EP1678546A1 - Laser scanning microscope comprising a non-descanned detection and/or observation beam path - Google Patents

Laser scanning microscope comprising a non-descanned detection and/or observation beam path

Info

Publication number
EP1678546A1
EP1678546A1 EP04790657A EP04790657A EP1678546A1 EP 1678546 A1 EP1678546 A1 EP 1678546A1 EP 04790657 A EP04790657 A EP 04790657A EP 04790657 A EP04790657 A EP 04790657A EP 1678546 A1 EP1678546 A1 EP 1678546A1
Authority
EP
European Patent Office
Prior art keywords
laser scanning
scanning microscope
detection
beam path
microscope according
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
EP04790657A
Other languages
German (de)
French (fr)
Inventor
Ralph Lange
Ralf Wolleschensky
Michael Goelles
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 EP1678546A1 publication Critical patent/EP1678546A1/en
Ceased legal-status Critical Current

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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

Definitions

  • the non-descanned detection method is often used to observe the fluorescence radiation. It is particularly important when samples with a high scatter are to be examined or large penetration depths are to be achieved and only closely adjacent mirrors with limited light conductance values are available for detection in the LSM scan head.
  • the excitation to be modulated is then detected, not descanned, externally in the vicinity of a pupil.
  • the excitation in the UV / VIS range is usually generated by a two-photon process with pulsed IR radiation [US 5034613].
  • the entire emitted fluorescence radiation is then assigned to the excitation in the confocal volume of the focus and can be detected in the incident or transmitted light channel after possibly multiple scattering in the sample. This requires a high light guide value in the detection channel.
  • a basic requirement of optimized structures is therefore the effective and near-sample detection of fluorescence radiation in order to maximize the effective field of view as a virtual source of scattered radiation.
  • accessibility in these rooms is often limited by the interfaces of the microscope stand itself and by the aids used, such as pipettes and electrodes.
  • An essential boundary condition is the detector itself, which has not only spectral, but also location and angle-dependent sensitivities.
  • the remaining microscope optics to be used are designed for the regular beam path and have limited free diameters.
  • excitation and emission should be separated in the incident light beam path.
  • Push &Click splitters are used as standard in the reflector level of the reflected light channel reflection in order to maintain the greatest possible flexibility.
  • the limitations of the following regularly designed optics have so far largely been accepted.
  • the location and size of such an interface determine essential boundary conditions for the scattered light transmission.
  • the aim of the claimed arrangement is to modify the beam path in relation to the regular beam path immediately after the separation of excitation and detection in such a way that the largest possible (bleed-free) field of view can be imaged on the detector, taking into account the following optics.
  • This is solved by the features of claim 1.
  • Preferred developments are the subject of the dependent claims.
  • An advantageous embodiment consists in the positioning of an additional optical system, preferably a converging lens in the reflector bracket of the reflected-light channel reflection. Their dimensioning is determined by the compromise between maximum main beam kinking and not too large resulting bundle diameters on the detector.
  • the optimization can be carried out so that the NDD module can be used on a TV port.
  • Figure 1 illustrates the effect of the arrangement using the beam path.
  • Part of the beam path of a laser scanning microscope see, for example, DE 197 02 753 A1
  • a scanning objective SO for transmitting the illuminating light beam
  • an illuminating tube lens 5 and a beam splitter 1 (main color splitter for separating Excitation and detection light) in the direction of the objective (here is only the objective pupil)
  • a non-descanned detection beam path extends over 1 and a mirror 2 as well as a detection tube lens 4 in the direction of the detection, wherein a further beam splitter ST 3 can be provided to mask out an observation beam path.
  • the regularly used diameter on the reflector (2) is reduced, thus allowing greater permeability to scattered light. It can be seen in particular that the regularly used diameters in front of the illumination tube lens (4) are significantly larger than those in front of the detection tube lens (5), which are each at the same distance from the
  • the aperture in the edge area can be increased by about 15%, which is good
  • the insertion of the converging lens directly on the reflector is advantageously implemented directly on the reflector housing, for example with an insertion point, and also existing ones
  • Insertion points for filters can be used.
  • the lens can advantageously also be exchangeable or can be pushed in and pushed out.
  • a second lens on the mirror 2 or integrated in the mirror housing can also be provided, individually or in combination with the lens on the beam splitter.
  • a deflecting mirror can also be formed in a deflecting element as a convex or concave mirror.

Abstract

The invention relates to a laser scanning microscope comprising a non-descanned detection and/or observation beam path. Said microscope is provided with a beam splitter for splitting the illumination and detection beam path, and at least one optical element arranged in the direction of the detection and used to regularly transmit the detected light. An additional optical element is provided between the beam splitter and the optical element, for reducing the diameter of the imaging bundle of rays.

Description

Laser-Scannin -Mikroskop mit einem non- escannten Detektions- und / oder BeobachtunqsstrahlenganqLaser scanning microscope with a non-scanned detection and / or observation beam
In der Laser-Scanning-Mikroskopie wird zur Beobachtung der Fluoreszenzstrahlung häufig das nicht-descannte Detektionsverfahren (NDD) benutzt. Es ist insbesondere dann von Bedeutung, wenn stark streuende Proben untersucht oder große Eindringtiefen erreicht werden sollen und zur Detektion im LSM-Scan-Kopf nur eng benachbarte Spiegel mit begrenztem Lichtleitwert zur Verfügung stehen. Die Detektion der zu modulierenden Anregung erfolgt dann nicht-descannt extern in der Nähe einer Pupille. Meist wird die Anregung im UV / VIS-Bereich über einen Zwei-Photonen-Prozeß mit gepulster IR-Strahlung generiert [US 5034613]. Die gesamte emittierte Fluoreszenzstrahlung wird dann der Anregung im konfokalen Volumen des Fokus zugeordnet und kann nach eventuell mehrmaliger Streuung in der Probe im Auflicht- oder Durchlichtkanal detektiert werden. Dazu wird ein hoher Lichtleitwert im Detektionskanal benötigt.In laser scanning microscopy, the non-descanned detection method (NDD) is often used to observe the fluorescence radiation. It is particularly important when samples with a high scatter are to be examined or large penetration depths are to be achieved and only closely adjacent mirrors with limited light conductance values are available for detection in the LSM scan head. The excitation to be modulated is then detected, not descanned, externally in the vicinity of a pupil. The excitation in the UV / VIS range is usually generated by a two-photon process with pulsed IR radiation [US 5034613]. The entire emitted fluorescence radiation is then assigned to the excitation in the confocal volume of the focus and can be detected in the incident or transmitted light channel after possibly multiple scattering in the sample. This requires a high light guide value in the detection channel.
Ein Grundanspruch optimierter Aufbauten besteht daher in der effektiven und probennahen Detektion der Fluoreszenzstrahlung, um das effektive Gesichtsfeld als virtuelle Quelle gestreuter Strahlung zu maximieren. Die Zugänglichkeit in diesen Räumen ist jedoch häufig durch die Schnittstellen des Mikroskopstatives selbst sowie durch die verwendeten Hilfsmittel, wie Pipetten und Elektroden, begrenzt. Eine wesentliche Randbedingung bildet der Detektor selbst, der nicht nur spektrale, sondern auch orts- und winkelabhängige Empfindlichkeiten aufweist.A basic requirement of optimized structures is therefore the effective and near-sample detection of fluorescence radiation in order to maximize the effective field of view as a virtual source of scattered radiation. However, accessibility in these rooms is often limited by the interfaces of the microscope stand itself and by the aids used, such as pipettes and electrodes. An essential boundary condition is the detector itself, which has not only spectral, but also location and angle-dependent sensitivities.
Ferner muß davon ausgegangen werden, daß die zu benutzende übrige Mikroskopoptik für den regulären Strahlengang ausgelegt ist, und über begrenzte freie Durchmesser verfügt. Um dennoch Streulichtbeschnitt an Berandungen der nachfolgenden Optiken, wie Tubuslinse oder Beleuchtungsoptik im Auflichtkanal, zu vermeiden, sollten im Auflichtstrahlengang Anregung und Emission objektivnah getrennt werden. In der Regel werden dafür standardmäßig „Push-&-Click"-Teiler in der Reflektorebene der Auflichtkanaleinspiegelung eingesetzt, um größtmögliche Flexibilität zu wahren. Die Begrenzungen der nachfolgenden regulär ausgelegten Optik werden bisher weitgehend hingenommen. Insbesondere ist es vorteilhaft, definierte Schnittstellen, wie den TV-Port, zur Ankopplung für das Detektionsmodul zu nutzen. Mit Lage und Größe einer solchen Schnittstelle werden jedoch wesentliche Randbedingungen für die Streulichttransmission festgelegt.It must also be assumed that the remaining microscope optics to be used are designed for the regular beam path and have limited free diameters. In order to avoid stray light trimming at the edges of the subsequent optics, such as tube lenses or illumination optics in the incident light channel, excitation and emission should be separated in the incident light beam path. Usually "Push &Click" splitters are used as standard in the reflector level of the reflected light channel reflection in order to maintain the greatest possible flexibility. The limitations of the following regularly designed optics have so far largely been accepted. In particular, it is advantageous to use defined interfaces such as the TV port to be used for coupling for the detection module However, the location and size of such an interface determine essential boundary conditions for the scattered light transmission.
Das Ziel der beanspruchten Anordnung besteht darin, unmittelbar nach der Trennung von Anregung und Detektion den Strahlengang derart gegenüber dem regulären Strahlengang zu modifizieren, daß ein größtmögliches (beschnittfreies) Sehfeld unter Berücksichtigung der nachfolgenden Optik auf den Detektor abgebildet werden kann. Dies wird durch die Merkmale des Anspruchs 1 gelöst. Bevorzugte Weiterbildungen sind Gegenstand der Unteransprüche. Eine vorteilhafte Ausführung besteht in der Positionierung eines optischen Zusatzsystems, vorzugsweise einer Sammellinse im Reflektorböckchen der Auflichtkanaleinspiegelung. Ihre Dimensionierung wird durch den Kompromiß zwischen maximaler Hauptstrahlabknickung und nicht zu großen resultierenden Bündeldurchmessern auf dem Detektor bestimmt. Insbesondere kann die Optimierung so erfolgen, daß das NDD-Modul an einem TV-Port genutzt werden kann.The aim of the claimed arrangement is to modify the beam path in relation to the regular beam path immediately after the separation of excitation and detection in such a way that the largest possible (bleed-free) field of view can be imaged on the detector, taking into account the following optics. This is solved by the features of claim 1. Preferred developments are the subject of the dependent claims. An advantageous embodiment consists in the positioning of an additional optical system, preferably a converging lens in the reflector bracket of the reflected-light channel reflection. Their dimensioning is determined by the compromise between maximum main beam kinking and not too large resulting bundle diameters on the detector. In particular, the optimization can be carried out so that the NDD module can be used on a TV port.
Abbildung 1 verdeutlicht anhand des Strahlengangs die Wirkung der Anordnung. Dargestellt ist ein Teil des Strahlenganges eines Laser- Scanning Mikroskopes (siehe z. B. DE 197 02 753 A1 ) dargestellt, von einer Scannerpupille SP über ein Scanobjektiv SO zur Übertragung des Beleuchtungslichtstrahls, eine Beleuchtungstubuslinse 5 und einen Strahlteiler 1 (Hauptfarbteiler zur Trennung von Anregungs-und Detektionslicht) in Richtung des Objektives (hier ist nur die ObjektivpupilleFigure 1 illustrates the effect of the arrangement using the beam path. Part of the beam path of a laser scanning microscope (see, for example, DE 197 02 753 A1) is shown, from a scanner pupil SP via a scanning objective SO for transmitting the illuminating light beam, an illuminating tube lens 5 and a beam splitter 1 (main color splitter for separating Excitation and detection light) in the direction of the objective (here is only the objective pupil)
3 dargestellt).3 shown).
Ein non descannter Detektionsstrahlengang erstreckt sich über 1 und einen Spiegel 2 sowie eine Detektionstubuslinse 4 in Richtung der Detektion, wobei ein weiterer Strahlteiler ST 3 zur Ausblendung eines Beobachtungsstrahlenganges vorgesehen sein kann. Durch Einbringen einer Sammellinse (6) unmittelbar nach der Einspiegelung anA non-descanned detection beam path extends over 1 and a mirror 2 as well as a detection tube lens 4 in the direction of the detection, wherein a further beam splitter ST 3 can be provided to mask out an observation beam path. By inserting a converging lens (6) immediately after the reflection
Reflektor (1 ) wird der regulär genutzte Durchmesser am Reflektor (2) verringert und damit eine größere Durchlässigkeit für Streulicht ermöglicht. Man erkennt insbesondere, daß die regulär genutzten Durchmesser vor der Beleuchtungstubuslinse (4) deutlich größer sind als die vor der Detektionstubuslinse (5), welche sich jeweils in gleichem Abstand zumReflector (1), the regularly used diameter on the reflector (2) is reduced, thus allowing greater permeability to scattered light. It can be seen in particular that the regularly used diameters in front of the illumination tube lens (4) are significantly larger than those in front of the detection tube lens (5), which are each at the same distance from the
Objektiv befinden und damit den Gewinn direkt veranschaulichen. Im ausgeführtenAre objective and thus illustrate the profit directly. In the executed
Beispiel läßt sich die Apertur im Randbereich um etwa 15% erhöhen, was einemFor example, the aperture in the edge area can be increased by about 15%, which is good
Helligkeitsgewinn von etwa 30% entspricht.Brightness gain of about 30%.
Realisiert wird das Einbringen der Sammellinse unmittelbar am Reflektor vorteilhaft direkt am Reflektorgehäuse, beispielsweise mit einer Einschubstelle, wobei auch vorhandeneThe insertion of the converging lens directly on the reflector is advantageously implemented directly on the reflector housing, for example with an insertion point, and also existing ones
Einschubstellen für Filter genutzt werden können. Die Linse kann vorteilhaft auch auswechselbar bzw. ein- und ausschiebbar sein.Insertion points for filters can be used. The lens can advantageously also be exchangeable or can be pushed in and pushed out.
Sie kann auch in Detektionsrichtung vor dem Reflektor 2 an seinem Reflektorgehäuse angeordnet sein. Eine zweite Linse am Spiegel 2 oder in das Spiegelgehäuse integriert kann ebenfalls vorgesehen sein, einzeln oder in Kombination mit der Linse am Strahlteiler. Es kann auch ein Umlenkspiegel in einem Umlenkelement als konvexer oder konkaver Spiegel ausgebildet sein. It can also be arranged in the detection direction in front of the reflector 2 on its reflector housing. A second lens on the mirror 2 or integrated in the mirror housing can also be provided, individually or in combination with the lens on the beam splitter. A deflecting mirror can also be formed in a deflecting element as a convex or concave mirror.

Claims

Patentansprüche claims
1.1.
Laser-Scanning-Mikroskop mit einem non-descannten Detektions und / oder Beobachtungsstrahlengang, wobei ein Strahlteiler zur Trennung von Beleuchtungs- undLaser scanning microscope with a non-descanned detection and / or observation beam path, with a beam splitter for the separation of illumination and
Detektionstrahlengang vorgesehen ist und in Richtung der Detektion mindestens eineDetection beam path is provided and at least one in the direction of detection
Optik zur regulären Übertragung des detektierten Lichtes vorgesehen ist, wobei zwischen dem Strahlteiler und der Optik eine Zusatzoptik zur Verringerung desOptics for regular transmission of the detected light is provided, with additional optics between the beam splitter and the optics to reduce the
Durchmessers des abbildenden Strahlenbündels vorgesehen ist. Diameter of the imaging beam is provided.
2.Second
Laser-Scanning-Mikroskop nach Anspruch 1 , wobei dieA laser scanning microscope according to claim 1, wherein the
Zusatzoptik eine Sammellinse ist.Additional optics is a converging lens.
3.Third
Laser-Scanning-Mikroskop nach Anspruch 1 , wobei die Zusatzoptik als diffraktiv optisches Element (DOE) ausgebildet ist.Laser scanning microscope according to claim 1, wherein the additional optics is designed as a diffractive optical element (DOE).
4.4th
Laser-Scanning-Mikroskop nach Anspruch 1 , 2 oder 3, wobei dieLaser scanning microscope according to claim 1, 2 or 3, wherein the
Zusatzoptik unmittelbar am Strahlteilergehäuse in Richtung der Detektion angebracht ist. Additional optics is attached directly to the beam splitter housing in the direction of the detection.
5.5th
Laser-Scanning-Mikroskop nach einem der Ansprüche 1-4, wobeiLaser scanning microscope according to any one of claims 1-4, wherein
Zusatzoptik ins Strahlteilergehäuse integriert ist.Additional optics is integrated in the beam splitter housing.
6.6th
Laser-Scanning-Mikroskop nach einem der Ansprüche 1-5, wobei die Zusatzoptik auswechselbar oder einschiebbar ist. Laser scanning microscope according to any one of claims 1-5, wherein the additional optics are exchangeable or insertable.
7.7th
Laser-Scanning-Mikroskop nach einem der Ansprüche 1-6, wobei eine zweite Linse an einem weiteren Umlenkelement oder in dieses integriert vorgesehen ist, einzeln oder in Kombination mit dem Zusatzelement am Strahlteiler.Laser scanning microscope according to one of claims 1-6, wherein a second lens is provided on a further deflecting element or integrated therein, individually or in combination with the additional element on the beam splitter.
8.8th.
Laser-Scanning-Mikroskop nach einem der Ansprüche 1-7, mit einer Ausführung eines Umlenkspiegels in einem Umlenkelement als konvexer oder konkaver Spiegel. Laser scanning microscope according to one of claims 1-7, with an embodiment of a deflecting mirror in a deflecting element as a convex or concave mirror.
EP04790657A 2003-10-30 2004-10-20 Laser scanning microscope comprising a non-descanned detection and/or observation beam path Ceased EP1678546A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10351414A DE10351414A1 (en) 2003-10-30 2003-10-30 Laser scanning microscope with a non-descanned detection and / or observation beam path
PCT/EP2004/011846 WO2005043212A1 (en) 2003-10-30 2004-10-20 Laser scanning microscope comprising a non-descanned detection and/or observation beam path

Publications (1)

Publication Number Publication Date
EP1678546A1 true EP1678546A1 (en) 2006-07-12

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Application Number Title Priority Date Filing Date
EP04790657A Ceased EP1678546A1 (en) 2003-10-30 2004-10-20 Laser scanning microscope comprising a non-descanned detection and/or observation beam path

Country Status (5)

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US (1) US20070121473A1 (en)
EP (1) EP1678546A1 (en)
JP (1) JP2007510176A (en)
DE (1) DE10351414A1 (en)
WO (1) WO2005043212A1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6249783B2 (en) 2014-01-14 2017-12-20 オリンパス株式会社 microscope
EP3538941A4 (en) 2016-11-10 2020-06-17 The Trustees of Columbia University in the City of New York Rapid high-resolution imaging methods for large samples

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US5034613A (en) * 1989-11-14 1991-07-23 Cornell Research Foundation, Inc. Two-photon laser microscopy
US5260828A (en) * 1992-03-27 1993-11-09 Polaroid Corporation Methods and means for reducing temperature-induced variations in lenses and lens devices
US5296703A (en) * 1992-04-01 1994-03-22 The Regents Of The University Of California Scanning confocal microscope using fluorescence detection
CA2231222C (en) * 1995-09-19 2001-12-11 Cornell Research Foundation, Inc. Multi-photon laser microscopy
US6631226B1 (en) * 1997-01-27 2003-10-07 Carl Zeiss Jena Gmbh Laser scanning microscope
US6167173A (en) * 1997-01-27 2000-12-26 Carl Zeiss Jena Gmbh Laser scanning microscope
DE19733194B4 (en) * 1997-08-01 2005-06-16 Carl Zeiss Jena Gmbh Laser Scanning Microscope
US6466040B1 (en) * 1997-08-01 2002-10-15 Carl Zeiss Jena Gmbh Three dimensional optical beam induced current (3-D-OBIC)
DE10024135B4 (en) * 2000-01-28 2004-07-08 Leica Microsystems Heidelberg Gmbh microscope
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Also Published As

Publication number Publication date
DE10351414A1 (en) 2005-06-23
WO2005043212A1 (en) 2005-05-12
US20070121473A1 (en) 2007-05-31
JP2007510176A (en) 2007-04-19

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