WO2007090591A1 - Système de microscopie pour l'observation de la fluorescence - Google Patents

Système de microscopie pour l'observation de la fluorescence Download PDF

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Publication number
WO2007090591A1
WO2007090591A1 PCT/EP2007/000958 EP2007000958W WO2007090591A1 WO 2007090591 A1 WO2007090591 A1 WO 2007090591A1 EP 2007000958 W EP2007000958 W EP 2007000958W WO 2007090591 A1 WO2007090591 A1 WO 2007090591A1
Authority
WO
WIPO (PCT)
Prior art keywords
light
microscopy
microscopy system
illumination
camera
Prior art date
Application number
PCT/EP2007/000958
Other languages
German (de)
English (en)
Inventor
Christoph Hauger
Joachim Steffen
Hans-Joachim Miesner
Petra Weinschenk
Helge Jess
Original Assignee
Carl Zeiss Surgical 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 Surgical Gmbh filed Critical Carl Zeiss Surgical Gmbh
Publication of WO2007090591A1 publication Critical patent/WO2007090591A1/fr

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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
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens
    • G02B21/20—Binocular arrangements
    • G02B21/22—Stereoscopic arrangements
    • 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
    • G02B21/365—Control or image processing arrangements for digital or video microscopes

Definitions

  • the invention relates to a microscopy system for observing fluorescence of a fluorescent dye in an object area
  • the microscopy system comprises a microscopy optics with an illumination system for providing illumination light for the object area with light. which contains wavelengths in the visible spectral region and which has wavelengths for exciting the fluorescence of the fluorescent dye, and has a microscope imaging unit.
  • a radiation flux of fluorescent light is supplied from the object area
  • a display system is provided for displaying an image of the object area. which contains a fluorescence-based partial image of the object area.
  • a microscopy system of the type mentioned is known from DE 103 39 784 Al. There, a surgical microscope is described . in which fluorescence dyes accumulated in a body tissue can be observed.
  • the surgical microscope has an illumination system that provides light in the visible spectral range and generates light that can be used to excite a fluorescent dye in body tissue to fluoresce. With a stereoscopic observation beam path, the surgical microscope allows an observer to observe an object area through a microscope main objective in an eyepiece view.
  • the surgical microscope comprises a camera which is sensitive to the visible spectral range and has a camera with which fluorescent body tissue can be detected.
  • a display unit is provided in which the images captured by the cameras can be displayed individually or in superposition.
  • the object of the invention is to provide a microscopy system which allows the observation of an object area optionally with illumination light in the visible spectral range and with fluorescence, wherein for an observation of the object area with fluorescent light, the system is automatically optimized for an optimal fluorescent light image.
  • a microscope image acquisition unit is to be understood as a unit for acquiring a microscope image.
  • Such a unit may, for example, contain an electronic sensor in the form of a camera or have conventional eyepieces for an observer.
  • the illumination system generates illumination light having wavelengths for exciting the fluorescence of the dye indocyanine green (ICG) or the dye protoporphyrin IX.
  • ICG dye indocyanine green
  • the dye protoporphyrin IX these dyes are suitable for clinical use on patients and thus allow a fluorescence observation of living body tissue.
  • the excitation band of the fluorescence of ICG is at about 78 nm and the fluorescence band at about 830 nm and thus outside the visible range in the near-infrared range.
  • the excitation band of the fluorescence is at about 400 nm and the fluorescence band between about 630 nm and 730 nm.
  • the microscope system control unit is connected to means for adjusting a luminous field diameter of the illumination light provided by the illumination system for the object region in order to maximize the illumination intensity in the object field.
  • the intensity of the Fluorescence light ie, the amount of fluorescent light that is generated in the object area to increase.
  • the microscope system has an observation beam path with adjustable diaphragm for adjusting the depth of field, which is connected to the microscope system control unit, wherein activation of the microscope system control unit causes the setting of a maximum opening of the diaphragm.
  • the illumination system in the microscope system has a lamp associated with means for adjusting the lamp intensity, wherein activation of the microscope system control unit effects the adjustment of the lamp intensity to a maximum value. In this way, the greatest possible radiation flux of fluorescence excitation light is made possible on the object area.
  • a zoom system and a coupling of the zoom system and illumination system are provided in the microscope system in order to allow an adjustment of the illumination intensity at an enlargement set by means of the zoom system for the purpose of a constant brightness impression for an observer, wherein the coupling of the zoom system and Lighting system is designed as a controllable coupling and activation of the microscope system control unit causes a decoupling of the zoom system and lighting system.
  • a color camera is provided in the microscope system for receiving the object area and the microscope system control unit causes in the case of activation in the color camera a color calibration of the means of
  • the color calibration weighted image data of the camera which correspond to a proportion of red light, more strongly than image data, which is based on green or blue light.
  • the microscope system has a camera with adjustable gain and adjustable exposure time
  • the microscope system control unit when activated put the camera in an operating mode by depending on the amount of light which is supplied to the camera, the exposure time is changed so that at decreasing amount of light increases the shutter speed set on the camera. In this way it is possible to operate the cameras in the microscopy system for optimum signal yield.
  • the single FIGURE shows a microscopy system operating microscope 1 for observing an object region 2 with stereoscopic observation beam paths 3, 4, which pass through a microscope main objective 5.
  • a zoom system 6, 7 is provided for setting the magnification in the observation beam paths 3, 4.
  • the zoom system 6, 7 motor actuators 8, 9 are assigned.
  • the surgical microscope 1 includes an illumination system 10 with a light source 1 1, which may be formed, for example, as a xenon lamp.
  • the light source 1 1 emits light 12, which is coupled via a collimating optics 13 in a light guide 14.
  • a diaphragm 16 is provided with an adjustable opening. The light from the diaphragm 16 is guided via a light field optical system 17 to the object area 2.
  • the field light optical system 17 comprises as a means for adjusting the size of the illuminated field 90 aumpsspankrat with a movable lens system 18, which is associated with an actuator 19.
  • a movable lens system 18 By adjusting the lens system 18, it is possible to vary the radiant flux from the illumination system 10 to the object area 2 defined and to focus.
  • a luminous field 90 changes and the irradiance, ie the radiation flux per unit area of illuminating light from the illumination system 10, can thus be set and maximized if necessary.
  • the light source 1 1 has a control unit 20 which allows control of the radiation flux indicated by the light source 1 1. Furthermore, a screen diaphragm 21 is provided in the beam path of the illumination system 10 in order to set the radiation flow through the illumination system 10 continuously. In the illumination system 10, there is also a controllable filter wheel 22, which makes it possible to turn filters 23, 24 and 25 into the illumination beam path. It is thus possible to filter out the spectral range from light emitted by the light source 11 and led to the object area 2, which corresponds to the wavelength of fluorescent light 26 of a dye 27 excited in the object area 2 for fluorescence.
  • the motor-driven actuators 8, 9 of the zoom system can be coupled to the actuators in the illumination beam path in such a way that the size of the light field 90 automatically adapts to the size of the observation field when the magnification setting in the surgical microscope 1 changes.
  • the fluorescent light 26 from the object region 2 passes through the microscope main objective 5 and the zoom system 6, 7 into a microscope imaging unit 100 of the surgical microscope 1.
  • the microscope imaging unit 100 contains eyepieces 28, 29, through which an observer can view the object region 2 and includes a documentation camera 32 and a camera 41 for detecting fluorescent light and a camera 42 for detecting an image signal corresponding to a superimposition of fluorescent light and non-fluorescent light.
  • a beam splitter 30 is provided, which decouples a partial beam path onto the documentation camera 32 via a lens system 31 from the observation beam path 3.
  • the microscope image acquisition unit 100 contains a further beam splitter 40.
  • This beam splitter 40 light is emitted from the observation beam path 4 of the camera 41 Detection of fluorescent light and the camera 42 for detecting an image signal corresponding to a superimposition of fluorescent light and non-fluorescent light supplied.
  • the cameras 41, 42 are assigned a beam splitter 43, which splits the light coupled out by means of the beam splitter 40 from the observation beam path of the surgical microscope 1.
  • the camera 41 is supplied with light through a filter 44 permeable to fluorescent light.
  • the camera 42 detects the light, the light having passed through an aperture and filter wheel 62 from the observation area 2 of the surgical microscope 1.
  • the camera 42 is associated with a calibration device 95.
  • the calibration device 95 contains the image data generated by the camera 42.
  • the calibration device 95 is connected to the microscopy system control unit 70.
  • the calibration device 95 has a first operating state in which it leaves the image data of the camera 42 unchanged. In a second operating state, the calibration device 95 effects a color calibration of the image data.
  • This color calibration ensures that, in operating the surgical microscope 1 for acquiring fluorescence images due to the dye protoporphyrin IX, the image data of the camera, which corresponds to a red light portion, for the purpose of display with the display units 51, 52 comparatively stronger than the corresponding green light and Blue light component of the camera signal are weighted.
  • the camera 42 is further connected to a device 96 for adjusting camera focus and camera shutter time.
  • This device 96 is supplied with a representative of the amount of light of the camera 42 camera signal. It has the effect that camera focus and shutter time are always adapted to the amount of light supplied to camera 42.
  • the device 96 can be switched to a fluorescence operating mode.
  • the device 96 causes the fluorescent light of the dye protoporphyrin IX compared to a normal operating mode of the surgical microscope 1 with unchanged from the amount of light that hits the camera 42, camera camera, but with a shutter time for the camera, that is Exposure time is detected, which increases with decreasing amount of light to a value of preferably up to 0.25 s or even more.
  • the surgical microscope 1 further contains in the left and right observation beam paths in each case a combined diaphragm and filter wheel 61, 62, which are associated with controllable drives 63, 64.
  • Apertures 65, 66 of differently sized passage openings and filters 67, 68 with different transmission characteristics are provided in the diaphragm and filter wheels 61, 62. If the diaphragm 65 is located in the observation beam paths, a comparatively faint image with great depth of field is produced with the surgical microscope. If the diaphragm 66 is switched into the observation beam paths, a maximum radiation flow to the cameras 32, 41, 42 and the eyepieces 28, 29 of the surgical microscope is ensured. For observation of the object region 2 under fluorescent light, it is favorable if the radiation flux to the eyepieces 28, 29 and the cameras 32, 41, 42 is maximum.
  • a signal processing and evaluation unit 50 is provided in the surgical microscope 1.
  • This signal processing and evaluation unit 50 is connected to the cameras 41, 42. It generates from the captured camera images an image signal which is optionally coupled to a display unit 51 whose image is coupled by means of a beam splitter 45 in the observation beam path of the surgical microscope. Alternatively or additionally, this image can be output to a display unit in the form of a monitor 52.
  • the signal processing and evaluation unit 50 is further connected to a microscope system control unit 70, which comprises an activation switch 71.
  • This microscopy control unit 70 has a memory 72 in which, for a fluorescence operating mode of the surgical microscope 1, optimum values for the lamp current of the illumination light source 1 1, for the adjustment of the screen diaphragm 21, the adjustment of the movable lens system 18 of the field light 17, for a gain the cameras 32, 41, 42 and the position of the aperture and filter wheels 61, 62 are stored in the illumination beam path.
  • the surgical microscope 1 Upon actuation of the activation switch 71, the surgical microscope 1 is automatically configured for a fluorescence operating mode in which a coupling of the actuators 19 in the illumination system 10 and the actuators 8, 9 of the zoom system is suppressed and the settings of the operating microscope 1, ie the setting of the illumination system 10, the Cameras 32, 41, 42 and the aperture and filter wheels 61, 62 are optimized for visualizing a fluorescent light based image of the object area 2.
  • the microscope system control unit 70 with a control unit 20 for the light source 1 1, the actuator 19 of the illumination system 10, the controllable filter wheel 22, the actuators 8, 9 for the zoom system 6, 7, the controllable drives for the aperture and filter wheels 63, 64 connected. If the microscope system control unit 70 is activated, the light source 1 1, the actuator 19, the zoom system and the filter wheel 22 and the aperture and filter wheels 61, 62 are automatically set to the values stored in the memory 72.

Abstract

L'invention concerne un système (1) de microscopie pour observer la fluorescence d'un colorant fluorescent (27) dans une zone (2) d'un objet. Le système (1) de microscopie comprend une optique (5, 6, 7) de microscopie munie d'un système d'éclairage (10) pour délivrer de la lumière (12) d'observation pour la zone (2) de l'objet, qui contient des longueurs d'onde dans la plage spectrale visible et présente des longueurs d'onde pour exciter la fluorescence du colorant fluorescent (27). Le système (1) de microscopie contient une unité (100) d'acquisition d'image de microscope à laquelle est acheminé un flux de rayonnement de lumière fluorescente depuis la zone de l'objet. Le système (1) de microscopie comprend un système d'affichage (51, 52) pour afficher une image de la zone (2) de l'objet qui contient une image partielle de la zone (2) de l'objet basée sur la lumière fluorescente. Conformément à l'invention, il est prévu une unité de commande (70) de système de microscopie qui peut être commutée par un opérateur dans un mode de fonctionnement en fluorescence et qui, en activant le mode de fonctionnement en fluorescence, positionne le système (1) de microscopie de telle sorte que dans le système (1) de microscopie, le flux de rayonnement de lumière fluorescente en provenance de la zone (2) de l'objet soit maximal dans l'unité (100) d'acquisition d'image de microscope.
PCT/EP2007/000958 2006-02-08 2007-02-05 Système de microscopie pour l'observation de la fluorescence WO2007090591A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200610006014 DE102006006014A1 (de) 2006-02-08 2006-02-08 Mikroskopiesystem zur Beobachtung von Fluoreszenz
DE102006006014.8 2006-02-08

Publications (1)

Publication Number Publication Date
WO2007090591A1 true WO2007090591A1 (fr) 2007-08-16

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WO (1) WO2007090591A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102008041290A1 (de) 2008-08-15 2010-02-25 Carl Zeiss Surgical Gmbh Mikroskopieanordnung mit Fokusversatz
US8144958B2 (en) 2008-09-11 2012-03-27 Carl Zeiss Meditec Ag Medical systems and methods
EP3422072A1 (fr) * 2017-06-30 2019-01-02 Leica Instruments (Singapore) Pte. Ltd. Système d'éclairage, microscope comprenant un système d'éclairage et procédé de microscope
WO2019053255A1 (fr) * 2017-09-15 2019-03-21 Leica Microsystems Cms Gmbh Procédé d'examen d'un échantillon multi-spectral, unité de commande et ensemble microscope
EP3961285A1 (fr) * 2020-08-28 2022-03-02 Leica Instruments (Singapore) Pte. Ltd. Microscope, système de microscope et procédé d'imagerie d'un objet à l'aide d'un microscope

Families Citing this family (7)

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DE102009011681A1 (de) 2009-02-23 2010-08-26 Obrebski, Andreas, Dr. Wechsler für optische Elemente
DE102009039434B4 (de) 2009-08-31 2023-08-10 Carl Zeiss Microscopy Gmbh Stereomikroskop
DE102010016264A1 (de) 2010-03-31 2011-10-06 Ludwig-Maximilians-Universität München Wellenlängenselektive Blende und Fluoreszenzendoskop
DE102010015691A1 (de) * 2010-04-21 2011-10-27 Carl Zeiss Microlmaging Gmbh Mikroskopiereinrichtung zur Vergleichs- und Mitbeobachtung
DE102015203844A1 (de) 2015-03-04 2016-09-08 Carl Zeiss Meditec Ag Optiksystem und Operationsmikroskop
DE102015216570A1 (de) * 2015-08-31 2016-11-03 Carl Zeiss Meditec Ag Mikroskopiesystem
DE102022121504A1 (de) 2022-08-25 2024-03-07 Carl Zeiss Meditec Ag Verfahren, Computerprogramm und Datenverarbeitungseinheit zum Erzeugen wenigstens eines Korrekturwertes für die Korrektur von Fluoreszenzintensitäten in einem Fluoreszenzbild und optisches Beobachtungssystem

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EP1533640A2 (fr) * 2003-11-21 2005-05-25 CARL ZEISS JENA GmbH Microsope à fluorescence stéréo pour l'éclairage incident

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102008041290A1 (de) 2008-08-15 2010-02-25 Carl Zeiss Surgical Gmbh Mikroskopieanordnung mit Fokusversatz
US8144958B2 (en) 2008-09-11 2012-03-27 Carl Zeiss Meditec Ag Medical systems and methods
US9129366B2 (en) 2008-09-11 2015-09-08 Carl Zeiss Meditec Ag Medical systems and methods
US9320438B2 (en) 2008-09-11 2016-04-26 Carl Zeiss Meditec Ag Medical systems and methods
US9351644B2 (en) 2008-09-11 2016-05-31 Carl Zeiss Meditec Ag Medical systems and methods
US9357931B2 (en) 2008-09-11 2016-06-07 Carl Zeiss Meditec Ag Medical systems and methods
EP3422072A1 (fr) * 2017-06-30 2019-01-02 Leica Instruments (Singapore) Pte. Ltd. Système d'éclairage, microscope comprenant un système d'éclairage et procédé de microscope
CN109212736A (zh) * 2017-06-30 2019-01-15 徕卡仪器（新加坡）有限公司 照明系统、包括照明系统的显微镜和显微镜方法
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CN109212736B (zh) * 2017-06-30 2022-06-03 徕卡仪器（新加坡）有限公司 照明系统、包括照明系统的显微镜和显微镜方法
WO2019053255A1 (fr) * 2017-09-15 2019-03-21 Leica Microsystems Cms Gmbh Procédé d'examen d'un échantillon multi-spectral, unité de commande et ensemble microscope
EP3961285A1 (fr) * 2020-08-28 2022-03-02 Leica Instruments (Singapore) Pte. Ltd. Microscope, système de microscope et procédé d'imagerie d'un objet à l'aide d'un microscope

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