WO2004008217A1 - Absorption confocale lors d'un balayage tridimensionnel - Google Patents

Absorption confocale lors d'un balayage tridimensionnel Download PDF

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Publication number
WO2004008217A1
WO2004008217A1 PCT/EP2003/007290 EP0307290W WO2004008217A1 WO 2004008217 A1 WO2004008217 A1 WO 2004008217A1 EP 0307290 W EP0307290 W EP 0307290W WO 2004008217 A1 WO2004008217 A1 WO 2004008217A1
Authority
WO
WIPO (PCT)
Prior art keywords
focus
sample
measuring beam
transmission
excitation
Prior art date
Application number
PCT/EP2003/007290
Other languages
German (de)
English (en)
Inventor
Erwin Thiel
Rainer Bornemann
Ingo Gregor
Original Assignee
Universität Siegen
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 Universität Siegen filed Critical Universität Siegen
Priority to AU2003246655A priority Critical patent/AU2003246655A1/en
Publication of WO2004008217A1 publication Critical patent/WO2004008217A1/fr

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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
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/59—Transmissivity
    • G01N21/5907—Densitometers
    • G01N21/5911—Densitometers of the scanning type
    • 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
    • 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
    • G02B21/0052—Optical details of the image generation
    • G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
    • 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
    • G02B21/008—Details of detection or image processing, including general computer control

Definitions

  • the invention relates to an arrangement and a method for determining the three-dimensional structure of an object.
  • a laser beam is focused in the object. Part of the laser light is absorbed by the object. The absorbed light energy is sometimes spontaneously emitted and verified by the sample. In this way, the entire object is systematically scanned.
  • Luminescent, in particular fluorescent or phosphorescent, samples or samples labeled with appropriate dyes are measured. Methods are also known which use the reflection of light on the surface of an object to clarify its topography.
  • the spatial resolution is improved by restricting the focus in front of the lens.
  • the light emitted by the sample is placed on a perforated aperture (pinhole) shown, which is arranged in a plane conjugate to the focal plane of the lens in the object.
  • the improved resolution results from the fact that two point imaging functions determine the imaging in the confocal microscope: the imaging function of the focus of the laser beam and the imaging function of the detector, which describes the imaging of the light emitted by the sample into the pinhole and the detector arranged behind it ,
  • the imaging function of the detector describes the probability with which a photon emitted in focus arrives at the detector.
  • the point mapping function of a confocal microscope is the product of the two probability distributions, that is to say the product of the point mapping functions for the excitation and the detection. This leads to a significantly narrower main maximum of the confocal point mapping function compared to a non-confocal microscope. This corresponds to a higher resolution of the confocal microscope and causes discrimination against all points that are not in the immediate vicinity of the focus. The latter is a prerequisite for creating three-dimensional images.
  • confocal optical arrangements with two beams so-called double confocal microscopes, are known, u. a. from DE 40 40 441 AI or WO 92/18850. Some of these are used for 2-photon excitation of the sample (cf. DE
  • the object of the invention is to enable the determination of the three-dimensional structure of an object without having to mark the object beforehand with dye molecules.
  • an arrangement for determining the three-dimensional structure of an object is specified.
  • objects are a. biological objects of interest, such as cells, the inner and / or outer structure of which is to be elucidated.
  • the arrangement has an excitation beam and a measuring beam with a common focus.
  • the foci can also be regarded as common if they do not overlap completely - which is difficult to implement anyway - but if they only overlap substantially. It is conceivable, for example, that the foci only partially overlap. According to the teaching according to the invention, they can be regarded as common if one there is sufficient overlap to carry out the invention.
  • the rays are i. d. R. electromagnetic rays, especially light rays, which are preferably generated by a laser. However, acoustic rays, neutron rays or other particle rays are also conceivable.
  • the beams should be focusable in a defined manner.
  • the focus is smaller than the object.
  • the i. d. R. three-dimensional ellipsoid called maximum radiation intensity.
  • the focus can be chosen to be relatively large. If the object is small, such as a biological cell, i. d. Usually a laser beam is used and the focus is chosen to be as small as possible, in particular diffraction limited.
  • the object can be fixed anywhere in the room.
  • the object can also be embedded in a medium that is essentially transparent to both the excitation and the measurement beam.
  • the arrangement has a device for the relative movement of the object and focus for scanning the object by the focus so that the structure of the object can be determined.
  • the electronically excited molecules have a different extinction coefficient or a different absorption spectrum compared to the molecules in the ground state.
  • the change is i. d.
  • a suitable measuring beam is selected, for example a laser beam with a wavelength that strikes an absorption band of the excited state of the molecules, but that does not hit an absorption band of the ground state.
  • the population of the excited state can also be demonstrated by stimulated emission.
  • the measuring beam is tuned to a wavelength that corresponds to an emission band of the excited state. If the stimulated emission occurs, the transmission of the measuring beam apparently increases. Furthermore, when the stimulated emission is used, the photostability (the number of possible excitation cycles on average) of any dyes used increases since these dwell a shorter time in electronically excited states, from which they i. d. R. can enter into chemical reactions that cancel their fluorescence ability.
  • the emptying of the basic state can be demonstrated in that the measuring beam has, for example, the same or similar wavelength as the excitation beam and then both are also generated by a laser. Both may be tuned to an absorption band of the ground state of the molecules. If the basic state is partially depopulated or faded, the absorption of the measuring beam depends on the intensity of the excitation beam, ie the transmission increases. In this case it is also possible that the measuring beam is tuned to a different absorption band of the basic state, that is to say it has a different wavelength than the excitation beam.
  • the excitation beam creates a volume within the object that has specific properties that can be selectively detected.
  • This volume is e.g. B. determined by the range of the maximum concentration of molecules in the excited state. This area is generated by the focus of the excitation beam. Without this targeted generation of a prepared volume in the object, an absorption measurement could only provide information about the totality of the molecules in the beam path of the measuring beam.
  • the excitation beam and the measuring beam if they are laser beams, have the same wavelength or different wavelengths. Accordingly, one or more radiation sources are required.
  • the arrangement has a device for determining the change in the transmission of the measurement beam when passing through the focus, depending on the change in the intensity of the excitation beam.
  • the change in transmission is proportional to the concentration of the molecules that have been converted into an excited state by the excitation beam.
  • the relative change in transmission is therefore usually proportional to the intensity of the excitation beam.
  • Typical values for the relative change in the transmission of the measuring beam are in the range of 1% or less. Relative transmission changes up to 3 * 10 ⁇ -8 can still be demonstrated with suitable equipment. With this technique, individual absorber molecules can be detected.
  • a great advantage of the invention is that the marking with dyes can be dispensed with. This means that a. to reduce the production of phototoxic substances in the sample, which is strongly catalyzed by fluorescent dyes.
  • the desired measurement signal can usually be detected free of background. If a laser beam with a narrow band in the wavelength is used as the measuring beam, the desired signal is also only detected in this narrow wavelength range. For this, e.g. B. a narrow-band color filter or monochromator can be used in front of the detector. Ambient light, background fluorescence or Raman light are thereby hidden. If fluorescence light is detected, as in the current state of the art, the emission light must be collected over a much broader wavelength range, which leads to a correspondingly increased background. Furthermore, the excitation light can be used to prepare a selective state, which is detected by the measuring beam, a state which may not occur naturally in the sample and is therefore free from the background. In addition to many applications, the invention can be used, inter alia, for ultra-trace analysis in a sample and for checking the homogeneity of the color of polymers or for checking the internal structure of the dye distribution in samples.
  • the measuring beam transmitted through the sample is advantageously detected with the aid of a confocal setup.
  • this confocal, two-beam absorption microscope has a spatial resolution that is improved by a factor of 2.
  • a microscope is used to focus the excitation beam and the measuring beam into the sample.
  • a modified confocal laser scanning microscope is most suitable for carrying out the invention.
  • the invention allows the spectral distribution of the change in the transmission of the measuring beam to be detected in a spatially resolved manner as it passes through the focus.
  • a lamp is used to generate the measuring beam, which emits a broad, as continuous as possible spectrum of light.
  • a narrow-band, tunable light source such as a tunable laser, can also be used.
  • the transmission of the measuring beam is then detected using a spectral detector.
  • the transmission or absorption spectra determined in this way can also be determined as a function of the wavelength of the excitation beam. This opens up a new field of multidimensional spectroscopy that is enormous Differentiated representation options ⁇ . B. of tissue or cell structures.
  • Fig. 1 is a schematic representation of the optical structure
  • Fig. 2 shows an exemplary measurement with confocal absorption microscopy.
  • a sample 10 e.g. B. a tissue preparation, attached to a positioning table 12.
  • a microscope objective 14 focuses a continuous beam of a helium-neon laser 16 into the sample 10 after it has been enlarged in diameter by a pair of lenses 17.
  • the helium-neon laser has a wavelength of 543.5 nm.
  • the transmitted light is collected by a second microscope objective 18 and focused on a pinhole 20 by means of a lens 22 via a spectral filter 19.
  • a silicon photodiode 24 is arranged behind the pinhole and detects the light passing through the pinhole.
  • the second microscope objective 18 is also used to focus the beam 26 of an argon ion laser 28 (with a wavelength of 514.5 nm) into the sample 10 after this was also enlarged in diameter by a pair of lenses 29 and was coupled into the microscope objective 18 by a dichroic beam splitter 30.
  • the intensity of the laser beam 26 is modulated at a frequency of 17 kHz.
  • the foci of the two laser beams coincide, as does the imaging of the pinhole 20 in the plane of the sample 10.
  • the beam 26 of the argon-ion laser 28 serves as an excitation beam.
  • the beam of the helium-neon laser 16 serves as the measuring beam 32.
  • the wavelength of the excitation beam 26 is selected such that it fits on an absorption band in the sample 10. Due to the absorption of the excitation beam 26 in the sample 10, a dynamic equilibrium results in a certain proportion of molecules being brought into an electronically excited state. This percentage is typically in the range of one percent or several orders of magnitude below.
  • the electronically excited molecules have a different extinction coefficient for the wavelength of the measuring beam 32 compared to the molecules in the ground state.
  • the transmission of the measuring beam 32 through the sample 10 thus changes as a function of the modulation of the excitation beam 26.
  • This periodic change in the transmission indicates that Presence of absorber molecules in sample 10.
  • the spatial distribution of the absorber molecules and thus the spatial structure of the sample 10 can be determined by three-dimensional scanning of the sample 10 with the aid of the positioning table 12.
  • the signal from the silicon photodiode 24 is amplified and evaluated with the aid of a lock-in amplifier 40 and a computer 42.
  • the lock-in amplifier 40 allows effective noise suppression and thus a high dynamic range of the measurement signal recording.
  • the lock-in amplifier 40 is tuned to the frequency and phase of the modulator 31.
  • the computer 42 also controls the positioning table 12 and thus the relative spatial position between the sample 10 and the focus. In this way, changes in the transmission of the measurement beam 32 through the sample 10 are determined as a function of the spatial positioning of the focus
  • FIG. 2 shows an exemplary measurement.
  • a specimen slide 34 for microscopy was coated with sample molecules as sample 10.
  • a 10 A -4 molar solution of rhodamine 6G in chloroform with three percent by mass of polycarbonate was prepared. This was applied as a layer 36 to the slide by spin coating at 2000 revolutions per minute.
  • FIG. 2 shows the change in the transmission delta I / I as a function of the coordinate z, which indicates the distance between the focal plane 38 of the excitation beam 26 and the measuring beam 32 from the plane of the dye layer 36.
  • the spatial localization of the layer 36 in the z direction is possible with the described absorption method, as can be clearly seen in FIG. 2.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Optics & Photonics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • General Engineering & Computer Science (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Selon l'invention, pendant l'absorption confocale se produisant lors d'un balayage tridimensionnel, pour la détermination de la structure tridimensionnelle d'un objet, un faisceau d'excitation (26) et un faisceau de mesure (32) sont focalisés dans un foyer sensiblement commun. L'objet (10) et le foyer sont déplacés l'un par rapport à l'autre pour que l'objet soit balayé par ledit foyer. La modification de la transmission du faisceau de mesure (32) pendant son passage à travers le foyer est déterminée en fonction de la modification de l'intensité du faisceau d'excitation (26). Par irradiation de l'échantillon avec de la lumière passant par un foyer bien défini, la transmission de l'échantillon dans ledit foyer est localement modifiée. Cette modification est mesurée à l'aide du faisceau de mesure. Cela permet, par exemple, de capter la structure intérieure de cellules de façon tridimensionnelle par des mesures d'absorption, sans que l'on ait à traiter ces cellules préalablement avec des colorants.
PCT/EP2003/007290 2002-07-11 2003-07-08 Absorption confocale lors d'un balayage tridimensionnel WO2004008217A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003246655A AU2003246655A1 (en) 2002-07-11 2003-07-08 Confocal 3d-scanning absorption

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2002131543 DE10231543B3 (de) 2002-07-11 2002-07-11 Konfokale 3D-Scanning Absorption
DE10231543.4 2002-07-11

Publications (1)

Publication Number Publication Date
WO2004008217A1 true WO2004008217A1 (fr) 2004-01-22

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AU (1) AU2003246655A1 (fr)
DE (1) DE10231543B3 (fr)
WO (1) WO2004008217A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007009812A1 (fr) * 2005-07-22 2007-01-25 Carl Zeiss Microimaging Gmbh Microscopie a luminescence a resolution amelioree
CN103115900A (zh) * 2013-01-21 2013-05-22 合肥知常光电科技有限公司 一种探测固体材料表面及亚表面光学吸收的方法及装置
US9341515B2 (en) 2011-02-11 2016-05-17 University Of Central Florida Research Foundation, Inc. Optical absorbance measurement apparatus, method, and applications

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006046369A1 (de) 2006-09-29 2008-04-03 Carl Zeiss Microimaging Gmbh Auflösungsgesteigerte Lumineszenzmikroskopie

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4631581A (en) * 1984-03-15 1986-12-23 Sarastro Ab Method and apparatus for microphotometering microscope specimens
WO1992000540A1 (fr) * 1990-06-29 1992-01-09 Arthur Edward Dixon Appareil et procede destines a l'imagerie par transmission ou par reflexion de lumiere
EP0491289A1 (fr) * 1990-12-18 1992-06-24 Stefan Dr. Hell Microscope double-confocal à balayage

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WO1992018850A1 (fr) * 1991-04-10 1992-10-29 Mayo Foundation For Medical Education And Research Systeme d'imagerie a foyer commun pour la lumiere visible et la lumiere ultraviolette
DE4324681C2 (de) * 1993-07-22 1997-09-04 Hell Stefan Verfahren zur optischen Anregung eines Energiezustands einer Probe in einem Probenpunkt und Vorrichtung zur Durchführung des Verfahrens
DE4331570C2 (de) * 1993-08-17 1996-10-24 Hell Stefan Verfahren zum optischen Anregen einer Probe
DE4416558C2 (de) * 1994-02-01 1997-09-04 Hell Stefan Verfahren zum optischen Messen eines Probenpunkts einer Probe und Vorrichtung zur Durchführung des Verfahrens

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4631581A (en) * 1984-03-15 1986-12-23 Sarastro Ab Method and apparatus for microphotometering microscope specimens
WO1992000540A1 (fr) * 1990-06-29 1992-01-09 Arthur Edward Dixon Appareil et procede destines a l'imagerie par transmission ou par reflexion de lumiere
EP0491289A1 (fr) * 1990-12-18 1992-06-24 Stefan Dr. Hell Microscope double-confocal à balayage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GREGOR I ET AL: "LASER-RASTER SPECTROMETER FOR TIME-RESOLVED RECORDING OF TRANSIENT ABSORPTION", APPLIED OPTICS, OPTICAL SOCIETY OF AMERICA,WASHINGTON, US, vol. 38, no. 36, 20 December 1999 (1999-12-20), pages 7468 - 7474, XP000893820, ISSN: 0003-6935 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007009812A1 (fr) * 2005-07-22 2007-01-25 Carl Zeiss Microimaging Gmbh Microscopie a luminescence a resolution amelioree
US9341515B2 (en) 2011-02-11 2016-05-17 University Of Central Florida Research Foundation, Inc. Optical absorbance measurement apparatus, method, and applications
CN103115900A (zh) * 2013-01-21 2013-05-22 合肥知常光电科技有限公司 一种探测固体材料表面及亚表面光学吸收的方法及装置

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Publication number Publication date
DE10231543B3 (de) 2004-02-26
AU2003246655A1 (en) 2004-02-02

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