WO2004113988A1 - Systeme optique confocal pour l'execution simultanee d'une mesure confocale et d'une mesure grand champ - Google Patents

Systeme optique confocal pour l'execution simultanee d'une mesure confocale et d'une mesure grand champ Download PDF

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Publication number
WO2004113988A1
WO2004113988A1 PCT/EP2004/006644 EP2004006644W WO2004113988A1 WO 2004113988 A1 WO2004113988 A1 WO 2004113988A1 EP 2004006644 W EP2004006644 W EP 2004006644W WO 2004113988 A1 WO2004113988 A1 WO 2004113988A1
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WO
WIPO (PCT)
Prior art keywords
light
detector
optical system
imaging
area
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Application number
PCT/EP2004/006644
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German (de)
English (en)
Inventor
Filipp Oesterhelt
Claus Seidel
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO2004113988A1 publication Critical patent/WO2004113988A1/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

Definitions

  • the invention relates to a confocal optical system comprising:
  • Coupling means for coupling light from a light source for at least area-wise illumination of an object
  • first imaging means for imaging light from a measurement area of the object in a plane conjugated to the level of the measurement area
  • Reflective separation means which are arranged in the area of the conjugate plane, for separating light from the measurement area and light from the object areas surrounding the measurement area, a first detector and a second detector for separately detecting light from the measurement area and light from the surrounding object areas.
  • Confocal optical systems are widely used in science, whereby imaging and non-imaging systems are known.
  • the particular advantage of a confocal system is that a measuring area in an object of interest can be defined very precisely in its three-dimensional extent. This advantage manifests itself, for example, in fluorescence microscopic examinations of “thick objects, ie objects whose spatial extent in the direction of the optical axis is significantly larger than the measuring region that is actually of interest. Even with focused illumination of the measuring area, it can generally not be avoided that object areas surrounding the measuring area are also illuminated become.
  • the separation means are designed as a pinhole perpendicular to the optical axis, the opening of the pinhole being positioned exactly at the point at which the light originating from the measuring area is focused.
  • Light that comes from areas above or below the measuring range level is not focused in the conjugate level, but has the shape of a large, unsharp spot. It is therefore almost completely absorbed by the screen material. Only the light from the three-dimensionally defined measuring range passes through the pinhole and can then be subjected to any further treatment.
  • FCS fluorescence correlation spectroscopy
  • DE 100 08 594 AI discloses a confocal optical system with two independent confocal detection arrangements. While one detection arrangement is designed as a confocal laser scanning microscope (CLSM), the other detection arrangement is an FCS arrangement, as explained above. To determine the position of the current measuring range in the object, a movable mirror unit is provided which can switch the beam path between the two detection arrangements.
  • CLSM confocal laser scanning microscope
  • FCS confocal laser scanning microscope
  • this system has two major disadvantages.
  • the spatial correspondence of the CLSM and FCS measurement depends on the reproducibility of the mechanical movement of the mirror, which is usually inadequate or can only be achieved with considerable technical effort.
  • DMD module digital micromirror device
  • a large number of miniaturized mirror elements are arranged area-wide on a chip, the angular position of which can be programmed individually. If the mirror element onto which the light originating from a measurement area is focused has a first angular position and the mirror elements surrounding it have a second angular position, the light from the measurement area and the light from the surrounding object areas can be reflected onto different imaging detectors.
  • This system uses the light rejected in the classic setup from the surrounding object areas to build up a (non-confocal) image (wide-field measurement) that has a dark spot at the location of the measurement area that can be used to localize the measurement area in the object.
  • This system enables confocal and non-confocal measurements to be carried out simultaneously without wasting time.
  • this system must be considered technically very complex, which is reflected in high costs for the device itself and its complex programming.
  • the system for simultaneous measurement of a large number of measurement areas in an object is disclosed with the aim of a particularly fast and sample-friendly, imaging measurement, the information from the surrounding object areas being used for a complex image analysis.
  • the technical effort required to implement the system seems justified for this complex application.
  • the high outlay must be regarded as a serious disadvantage.
  • the object of the present invention to develop a generic system in such a way that a simultaneous carrying out of a confocal as well as a wide field measurement is made possible in a simple manner.
  • This object is achieved in connection with the features of the preamble of claim 1 in that the reflective separation means in the conjugate plane have at least one optical opening through which light can pass from the measurement area and be imaged on the first detector, while light from the surrounding object areas can be reflected for imaging on the second detector.
  • the light that comes from the object areas surrounding the measurement area can be used to build up a non-confocal image, while at the same time the light from the measurement area can be captured confocally and used in a suitable manner.
  • this system can be implemented cost-effectively in a particularly simple manner. It is also comparatively easy to retrofit classic confocal systems according to the invention and thus to considerably expand their range of applications.
  • the system according to the invention can be implemented particularly cost-effectively by the reflective separation means being designed as a perforated diaphragm which is inclined at an angle with respect to the conjugate plane and whose surface facing the object is mirrored.
  • the diaphragm is inclined for the purpose of directing light from the surrounding object areas onto a detector outside the rest of the detection beam path. Pinholes with one or more holes can be provided to parallelize the measurement.
  • Another advantage of the device according to the invention over the generic state of the art is that a particularly sensitive FCS measurement can be carried out in the confocal beam path with a minimized number of optical elements.
  • a mechanically movable mirror element in the direct vicinity of a large number additional movable mirror element required, so that a minimum susceptibility to interference with maximum vibration resistance is achieved.
  • a transparent pane instead of the perforated diaphragm, which contains a mechanical passage opening, a transparent pane is used, the surface of which facing the object is mirrored around at least one transparent passage area.
  • An optical opening with a mirror surface surrounding it is thus formed in a particularly simple manner in terms of production technology.
  • the transparent support substrate e.g. Glass, quartz, sapphire or the like, suitably selected so that the confocal measurement is not impaired, this variant represents a particularly cost-effective alternative to a real pinhole.
  • Panels with one or more transparent passages can be provided to parallelize the measurement.
  • an optical opening in the diaphragm plane is circular, this leads to an oval projection in the conjugate plane.
  • an optical opening is oval, in such a way that a projection into the conjugate plane is circular.
  • the stated object is achieved in connection with the features of the preamble of claim 5 in that the reflective separation means in the conjugate plane have at least one optical transmission area through which light can pass from the object areas surrounding the measurement area and can be imaged on the first detector , while light from the measuring range can be reflected for imaging on the second detector.
  • This variant of the system according to the invention can be implemented in a particularly cost-effective manner by the reflective separating means being arranged as one against the conjugate plane an obliquely standing, transparent pane are formed, the surface of which facing the object has at least one mirrored surface area within a transparent passage area.
  • the purpose of the inclination arises analogously from the above. Disks with one or more mirrored surface areas can be provided to parallelize the measurement.
  • a mirrored surface area is circular in the pane plane, this leads to an oval projection in the conjugate plane.
  • a mirrored surface area is oval, such that a projection into the conjugate plane is circular.
  • the second detector is an imaging detector.
  • the person skilled in the art has a large number of variants, for example CCD, CID, or the like. available, which can be selected appropriately depending on the specific application.
  • the second detector — imaging or non-imaging — consists of a plurality of detector units, the optical input surfaces of which are parallel to one Imaging plane are spatially offset from each other.
  • This can take place, for example, in the form of a multiple, for example two or four, divided photodiode or a plurality of CCD detectors, to which the individual diode surfaces are suitably connected in order to detect a point of light migrating.
  • the system can be set up so that in the event that there is a light-emitting object of interest in the measurement volume, no signal is detected on the second detector, since all of the emitted light is directed to the first detector.
  • the molecule of interest begins to migrate out of the measuring range, it can be detected with the second detector, the spatially offset arrangement of individual detector units described allowing the direction of emigration to be determined.
  • the second detector is only able to recognize the direction of emigration in the plane of the measuring range.
  • An emigration perpendicular to this would only lead to an enlargement of the light spot on the second detector, which does not allow any information about the sign of the emigration direction.
  • the detector can be arranged somewhat outside the 'conjugate plane, so that the image of a fluorophore is larger than its minimal, diffraction-limited image, but a change in position of the fluorophore out of the measurement plane then causes - depending on the direction of movement - a smaller or growing image, from which the direction of movement can be clearly determined.
  • the optical input surfaces of the individual detector units spatially staggered perpendicular to an imaging plane.
  • an emigration of the fluorophore of interest perpendicular to the measuring range plane results in an enlargement of the partial light spot on one detector unit, while an additional partial light spot is located on another, spatially further forward or would reduce or disappear detector unit located further back.
  • the sign of the emigration direction perpendicular to the measuring range level can also be recognized.
  • the optical input surfaces of the individual detector units are designed as the end faces of optical fibers or fiber bundles. These can then preferably direct the incident light to a non-imaging sensor element.
  • a particularly sensitive avalanche photodiode (APD) or the like can be used as the sensor element.
  • the individual detector units are each imaging, e.g. as independent CCD elements.
  • the imaging means for imaging the light from the surrounding object areas onto the second detector comprise an astigmatic imaging unit.
  • astigmatic imaging units in particular astigmatic lenses, do not have a focal point, but two focal lines that are essentially perpendicular to one another at different distances in front of and behind the lens plane.
  • a light spot emanating from the measurement area perpendicular to the measurement area plane is expressed in a longitudinal deformation of the light spot in the input plane of the detector, the orientation of the change in shape being an indication of the sign of the direction of emigration from the measurement area ,
  • the astigmatic imaging and the spatial staggering of detector units perpendicular to an imaging plane are primarily intended as an alternative, a combination of both measures is of course possible.
  • tracking means are provided, by means of which the colocalization of a given object element and the measurement range can be maintained.
  • Such tracking means can either change the optical beam path (illumination and / or detection beam path) of the system or move the object itself accordingly.
  • a data processing unit for recording and evaluating measurement signals of the second detector on the one hand and for controlling the tracking means on the other hand is preferably provided, which is set up in terms of program technology in such a way that the tracking means can be controlled as a function of evaluation results of the measurement signals of the second detector.
  • Figure 1 a schematic beam path of an embodiment of the system according to the invention
  • Figure 2a is a schematic representation of a first embodiment of the image on the second detector;
  • Figure 2b a schematic representation of a first
  • Figure 3a a schematic representation of a second
  • Figure 3b a schematic representation of a second embodiment of the image of the second detector.
  • Figure 1 shows schematically an inventive confocal optical system.
  • the orientation of the measuring range plane 10 is defined by the optical axis 13 of the detection beam path of the system, which is perpendicular to the measuring range plane 10.
  • a light source 14 in particular a laser unit, is arranged in the detection beam path, the light of which is parallelized by means of the optics 15 or the laser beam of which is suitably expanded.
  • the excitation light is focused with the objective 17 in the measuring region 11 via a beam splitter 16 which is reflective for the excitation light.
  • the fluorescence light emitted by the measuring range and longer-wave due to the Stokes shift passes the beam splitter 16 essentially in parallel and is refocused by the first imaging optics 18 in a plane 20 which is conjugated to the measuring range plane 10.
  • the light is parallelized again via a further imaging optics 31 ′ and focused in the input plane 30 ′ of the detector 32 ′.
  • object areas surrounding the measuring area 11 are also illuminated by the excitation light, so that fluorophores also emit fluorescent light in these areas.
  • Light from surrounding object areas in the measuring area level 10 are imaged laterally offset in the conjugate plane 20 from the light from the measuring area 11, while light from surrounding object areas outside the measuring area level 10 are focused above or below the conjugate plane 20.
  • a mirrored perforated diaphragm 40 is arranged in the region of the conjugate plane 20, which is inclined by an angle ⁇ with respect to the conjugate plane 20.
  • the opening of the pinhole is colocalized with the imaging point of the light from the measuring area 12. This means that this light, as indicated by the solid lines in FIG. 1, can pass through unhindered and be imaged on the detector 32 '.
  • the light from the surrounding object areas is reflected by the reflecting surface 41 of the pinhole 40 from the detection beam path of the detector 32 ′ into the detection beam path of the second detector 32.
  • Suitable imaging optics 31 are used for imaging on the detector 32.
  • the input plane of the detector 32 provided with the reference symbol 30 in FIG. 1 is an imaginary plane which is conjugated to the conjugate plane 20 and the measuring range plane 10. In this plane, only light from the surrounding object areas is sharply imaged in the measuring area plane 10. In contrast, light from surrounding object areas outside the measuring area level 10 are sharply imaged in front of or behind the level 30.
  • FIG. 2a shows the detection beam path of the second detector 32 in a first embodiment, in which an imaging lens 311 is designed as a conventional spherical lens.
  • the image of the light from the surrounding object areas that arises in the focal plane of the lens 311 is shown schematically as a light spot 312 shown, which has a darkening 313 in its interior, which corresponds to the measuring range.
  • the area 313 is a darkening area because the corresponding light from the
  • Measuring range according to the invention was directed to the first detector 32 '.
  • Figure 2b represents a with the imaging optics.
  • Figure 2a shows a particularly advantageous interacting embodiment of the detector 32.
  • the detector has four individual detector units 321, 322, 323 and 324, which are located both in the imaging plane and perpendicularly thereto, i.e. are arranged spatially staggered parallel to the optical axis.
  • the detector units 321 and 322 lie in an upstream plane in comparison to the detector units 323 and 324.
  • This embodiment is particularly suitable for measuring individual fluorophores. If the fluorophore of interest is exactly in the measuring range, no signal is detected on the detector 32. However, if the fluorophore begins to migrate out of the measuring range, the two-tiered arrangement of the detector units can detect both the migration in the measuring range plane and perpendicular to it.
  • FIG. 3a shows a further embodiment of the detection beam path of detector 32.
  • An imaging lens 314 is designed as an astigmatic lens.
  • the reference numerals 315 and 317 denote the elongated light spots which result as an image of the light from the surrounding object areas around the respective focal lines of the lens 314.
  • the light spots 315 and 317 also have central obscurations 316 and 318 which result from the redirection of the light from the measurement area to the first detector 32 ' .
  • FIG. 3b shows an embodiment of the detector 32, which interacts in a particularly advantageous manner with the astigmatic arrangement of FIG. 3a.
  • the Detector 32 is composed of four individual detector elements, the input surfaces 325, 326, 327 and 328 of which are arranged in a common imaging plane.
  • the input surfaces are the end surfaces of optical fibers, which each feed the incident light to a highly sensitive sensor unit.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un système optique comprenant : des éléments d'entrée (15) servant à l'entrée de lumière provenant d'une source lumineuse (14) pour l'éclairage, au moins partiel, d'un objet ; des premiers éléments de production (18) servant à projeter la lumière d'une zone mesurée (11) de l'objet (12) dans un plan (20) conjugué avec le plan (10) de la zone mesurée ; des éléments de séparation réflecteurs (40), qui sont placés à proximité du plan conjugué (20) pour séparer la lumière provenant de la zone mesurée (11) et la lumière provenant des zones de l'objet entourant la zone mesurée (11) ; et un premier détecteur (32') et un second détecteur (32) servant à la captation séparée de la lumière provenant de la zone mesurée (11) et de la lumière provenant des zones de l'objet entourant la zone mesurée. Selon l'invention, les éléments de séparation réflecteurs (40), dans le plan conjugué (20), présentent au moins une ouverture optique par laquelle la lumière provenant de la zone mesurée (11) peut passer et venir frapper le premier détecteur (32'), tandis que la lumière provenant des zones entourant la zone mesurée de l'objet peut être réfléchie pour venir frapper le second détecteur (32) ; ou bien, les éléments de séparation réflecteurs (40), dans le plan conjugué (20), présentent au moins une zone de transmission optique par laquelle la lumière provenant des zones entourant la zone mesurée (11) de l'objet peut passer et venir frapper le premier détecteur (32'), tandis que la lumière provenant de la zone mesurée (11) peut être réfléchie pour venir frapper le second détecteur (32).
PCT/EP2004/006644 2003-06-21 2004-06-18 Systeme optique confocal pour l'execution simultanee d'une mesure confocale et d'une mesure grand champ WO2004113988A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003127987 DE10327987A1 (de) 2003-06-21 2003-06-21 Konfokales optisches System
DE10327987.3 2003-06-21

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WO2004113988A1 true WO2004113988A1 (fr) 2004-12-29

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013149731A1 (fr) * 2012-04-05 2013-10-10 Carl Zeiss Microscopy Gmbh Dispositif et procédé de microscopie

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102012009836A1 (de) 2012-05-16 2013-11-21 Carl Zeiss Microscopy Gmbh Lichtmikroskop und Verfahren zur Bildaufnahme mit einem Lichtmikroskop

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EP0911667A1 (fr) * 1997-10-22 1999-04-28 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Microscope programmable à modulation spatiale de la lumière et méthode de microscopie
DE10008594A1 (de) * 2000-02-22 2001-08-23 Zeiss Carl Jena Gmbh Verfahren und Anordnung zur Detektion des von einer Probe kommenden Lichtes

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DE19537586C2 (de) * 1995-10-09 2002-03-28 Schleifmittelwerk P Lapport & Messgerät zur Bestimmung von Oberflächen, Oberflächenprofilen und Volumina
DE19650391C2 (de) * 1996-12-05 2001-07-26 Leica Microsystems Anordnung zur simultanen polyfokalen Abbildung des Oberflächenprofils beliebiger Objekte
DE19842153C2 (de) * 1998-09-15 2003-07-31 Leica Microsystems Fluoreszenzmikroskop

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Publication number Priority date Publication date Assignee Title
EP0911667A1 (fr) * 1997-10-22 1999-04-28 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Microscope programmable à modulation spatiale de la lumière et méthode de microscopie
DE10008594A1 (de) * 2000-02-22 2001-08-23 Zeiss Carl Jena Gmbh Verfahren und Anordnung zur Detektion des von einer Probe kommenden Lichtes

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013149731A1 (fr) * 2012-04-05 2013-10-10 Carl Zeiss Microscopy Gmbh Dispositif et procédé de microscopie
GB2514735A (en) * 2012-04-05 2014-12-03 Zeiss Carl Microscopy Gmbh Device and method for microscopy
US9494782B2 (en) 2012-04-05 2016-11-15 Carl Zeiss Microscopy Gmbh Device and method for microscopy using light with differing physical properties
GB2514735B (en) * 2012-04-05 2016-12-14 Zeiss Carl Microscopy Gmbh Device and method for microscopy

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