573,435 patent/US7480046B2/en not_active Expired - Fee Related
- 2004-09-23 DE DE502004008546T patent/DE502004008546D1/de active Active
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DE19902234A1 (de) * | 1998-08-21 | 2000-02-24 | Witec Wissenschaftliche Instr | Kombinationsmikroskop |
US6255642B1 (en) * | 1999-06-23 | 2001-07-03 | Massachusetts Institute Of Technology | Standing wave total internal reflection imaging |
US20030058530A1 (en) * | 2001-09-25 | 2003-03-27 | Yoshihiro Kawano | Microscope switchable between observation modes |
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KAWATA S ET AL: "NEAR-FIELD SCANNING OPTICAL MICROSCOPE WITH A LASER TRAPPED PROBE", JAPANESE JOURNAL OF APPLIED PHYSICS, PUBLICATION OFFICE JAPANESE JOURNAL OF APPLIED PHYSICS. TOKYO, JP, vol. 33, no. 12A, 1 December 1994 (1994-12-01), pages L1725 - L1727, XP000682439, ISSN: 0021-4922 * |
V. PROTASENKO, A. GALLAGHER, M. LABARDI, D.J. NESBITT: "Enhancement and quenching of the fluorescence of single CdSe/ZnS quantum dots studied by confocal apertureless near-field scanning optical microscope", PROCEEDINGS OF SPIEADVANCED CHARACTERIZATION TECHNIQUES FOR OPTICS, SEMICONDUCTORS, AND NANOTECHNOLOGIES, vol. 5188, November 2003 (2003-11-01), pages 254 - 263, XP002312031 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1698929A1 (de) * | 2005-03-01 | 2006-09-06 | Leica Microsystems CMS GmbH | Objektiv und Mikroskop |
US7486441B2 (en) | 2005-03-01 | 2009-02-03 | Leica Microsystems Cms Gmbh | Objective and microscope |
Also Published As
Publication number | Publication date |
---|---|
JP2007506955A (ja) | 2007-03-22 |
US20070052958A1 (en) | 2007-03-08 |
DE10344410A1 (de) | 2005-04-28 |
CN1856725A (zh) | 2006-11-01 |
US7480046B2 (en) | 2009-01-20 |
EP1664888B1 (de) | 2008-11-26 |
EP1664888A1 (de) | 2006-06-07 |
DE502004008546D1 (de) | 2009-01-08 |
ATE415641T1 (de) | 2008-12-15 |
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