EP1664886A1 - Microscope a fluorescence multiphotonique a detecteur plan - Google Patents

Microscope a fluorescence multiphotonique a detecteur plan

Info

Publication number
EP1664886A1
EP1664886A1 EP04765183A EP04765183A EP1664886A1 EP 1664886 A1 EP1664886 A1 EP 1664886A1 EP 04765183 A EP04765183 A EP 04765183A EP 04765183 A EP04765183 A EP 04765183A EP 1664886 A1 EP1664886 A1 EP 1664886A1
Authority
EP
European Patent Office
Prior art keywords
radiation
sample
excitation
detector
lurninescence
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Withdrawn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
EP04765183A
Other languages
German (de)
English (en)
Inventor
Dan Davidovici
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 EP1664886A1 publication Critical patent/EP1664886A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the invention relates to a multi-photon luminescence microscope with an excitation beam path that has an objective that bundles excitation radiation in a focal point in the sample, a scanning device that adjusts the focal point at least one-dimensionally, and a detector device that in the sample luminescent radiation stimulated by multi-photon excitation.
  • the invention further relates to a method for multi-photon luminescence microscopy, in which excitation radiation is concentrated in a focus point lying in a sample, thereby stimulating luminescence radiation in the sample by multi-phone excitation, the focus point is adjusted for scanning the sample and the Luminescence radiation is detected.
  • a bundled excitation radiation usually laser radiation, which is matched to maximum luminescence, is usually used for this purpose.
  • the excitation takes place in the focus area, luminescence also being stimulated in the incident or emerging light cone of the focused beam.
  • the luminescence radiation is recorded by confocal detection only from the area of the focus of the excitation radiation. An image is created by scanning a sample.
  • the excitation radiation is selected spectrally in such a way that at least two photons are required to effect an excitation. Since the probability of excitation is thus greatly reduced, effective excitation can only take place at a very high flux density, which is only given exactly in the focus of the bundled excitation radiation. Therefore, emission of luminescence or fluorescence radiation is only stimulated at the focal point.
  • the confocal detection required in conventional luminescence microscopy can be dispensed with, since it is not necessary to emit lurninescence radiation that was emitted outside the focus of the excitation radiation. Multi-photoneh luminescence microscopy works with it without confocal stray light suppression during detection.
  • the detectors used are called direct detectors.
  • the document http: Wmicroscopy.bio- rad.com/faqs/multophotone/faqs2.htm available on the Internet proposes as a direct detector a photomultiplier unit that is also customary for confocal microscopy, which is coupled into the excitation beam path via a chromatic beam splitter and absorbs fluorescent radiation, which runs in the opposite direction to the radiation of the excitation radiation.
  • a corresponding converging lens is connected upstream of the photomultiplier tube used in the unit, which together with an objective lens present in the excitation beam path completely images the sample field onto the relatively small window of the highly sensitive photomultiplier tube.
  • the invention has for its object to develop a multi-photon luminescence microscope of the type mentioned and a corresponding method for multi-photon luminescence microscopy so that radiation detection is possible with reduced effort.
  • This object is achieved with a microscope of the type mentioned at the outset, in which the detector device has an area detector which is located on the side of the sample opposite the objective.
  • the object is further achieved by a method of the type mentioned at the outset, in which the luminescent radiation is areally detected on the side opposite the radiation of the excitation radiation.
  • a so-called “direct” detector is used, which is now designed as an area detector that is located on the side of the sample opposite the objective.
  • Area detector is understood to mean any detector whose detector area is larger than the light path to the sample in The arrangement of such an area detector in the transmit mode makes it possible, on the one hand, to dispense with chromatic beam splitters which reduce the intensity, and on the other hand, the area detector can be arranged at an extremely short distance from the sample, so that it has a large clearing angle with respect to the Covers the sample of lurninescence radiation
  • Area detector used in transmisive operation receives much more luminescence radiation intensity and thus achieves a better signal / noise ratio; this is particularly so because there are no losses through intermediary optics, such as imaging optics or dichroic beam splitters, which are also used for irradiation of the excitation radiation. The detection of the lurninescence radiation no longer has to take place through the objective of the excitation beam path.
  • the area detector In order to cover the largest possible solid angle, it is advantageous for the area detector to be at a distance from the focal point that is very much smaller than the extent of the area detector, for example only one tenth of it.
  • the optical element can be designed as a grating, preferably as a holographic grating.
  • such an optical element can also be attached directly to the underside of a sample carrier that is used in the luminescence microscope.
  • luminescence microscopy it is possible to identify biological samples on the basis of their own luminescence spectrum. This procedure is also possible in the luminescence microscope according to the invention if a spatially resolving surface detector is used and a spectral analyzer is connected between the surface detector and the sample, which spectrally decomposes the radiation emanating from the sample.
  • the grating already mentioned is arranged between the sample and the area detector for spectral decomposition.
  • the grating or the area detector is coupled with a suitable mechanism which carries out a one- or two-dimensional transverse displacement (in relation to the areal formation of the specimen to be examined).
  • Fig. 1 is a schematic representation of a section of a microscope for multi-photon fluorescence microscopy
  • FIG. 2 shows a schematic representation of the laser beam which excites a multi-photon fluorescence.
  • a microscope M is shown schematically, which allows multi-photon fluorescence or luminescence microscopy. 1 shows only the area of the microscope in which the sample is located.
  • the microscope M has a beam source (not shown) which emits a laser beam 1 with a wavelength around 700 nm.
  • the laser beam 1 passes through an objective 2, which emits a focused beam 3.
  • the focus 4 lies in a sample 5, which is located under the lens 2 behind a cover glass 6 on a sample holder 7.
  • the laser beam 1 focused in this way in the sample 5, as shown in FIG. 2, causes a multi-photon excitation in the sample 5.
  • Either an inherent fluorescence of the biological material of the sample 5 or a fluorescence specifically provided in the sample 5 can be provided Fluorophores are stimulated.
  • the laser beam 1, which is focused in a beam waist T by the objective 2, which is only shown schematically in FIG. 2, only reaches a beam density in the area of the focus 4, which is sufficient to excite multi-photon fluorescence. No more photon fluorescence can be excited with sufficient probability outside the beam waist T. Therefore, fluorescence radiation only arises in the area of focus 4. No fluorescence occurs at other points in the focused beam 3.
  • a grating 8 is arranged under the sample carrier 7, which redirects radiation emanating within a beam cone K to a CCD sensor 9 in such a way that the radiation falls as perpendicularly as possible onto the sensor 9 ,
  • the optional grating is located at a very small distance d below the focus 4, so that in combination with the comparatively large extent of the in FIG. 1 only as The sectional view shown sensor 9 covers a very large solid angle based on the focus 4.
  • the unit comprising the grating 8 and the sensor 9, which embodies the area detector, collects almost all fluorescence radiation emitted in a half space. This greatly improves the signal-to-noise ratio.
  • the CCD sensor 9 which in the present example is designed as a back-illuminated CCD sensor, supplies the corresponding image information to a control device 10. This carries out the signal evaluation.
  • the reading of the sensor 9 can be limited to periods in which no excitation radiation 1 is emitted. It is also possible to hide the relatively small area of the area detector in which excitation radiation falls on the sensor 9. Either a spatially resolving detector can be used for this, which is not read out in the area concerned, or sensor 9 becomes a suitable one
  • a filter for excitation radiation is attached to the underside of the sample carrier 7 and / or to the grating 8. It is an infrared cut filter that blocks at 700 nm.
  • the control device 10 reads out the (location-detecting) sensor 9 in a suitable manner and identifies a sample 5 on the basis of its own fluorescence spectrum.
  • the spectral activity of the grating 8 also opens up an additional spectral possibility of masking the excitation radiation 1, since it differs significantly from the fluorescence radiation. As a rule, the grating 8 will generate an interference pattern on the sensor 9.
  • control device 10 effects a relative shift of the grating 8 and the sensor 9, so that the interference pattern, which indicates the spectral composition of the fluorescent radiation entering the beam cone K (optionally together with excitation radiation 1), changes.
  • the change then enables the control unit 10 to make a statement about the spectral composition of the fluorescent radiation from the focus 4 using known algorithms.
  • the distance d should of course be as small as possible.
  • the grid 8 is therefore attached directly to the underside of the sample carrier 7. Without the grating 8, the distance d (now between the focus 4 and the sensor 9) should be minimized by the sensor being as close as possible to the sample carrier 7.

Landscapes

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

Abstract

L'invention concerne un microscope à fluorescence multiphotonique (M) présentant une trajectoire de faisceau d'excitation sur laquelle un objectif (2) est placé, lequel objectif focalise le faisceau d'excitation (1) sur un point de focalisation (4) situé dans l'échantillon (5), un dispositif de balayage qui ajuste le point de focalisation (4) au moins de façon unidimensionnelle ainsi qu'un dispositif de détection absorbant le rayonnement de fluorescence induit dans l'échantillon par excitation multiphotonique. Ladite invention se caractérise en ce que le dispositif de détection comprend un détecteur plan (9) situé sur un côté de l'échantillon (5), opposé à l'objectif (2).
EP04765183A 2003-09-18 2004-09-14 Microscope a fluorescence multiphotonique a detecteur plan Withdrawn EP1664886A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10343276A DE10343276A1 (de) 2003-09-18 2003-09-18 Mehr-Photonen-Fluoreszenzmikroskopie
PCT/EP2004/010269 WO2005029148A1 (fr) 2003-09-18 2004-09-14 Microscope a fluorescence multiphotonique a detecteur plan

Publications (1)

Publication Number Publication Date
EP1664886A1 true EP1664886A1 (fr) 2006-06-07

Family

ID=34305893

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04765183A Withdrawn EP1664886A1 (fr) 2003-09-18 2004-09-14 Microscope a fluorescence multiphotonique a detecteur plan

Country Status (5)

Country Link
US (1) US20060245021A1 (fr)
EP (1) EP1664886A1 (fr)
JP (1) JP2007506123A (fr)
DE (1) DE10343276A1 (fr)
WO (1) WO2005029148A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011056658A1 (fr) * 2009-10-27 2011-05-12 Duke University Microscopie multiphotonique par l'intermédiaire d'une lentille d'objectif à interface hertzienne
CN113594054A (zh) * 2021-05-24 2021-11-02 厦门大学 一种自带位置监测的微镜系统

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19801139B4 (de) * 1998-01-14 2016-05-12 Till Photonics Gmbh Punktabtastendes Luminiszenz-Mikroskop
US6169289B1 (en) * 1998-01-27 2001-01-02 Wisconsin Alumni Research Foundation Signal enhancement for fluorescence microscopy
US6403332B1 (en) * 1999-07-30 2002-06-11 California Institute Of Technology System and method for monitoring cellular activity
DE19957418B4 (de) * 1999-11-29 2016-02-04 Leica Microsystems Cms Gmbh Verfahren zur lichtoptischen Abtastung eines Objekts und Rastermikroskop zur Anwendung des Verfahrens
JP3797874B2 (ja) * 2000-12-26 2006-07-19 オリンパス株式会社 走査型光学顕微鏡
EP1294004B1 (fr) * 2001-09-12 2004-12-01 Kabushiki Kaisha Meidensha Contact pour un interrupteur à vide et interrupteur à vide avec un tel contact
DE10151216A1 (de) * 2001-10-16 2003-04-24 Zeiss Carl Jena Gmbh Verfahren zur optischen Erfassung von charakteristischen Größen einer beleuchteten Probe

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005029148A1 *

Also Published As

Publication number Publication date
JP2007506123A (ja) 2007-03-15
DE10343276A1 (de) 2005-04-14
WO2005029148A1 (fr) 2005-03-31
US20060245021A1 (en) 2006-11-02

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