EP1664887A1 - Microscope confocal a balayage laser - Google Patents

Microscope confocal a balayage laser

Info

Publication number
EP1664887A1
EP1664887A1 EP04765251A EP04765251A EP1664887A1 EP 1664887 A1 EP1664887 A1 EP 1664887A1 EP 04765251 A EP04765251 A EP 04765251A EP 04765251 A EP04765251 A EP 04765251A EP 1664887 A1 EP1664887 A1 EP 1664887A1
Authority
EP
European Patent Office
Prior art keywords
spots
spot
along
microscope
excitation
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.)
Withdrawn
Application number
EP04765251A
Other languages
German (de)
English (en)
Inventor
Dieter GRÄFE
Martin KÜHNER
Frank Eismann
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.)
Jenoptik AG
Original Assignee
Carl Zeiss Jena 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 Carl Zeiss Jena GmbH filed Critical Carl Zeiss Jena GmbH
Publication of EP1664887A1 publication Critical patent/EP1664887A1/fr
Withdrawn legal-status Critical Current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
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers

Definitions

  • the invention relates to a conf ⁇ cal laser scanning microscope with an excitation beam path that bundles the excitation beam in several spots located in an object plane, and a detection beam path that confocally images the spots by means of pinhole diaphragms on a multi-channel detector, and with a scanning device that causes a two-dimensional relative movement between an object located in the object plane and the spots.
  • Another confocal laser scanning microscope of the type mentioned is known from US ⁇ .028.306.
  • a spot distribution with several spots is imaged in an object plane by means of a laser light source and a microlens array.
  • the spots are imaged confocally using an aperture array.
  • An x / y beam scanner scans the area to be examined, in one embodiment the spots being shifted over a path length which is the same as the distance between neighboring spots. This allows a large area to be scanned with a small beam deflection, since the each of the adjacent individual spots covers a small area and all these areas together fill the scanned area.
  • a disadvantage of this arrangement is that the small scanned areas must meet seamlessly with tolerances in the micrometer range. In some applications, overexposure would cause bleaching and saturation effects of fluorophores that cannot be compensated for.
  • the invention has for its object to provide a laser scanning microscope of the type mentioned, with which an object can be scanned quickly.
  • This task is accomplished in a confocal laser scanning microscope with an excitation beam path that bundles excitation radiation in several spots 30 located in an object plane (11) and a detection beam path that confocates the spots (30) onto a multichannel by means of pinhole diaphragms (14.2).
  • the object is thus scanned in strips, each strip being detected in that all spots are passed over it.
  • the object area to be detected is therefore not divided into individual areas to be seamlessly joined together, each of which is sensed by a spot, but rather all spots detect fluorescent radiation from the strip.
  • the next strip of the object is imaged by a subsequent displacement of the spots in a second direction, which is preferably orthogonal to the first direction.
  • the object area is thus divided into strips, with all spots being guided over each strip.
  • the generation of the spot pattern is expediently carried out by means of a microlens array not used for the detection in the excitation beam path, the microlens array bringing about a line-shaped or a rectangular or square arrangement of the spots.
  • the pinhole covers are of course adapted to the spot pattern; in the case of a cell-shaped microlens array, an aperture row will be used; in the case of a rectangular or square spot pattern, a corresponding aperture array is provided.
  • the pinhole diaphragms are advantageously not located in the excitation beam path, but are, for example, arranged upstream of the multi-channel detector, since then no disturbing excitation beam reflections occur. There are separate diffraction-limiting ones for the generation and for the detection of the spots Objects provided and a central aperture, which is part of both the excitation and the detection beam path, can be disregarded.
  • This distance should preferably be at least ten times the spot diameter.
  • a large distance between adjacent spots can be realized particularly easily if the spot pattern is tilted with respect to the first direction in such a way that the spots have a distance perpendicular to the direction equal to or less than the spot diameter.
  • This configuration ensures, on the one hand, that the strip of the object is scanned without gaps during the displacement along the first direction and, on the other hand, that an almost arbitrarily large distance between adjacent spots can be set.
  • the tilt or inclination of the spot pattern with respect to the first direction with which the scanning device moves the beam relatively can be achieved in an optical scanning device in that the element producing the optical spots in the excitation beam path, for example the aforementioned microlens array, as well as the pinhole diaphragms and the multi-channel detector can be rotated about the optical axis with respect to the first direction in the beam path.
  • the element producing the optical spots in the excitation beam path for example the aforementioned microlens array, as well as the pinhole diaphragms and the multi-channel detector can be rotated about the optical axis with respect to the first direction in the beam path.
  • the microscope according to the invention can particularly preferably choose the path of displacement along the first direction significantly larger than the distance between adjacent spots, so that the problem mentioned with respect to US Pat. No. 6,028,306 that small areas must be joined together seamlessly is avoided.
  • FIG. 3 shows a schematic representation of spot distribution and scanning movement in a spot line
  • FIG. 5 shows a scanning movement with a square spot distribution
  • Fig. 6 shows a laser scanning microscope similar to that of Figure 2, but with a table scanner.
  • FIG. 1 shows a conventional laser scanning microscope with an optical beam scanner, an object being scanned with a beam.
  • the radiation from a laser 1 is adapted to the requirements of the microscope with regard to the beam parameters such as waist position and beam cross section with an optical arrangement 2.
  • the excitation or illumination radiation is coupled into the general beam path via a divider 3 and directed to beam scanners 4 and 5.
  • the beam scanners are closely adjacent and are arranged in the immediate vicinity of a pupil of the beam path can be controlled separately.
  • a downstream scanning optics 6 generates a spot image in all the different beam deflections generated by the scanners in an image plane 7.
  • a tube lens 8 collects the radiation in an aperture plane 9, from which an objective 10 generates a reduced spot image in an object plane 11.
  • sample parts at the spot emit fluorescence radiation with radiation which is shifted longer than the excitation radiation, which is collected again by the objective 10 and which runs the same way back through the arrangement described.
  • the beam splitter 3 effects a separation of the fluorescent radiation into a detection beam path.
  • An interference filter 12 separates portions of the short-wave excitation radiation that are still present in the beam path.
  • a lens 13 generates a spot image in a pinhole plane 13 from the object spot 11 that is just illuminated or fluorescent.
  • a detector 15 downstream of this in this case a single-point receiver, delivers a radiation-intensity-dependent video signal, which is converted into an image signal by a connected evaluation unit ,
  • the splitter 3 is not a wavelength-selective, dichroic beam splitter, but a simple neutral beam splitter.
  • the emission filter 12 is then omitted.
  • the size of the pinhole diaphragm With the size of the pinhole diaphragm, the size of the object structure to be detected can be adjusted and with smaller diaphragm diameters, a greater depth discrimination in the Object level is set, ie the depth range is set from which radiation is taken to form the image. In this way, disruptive beam components from other depth ranges can be eliminated. This is the decisive advantage of laser scanning microscopy over conventional light microscopy.
  • FIG. 2 shows a confocal multichannel laser scanning microscope which, except for the deviations described below, corresponds to the design according to FIG. 1.
  • the arrangement is equipped for multi-channel operation.
  • a collimated laser beam is expanded accordingly by a telescope 2.2, so that it illuminates a lens array 16 as completely and uniformly as possible.
  • the lens array 16 to be selected depends on the geometry and the number and distribution of the channels according to the detector array used, for , B.
  • the corresponding multianode photomultiplter tubes from Hamamatsu for example the type H7546 with 8 x 8 individual receivers or H7260 a linearly arranged detector array with 1 x 32 individual receivers.
  • the lens array 16 In the first case a lens array (square arrangement) with 8 x 8 microlenses is required, in the second case a linear array (row) with 32 microlenses in a row.
  • the individual lenses of the lens array 16 have a sufficiently uniform focal length, which e.g. in the case of production using lithographic processes.
  • the expansion optics 2.2 for the laser beam are dimensioned accordingly for the illumination of the respective lens array 16.
  • the homogeneity of the illumination must be taken into account here.
  • corresponding holographic optical elements (HOE) can also be used to improve the illumination.
  • the expanded and collimated beam is broken down into several partial beams by the lens array 16.
  • Fan-shaped collimated bundles of rays emanate from the aperture image, one bundle for each spot.
  • the size of the scanners are dimensioned in such a way that they detect all beams even when fully deflected.
  • a scanning optical system detects the beam bundle and generates a spot distribution in an image plane 7, ie an arrangement of several individual spots, which moves with the scanner movement.
  • a fixed or adjustable diaphragm arrangement 7 is attached in the image plane 7, which marks exactly the area to be scanned so that spots, which are due to the measuring regime, lie outside the desired image area, do not reach the object field and there fluorescence bleaching, saturation or other irreversible Can cause sample changes.
  • the spot distribution is imaged into the object plane 11 in a reduced manner via a tube lens 8 and an objective 10.
  • the fluorescent structure or sample located in the object plane is excited by the migrating spot distribution to emit generally longer-wave fluorescent radiation. This takes the same way back to the main color splitter due to the optical arrangement as the excitation radiation.
  • the beam passage is scanned, ie canceled, by the passage twice over the scanner, so that a stationary beam is created in the section between scanner 4 and detector, which is now designed as a detector array 15.2.
  • the dichroic beam splitter 3 separates the detection beam path from the excitation beam path, an emission filter 12 blocking off residual residues of the excitation light.
  • a lens system 18 and 13 produces a focus in a further image plane located immediately in front of the detector array 15.2.
  • a confocal pinhole array 14.2 lies in this image plane. It is adjusted to the position of the spot distribution generated by the lens array 16 and acts analogously to the pinhole diaphragm 14 and separates light from different depth planes of the sample attached to the object plane 11.
  • the individual channels of the detector array 15.2 simultaneously assign each spot, coupled with the scanner movement, time signals which can be assembled into an image in an electronic evaluation.
  • FIG. 3 shows the spot distribution in a linear (row) arrangement of lens array 16, detector array 15.2 and pinhole array 14.2.
  • the scanning process is shown over an area 34 to be scanned.
  • the starting point for the scanning process is, for example, a position of an inclined spot row to the right of an area 34.
  • the first scanner moves the spot row in a direction 32 and shifts the spots 30 over a strip of the object field.
  • the second scanner then takes action and shifts all spots 30 in the direction of 33.
  • the first scanner then moves back in the direction of 32 and a second adjacent strip is imaged. This continues to scan the entire area.
  • Each spot 30 runs on a track 31 and all tracks 31 cover a strip together.
  • the scan length in direction 32 is determined by the length of area 34, increased by the length of the spot distribution along direction 32.
  • the row of spots is shown much longer than the corresponding dimensions of area 34.
  • the length of the spot row if the spot distance is 10 times the diameter, is 100 ⁇ m.
  • the spots 30 lie on an inclined straight line 34 with respect to the direction 32 or the tracks 31.
  • the spot radius 35 is dimensioned to match the resolution of the objective 10. At a given wavelength and diffraction-limited optical design, this is only determined by the reciprocal numerical aperture.
  • the spots 30 have at least a distance 36 in the projection perpendicular to the scanning direction 32 or path 31, the size of a spot radius 35.
  • the distance 36 is determined by the crosstalk between neighboring spots 30 and is calculated according to the image function (point spread function PSF).
  • the lens array 16 is set up at this angle at an angle to the direction 32 or path 31.
  • the spot array 30.5 is not detailed in the illustration.
  • the inclination specified between the individual spots, which is now expressed as an array inclination, is also set here, and the inclined image is scanned over the sample area 34.
  • FIG. 6 shows an arrangement with an x / y table scanner.
  • the optical structure is analog here. Light microscope.
  • the image of the spot distribution arises in the image plane in front of the receiver 15.2, in which the confocal pinhole array 14.2 is arranged.
  • the sample is moved with the x / y scanning table in the directions indicated, analogously to 32 and 33 or 32.5 and 33.5 in FIGS. 3 and 5.
  • such an arrangement is advantageous in order to quickly also detect larger sample areas 34.

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Mechanical Optical Scanning Systems (AREA)

Abstract

L'invention concerne un microscope confocal à balayage laser doté d'une trajectoire de faisceau d'excitation, qui focalise le faisceau d'excitation en plusieurs points (30) situés sur un même plan objet (11), d'une trajectoire de faisceau de détection, qui reproduit les points (30) de manière confocale au moyen d'écrans à microperforations (14.2) sur un détecteur (15.2) à plusieurs canaux, ainsi que d'un dispositif de balayage (4, 5, 37), qui effectue un mouvement relatif en deux dimensions entre un objet situé dans le plan objet (11) et les points (30). L'invention est caractérisée en ce que le dispositif de balayage (4, 5, 37), lors de son mouvement relatif, déplace les points (30) dans une première direction (32) et balaie ainsi avec les points (30) une traînée de l'objet (34), puis il déplace les points (30) dans une deuxième direction (33), pour balayer ensuite une traînée voisine par déplacement dans la première direction (32).
EP04765251A 2003-09-23 2004-09-15 Microscope confocal a balayage laser Withdrawn EP1664887A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10344060A DE10344060A1 (de) 2003-09-23 2003-09-23 Konfokales Laser-Scanning-Mikroskop
PCT/EP2004/010344 WO2005033767A1 (fr) 2003-09-23 2004-09-15 Microscope confocal a balayage laser

Publications (1)

Publication Number Publication Date
EP1664887A1 true EP1664887A1 (fr) 2006-06-07

Family

ID=34398873

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04765251A Withdrawn EP1664887A1 (fr) 2003-09-23 2004-09-15 Microscope confocal a balayage laser

Country Status (5)

Country Link
US (1) US20070041090A1 (fr)
EP (1) EP1664887A1 (fr)
JP (1) JP2007506146A (fr)
DE (1) DE10344060A1 (fr)
WO (1) WO2005033767A1 (fr)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1943376A4 (fr) 2005-11-04 2009-10-28 Ceracomp Co Ltd Monocristal piezo-electrique et son procede de production, et elements piezo-electriques et dielectriques associes
CN101779155A (zh) * 2007-08-16 2010-07-14 皇家飞利浦电子股份有限公司 对样本成像的方法
JP2009181088A (ja) * 2008-02-01 2009-08-13 Nikon Corp 共焦点ユニット、共焦点顕微鏡、および共焦点絞り
JP2011515710A (ja) * 2008-03-20 2011-05-19 コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ 光学式走査装置の放射線スポットの２次元アレイ
BRPI0913786A2 (pt) * 2008-09-25 2015-10-20 Koninkl Philips Electronics Nv sistema de detecção e método de detecção
US8773760B2 (en) 2009-04-27 2014-07-08 The Arizona Board Of Regents On Behalf Of The University Of Arizona Multi-point scan architecture
DE102011104379B4 (de) 2011-06-18 2021-11-25 Carl Zeiss Microscopy Gmbh Konfokales Rastermikroskop und Verwendung, Steuerverfahren sowie programmierbare Steuereinheit für ein solches Mikroskop
DE102013021482A1 (de) 2013-12-17 2015-06-18 Carl Zeiss Microscopy Gmbh Verfahren zur Scanning-Mikroskopie und Scanning-Mikroskop
DE102014107606A1 (de) 2014-05-28 2015-12-03 Carl Zeiss Ag Funktionsintegriertes Laser-Scanning-Mikroskop
CN105954194B (zh) * 2016-04-28 2019-08-13 大连理工大学 一种基于光锥的便携式光流控显微成像装置及系统
CN106802233B (zh) * 2017-04-07 2023-04-25 上海汇珏网络通信设备股份有限公司 一种微透镜阵列测试装置及方法
DE102017125688A1 (de) * 2017-11-03 2019-05-09 Leica Microsystems Cms Gmbh Verfahren und Vorrichtung zum Abrastern einer Probe
US10989903B2 (en) * 2018-02-26 2021-04-27 Washington State University Modular scanning confocal optical profile microscopy with digital imaging processing

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9218482D0 (en) * 1992-09-01 1992-10-14 Dixon Arthur E Apparatus and method for scanning laser imaging of macroscopic samples
DE69530072T2 (de) * 1994-12-08 2004-03-04 Molecular Dynamics, Sunnyvale System zur fluoreszenzabbildung unter verwendung eines objektivs mit makroabtastung
EP0991959B1 (fr) * 1996-02-28 2004-06-23 Kenneth C. Johnson Scanner a microlentilles pour la microlithographie et la microscopie confocale a champ large
US6038067A (en) * 1996-05-23 2000-03-14 The Regents Of The University Of California Scanning computed confocal imager
DE19653413C2 (de) * 1996-12-22 2002-02-07 Stefan Hell Rastermikroskop, bei dem eine Probe in mehreren Probenpunkten gleichzeitig optisch angeregt wird
JP3816632B2 (ja) * 1997-05-14 2006-08-30 オリンパス株式会社 走査型顕微鏡
US6856457B2 (en) * 2001-03-27 2005-02-15 Prairie Technologies, Inc. Single and multi-aperture, translationally-coupled confocal microscope
WO2003025838A1 (fr) * 2001-09-14 2003-03-27 The Arizona Board Of Regents On Behalf Of The University Of Arizona Systeme d'ecran entre objectifs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005033767A1 *

Also Published As

Publication number Publication date
JP2007506146A (ja) 2007-03-15
US20070041090A1 (en) 2007-02-22
WO2005033767A1 (fr) 2005-04-14
DE10344060A1 (de) 2005-05-04

Similar Documents

Publication Publication Date Title
EP0961945B1 (fr) Dispositif de balayage lumineux
DE102010060121B4 (de) SPIM-Mikroskop mit sequenziellem Lightsheet
DE10004191B4 (de) Fluoreszenz-Scanmikroskop
EP3033645B1 (fr) Microscopie à balayage haute résolution
EP0898783B1 (fr) Microscope a balayage dans lequel un echantillon est excite optiquement simultanement en plusieurs points
DE102013001238B4 (de) Lichtmikroskop und Mikroskopieverfahren
EP3033646B1 (fr) Microscopie à balayage haute résolution
DE102012204128B4 (de) Hochauflösende Scanning-Mikroskopie
EP2904442B1 (fr) Microscope à foyer commun avec balayage d'échantillon librement réglable
DE102011109653B4 (de) Laser-Scanning-Mikroskop mit einem Beleuchtungsarray
EP1178345A1 (fr) Dispositif de spectrométrie dans une microscope confocal
EP1664887A1 (fr) Microscope confocal a balayage laser
DE102004016253B4 (de) Rastermikroskop und Verfahren zur rastermikroskopischen Untersuchung einer Probe
DE102020209889A1 (de) Mikroskop und Verfahren zur mikroskopischen Bildaufnahme mit variabler Beleuchtung
DE102007038579A1 (de) Mikroskop mit Innenfokussierung
WO2008037346A1 (fr) Microscope à balayage laser muni d'un élément de manipulation de pupille
DE102020213714A1 (de) Mikroskop und Verfahren zur Lichtfeldmikroskopie mit Lichtblattanregung sowie zur konfokalen Mikroskopie
EP2784564A1 (fr) Microscope lumineux et procédé d'analyse d'un échantillon microscopique
WO2017174792A1 (fr) Procédé et dispositif d'analyse d'un échantillon
DE102006011277A1 (de) Laser-Scanning-Mikroskop und Laser-Scanning-Mikroskopierverfahren
WO2013124107A1 (fr) Microscope à balayage optique avec deux unités de scanner
DE10206004A1 (de) Vorrichtung zur konfokalen optischen Mikroanalyse
DE102017105719A1 (de) Multi-Pinhole-Lichtrastermikroskop und -mikroskopieverfahren
DE102005007756B4 (de) Scanmikroskop
DE102013021222B4 (de) Mikroskop und Mikroskopieverfahren

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20060307

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20060630

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20060630

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20070922