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EP0706671A1 - Procede de microscopie a balayage a luminescence et microscope a balayage a luminescence - Google Patents

Procede de microscopie a balayage a luminescence et microscope a balayage a luminescence

Info

Publication number
EP0706671A1
EP0706671A1 EP95916571A EP95916571A EP0706671A1 EP 0706671 A1 EP0706671 A1 EP 0706671A1 EP 95916571 A EP95916571 A EP 95916571A EP 95916571 A EP95916571 A EP 95916571A EP 0706671 A1 EP0706671 A1 EP 0706671A1
Authority
EP
European Patent Office
Prior art keywords
laser
luminescence
luminescent
scanning microscope
light emitted
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
EP95916571A
Other languages
German (de)
English (en)
Inventor
Pekka Hänninen
Stefan Dr. Hell
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.)
Individual
Original Assignee
Individual
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
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=6516737&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP0706671(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Individual filed Critical Individual
Publication of EP0706671A1 publication Critical patent/EP0706671A1/fr
Ceased 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes

Definitions

  • the invention relates to a method for luminescence scanning microscopy according to the preamble of the main claim and a luminescence scanning microscope for performing this method.
  • REPLACEMENT LEAF luminescent, especially fluorescent dye The dye in the object is excited by the joint action of two photons and the light emitted by the dye is registered and evaluated by means of a detector. In order to capture the entire sample, the laser beam is scanned over the object. The values of all points of the object are registered, which enables a three-dimensional representation of the evaluation results.
  • the two-photon excitation in connection with the scanning leads to a direct three-dimensional image of the object.
  • laser pulses are used, the duration of which is in the sub-pico second range. Lasers that generate such extremely short pulses are very expensive.
  • biological objects are often destroyed by the pulses of high power. Excitation of the object with laser pulses in the sub-pico-second range often leads to the luminescence signal "breaking down". This "collapse” can be clearly illustrated as a "micro-explosion” that destroys the object material.
  • the object of the invention is to develop a generic method in such a way that the disadvantages mentioned above do not occur.
  • Measuring times are usually applied, the sensitivity of the detector used being adapted to the measuring conditions. It has been found that if the
  • lasers with continuous or pulsed radiation can be used as the light source for carrying out the measuring method according to the invention. These are not expensive.
  • Conventional semiconductor or gas lasers can be used. HeNe lasers and krypton ion lasers are particularly suitable as gas lasers.
  • the laser arrangement can also be designed in the form of an array. The use of two or three lasers for irradiating the same object point has proven to be particularly advantageous.
  • a detector with a high signal-to-noise ratio is used to record and evaluate the luminescent light. It is particularly important that the detector used has a correspondingly high sensitivity and a has low self-noise.
  • the array-like arrangement of the lasers and detectors and the resulting irradiation and measurement of several raster points makes it possible to shorten the duration of the method.
  • a beam screening device is arranged upstream of the object, the beam being scanned over the object.
  • Filters are also provided in the luminescence scanning microscope according to the invention for separating the light emitted by the sample from the laser light.
  • a detector can also be used, which filters out the corresponding wavelength.
  • FIG. 1 A schematic representation of a luminescence scanning microscope for carrying out the method according to the invention is shown in FIG. 1.
  • the luminescence scanning microscope has a laser 1 for generating the laser light, a photo detector 2 for evaluating the measurement results and a lens 3 for focusing the laser beam 4 emitted by the laser light source 1 onto the object 5 to be examined.
  • the laser beam 4 is controlled with a beam raster device 6 for scanning the object 5.
  • the luminescence scanning microscope has filters 7 and 8 for separating the laser light 4 from the luminescent light.
  • the laser light source 1 is designed as a conventional laser with continuous or pulsed radiation.
  • the laser light source 1 can also be formed by an array-like arrangement of lasers.

Landscapes

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

Abstract

L'invention concerne un procédé de microscopie à balayage à luminescence avec excitation par deux photons, notamment pour l'examen d'objets biologiques (5). Une impulsion laser excite des molécules luminescentes, en particulier fluorescentes, puis la lumière luminescente émise par l'objet (5) est mesurée et évaluée. Ce procédé est caractérisé en ce que les molécules luminescentes présentes dans l'objet (5) sont excitées par des impulsions laser de durée supérieure à 10-12 seconde. Un microscope à balayage à luminescence destiné à appliquer ce procédé présente un détecteur (2), un filtre (7, 8) conçu pour séparer la lumière émise par l'échantillon de la lumière laser (4), ainsi qu'une source de lumière laser constituée par un laser (1) à rayonnement pulsé ou continu.
EP95916571A 1994-04-28 1995-04-27 Procede de microscopie a balayage a luminescence et microscope a balayage a luminescence Ceased EP0706671A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4414940 1994-04-28
DE4414940A DE4414940C2 (de) 1994-04-28 1994-04-28 Lumineszenz-Rastermikroskop mit zwei Photonen Anregung
PCT/DE1995/000566 WO1995030166A1 (fr) 1994-04-28 1995-04-27 Procede de microscopie a balayage a luminescence et microscope a balayage a luminescence

Publications (1)

Publication Number Publication Date
EP0706671A1 true EP0706671A1 (fr) 1996-04-17

Family

ID=6516737

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95916571A Ceased EP0706671A1 (fr) 1994-04-28 1995-04-27 Procede de microscopie a balayage a luminescence et microscope a balayage a luminescence

Country Status (4)

Country Link
US (1) US5777732A (fr)
EP (1) EP0706671A1 (fr)
DE (1) DE4414940C2 (fr)
WO (1) WO1995030166A1 (fr)

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EP1207387A1 (fr) * 2000-11-20 2002-05-22 Institut Curie Dispositif d'imagerie multiphotonique
WO2002043574A2 (fr) 2000-11-29 2002-06-06 Xy, Inc. Systeme de dissociation de spermatozoides congeles-decongeles porteurs d'un chromosome x de ceux porteurs d'un chromosome y
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Also Published As

Publication number Publication date
DE4414940A1 (de) 1995-11-02
DE4414940C2 (de) 1998-07-02
US5777732A (en) 1998-07-07
WO1995030166A1 (fr) 1995-11-09

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