WO1994018592A1 - Microscope confocal - Google Patents

Microscope confocal Download PDF

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Publication number
WO1994018592A1
WO1994018592A1 PCT/AU1994/000053 AU9400053W WO9418592A1 WO 1994018592 A1 WO1994018592 A1 WO 1994018592A1 AU 9400053 W AU9400053 W AU 9400053W WO 9418592 A1 WO9418592 A1 WO 9418592A1
Authority
WO
WIPO (PCT)
Prior art keywords
transmitter
light
light beam
microscope
objective lens
Prior art date
Application number
PCT/AU1994/000053
Other languages
English (en)
Inventor
Martin Russell Harris
Original Assignee
Optiscan Pty Ltd Acn 060 658 754
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 Optiscan Pty Ltd Acn 060 658 754 filed Critical Optiscan Pty Ltd Acn 060 658 754
Priority to AU59964/94A priority Critical patent/AU672876B2/en
Publication of WO1994018592A1 publication Critical patent/WO1994018592A1/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
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers

Definitions

  • This invention relates to the field of microscopy and more particularly to confocal microscopes. Background of the Invention
  • these microscopes use a laser as the high illumination light source and have a computer or video system to process and store or display the detected image signals.
  • Confocal microscopes have better resolution that conventional microscopes and sharper definition in that out of focus signals and interference are much reduced. They have found particular application in the examination of biological specimens by epi-fluorescence where the reduction of out of focus interference is a major advantage.
  • a confocal microscope can have a light transmitter and detection system and an objective lens which are physically independent of one another so that the objective lens can be mounted on or fixed relative to the object to be examined and interrogated from a remote location by the light transmitted detection system.
  • optic fibres for the transmission of light between the light source and the transmitter and detection components of the microscope enables a construction which has a freely mobile light transmitter which may incorporate a beam scanning system.
  • the invention is not limited in its application to microscopes employing optical fibres and it is applicable to microscopes with bulk optical transmission components. It may also be applied to microscopes which do not move alight beam in a scanning pattern but rely on movement of the object to generate an appropriate scan. Disclosure of the Invention
  • a confocal microscope comprising: a light source for generating a light beam; a light beam transmitter to receive said light beam and to transmit the beam out from the transmitter; an objective lens for receiving the light beam from the transmitter and to focus the light from the beam onto an object to illuminate a confined observational field on or within the object and for receiving light emanating from that confined observational field on or within the object and for transmitting that emanated light as a return beam back to the transmitter; and a detector for detecting the return beam of emanated light transmitted back to the transmitter; wherein the objective lens is physically independent of the transmitter so as to be positionable adjacent an object to be viewed at a location separated from the transmitter by free space and there is beam aiming means for aiming the light beam transmitted to the objective lens through said free space.
  • the objective lens may be one of plurality of such lenses all physically independent of the transmitter and positionable at a series of locations separated from the transmitter by free space and the beam aiming means may be operable to aim the light beam transmitted from the transmitter alternatively onto each of the objective lenses.
  • the aiming means may be operable adjustably to move the transmitter thereby to vary the aim of the light beam through said free space.
  • the aiming means may comprise moveable reflector means associated with the transmitter so as to reflect the light beam and moveable to change the direction of refection thereby to vary the aim of the light beam through said free space.
  • Figure 1 illustrates one form of confocal microscope which is constructed in accordance with the invention and which employs fibre optics to produce a freely mobile scanning transmitter head
  • Figure 2 is a perspective view of the transmitter head
  • FIG 3 illustrates an alternative confocal microscope which is also constructed in accordance with the invention but which has bulk optical transmission elements.
  • the microscope illustrated in Figures 1 and 2 has many components which are common to the confocal microscope illustrated in Figure 1 of International Patent Application PCT/AU89/00298 and these common elements have been identified by the same reference numerals .
  • This microscope comprises a high intensity light source in the form of a laser generator 1 to supply a light beam 2 which is focussed by a lens 3 into one end of flexible optical fibre .
  • the other end of optical fibre 4 runs into one side of a directional coupler 5 which may be a fused biconical taper coupler or other coupler for separating light rays travelling in opposite directions.
  • the light going into one of the outgoing limbs 6 at the other side of the coupler is absorbed with minimum Fresnel reflection by an indexing matching media body 7 while light going into the other leg of the coupler at that side is transmitted by flexible optical fibre 8 to an optical transmitter head denoted generally as 10.
  • Optical transmitter head 10 comprises a hollow casing 11 which houses components of a moving mirror scanning system. These components comprise a lens 14 to receive light 15 diverging from the end 9 of fibre 8, a pair of mirrors 16, 17 by which the light transmitted through lens 14 is successively reflected to a beam converging lens 19 whence the beam is transmitted outwardly from transmitter head 10 to pass through free space onto an objective lens 18 which condenses or focuses the light onto a spot or point observational field in a specimen 20 to be examined. Lens 18 is mounted in a clamp structure 30 whereby it is clamped to the specimen 20. Clamp structure 30 .may be adjustable to enable the distance between the lens 18 and the surface of the specimen 20 to be varied.
  • Mirrors 16, 17 can be moved by transducers 21, 22 installed within the transmitter head casing 11 in response to signals supplied through electrical connections 23, 24 from an electronic scanning signal generator 25 such that the reflected light beam is moved in X and Y directions to cause the illuminated space to traverse the specimen in a scanning pattern. Scanning means of this kind is used in conventional scanning confocal microscopes. As well as focusing high intensity light onto the specimen to produce an illuminated spot, the objective lens 18 also receives light emanating from the specimen which is transmitted back through the optical train of transmitter head 10 to the optical fibre 8. Depending on the nature of the specimen, this light emanating from the specimen may comprise reflected light, Raman scattered light or fluorescent light.
  • the term "emanating" as used in this specification is to be construed in a broad sense as covering any light transmitted from the object back through the objective lens. This light reconverges to a focus back at the tip 9 of optical fibre 8 and travels back through that fibre to the coupler 5 where a portion of that light is transmitted via the fourth leg of the coupler and a further optical fibre 31 then via a filter 32 and lens 33 to a photo detector 34.
  • the signal from photo detector 34 passes through an electrical connection 35 to the signal processor 36 of a video display system which produces an image on a display screen 37.
  • the signal from photo detector 34 modulates the intensity of the image signal transmitted from the processing circuit 36 through output line 38 to the display screen 37 and the mechanical scanning movements of the mirrors 16, 17 are synchronised with the electronic raster scanning movements of the display system through an interconnection 39 between the electronic scanning signal generator 25 and the signal processing means 36 of the video display unit 37.
  • the optical scan and transmitter head 10 is mounted on a turret 40 on which it can be rotated about an axis 41 and tilted back and forth in the direction of arrows 42 whereby to adjust the direction in which the light beam is transmitted outwardly from the head.
  • the objective lens 18 can be set adjacent the specimen and the transmitter head can be set at any convenient position for remote scanning and interrogation of the objective lens. Because of the confocality principle virtually the only light the detector "sees" through the fibre is that from the spot on the specimen when it is in focus. The detector is virtually unaffected by moderate ambient light even if the fibre core "field of view" does spill over the objective lens pupil.
  • FIG. 1 employs fibre optics to allow the possibility of an optical scanning and transmitter head 10 which is freely mobile and isolated from the laser source and detection components.
  • the principle of a free-space beam confocal microscope is not limited to optic fibre confocal systems and can also be applied to bulk optical systems.
  • Figure 3 illustrates a development by which a series of objective lenses may be scanned and interrogated by a single transmitter and scanning system.
  • a laser generator 51 supplies a light beam 52 to a beam splitting mirror 53 which reflects it via scanning mirrors 54, 55 to a pivotable aiming mirror 56 whence it is directed via a field lens 57 onto an objective lens 58 located adjacent the specimen to be examined.
  • Return light emanating from the specimen passes back through the system to the beam splitter mirror 53 whence it is split to form a return light beam 59 which is detected by a photo multiplier tube 60.
  • Scanning mirrors 54, 55 can be moved by transducers 61, 62 in response to signals supplied through electrical connections 63, 64 from an electronic scanning signal generator 65 such that the reflected light beam is moved in X and Y directions to cause the illuminated spot to traverse the specimen in a scanning pattern.
  • the signal from photo multiplier tube detector 60 can be passed through an electrical connection 66 to the signal processor 67 of a video display system which produces an image on a display screen 68.
  • the objective lens may be one of a series of objective lenses to be scanned and interrogated by the one scanning and detector system.
  • additional objective lenses 58A, 58B, etc can be scanned and interrogated by the one light beam by appropriate movement by aiming mirror 56.
  • a single field lens 57 may move with the aiming mirror to focus the light beam on each of the objective lenses in turn or additional field lenses 57A, 57B, etc may be set up in association with each of the additional objective lenses.
  • the light beam transmitter also serves to move the light beam in a scanning pattern there are applications in which the object to be examined is moving at a regular speed and the scanning movements may not be required.
  • the light source generator may be provided with a cylindrical lens or other means to provide a line illumination of the moving sheet transverse to the direction of movement.
  • Such line illumination could be moved across the sheet by having a series of objective lens spaced across the sheet and interrogated by the one illumination and detection system and in this case it would not be necessary to move the beam with an X and Y scanning patte.rn.
  • the light detector composed of a multiplicity of separate elements arranged in a line.
  • This line would be oriented so as to be at the confocal position to the line of light projected on the specimen.
  • Each photoreceptive element would receive light from only one "point" on the specimen at a time and the scanning could be carried out electronically by individually addressing the electrical output from each of the elements.
  • a linear charge coupled device is one well known way to achieve this.

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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

Un microscope confocal comprend une source de lumière (1) qui peut être un laser, pour générer un faisceau lumineux, et un transmetteur de faisceau lumineux (10) pour recevoir le faisceau lumineux et le transmettre à une lentille d'objectif (18) qui focalise la lumière sur un champ d'observation confiné sur un objet (20) devant être examiné. La lumière émanant de l'objet (20) ainsi illuminé retourne à travers la lentille d'objectif (18) et le transmetteur (10), et elle est détectée par un détecteur (34). La lentille d'objectif (18) est physiquement indépendante du transmetteur (10) de manière à être positionnable à un emplacement séparé du transmetteur (10) par un espace libre et un dispositif de pointage du faisceau (40) est prévu, pour pointer le faisceau lumineux provenant du transmetteur (10) vers la lentille d'objectif (18) à travers ledit espace libre.
PCT/AU1994/000053 1993-02-08 1994-02-08 Microscope confocal WO1994018592A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU59964/94A AU672876B2 (en) 1993-02-08 1994-02-08 Confocal microscope

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPL713993 1993-02-08
AUPL7139 1993-02-08

Publications (1)

Publication Number Publication Date
WO1994018592A1 true WO1994018592A1 (fr) 1994-08-18

Family

ID=3776690

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1994/000053 WO1994018592A1 (fr) 1993-02-08 1994-02-08 Microscope confocal

Country Status (1)

Country Link
WO (1) WO1994018592A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0862055A2 (fr) * 1997-02-27 1998-09-02 Basf Aktiengesellschaft Procédé et dispositif pour déterminer le degré de réticulation de couches d'adhésif à contact réticulé
WO1998038542A1 (fr) * 1997-02-24 1998-09-03 Bodenseewerk Perkin-Elmer Gmbh Dispositif de balayage lumineux
EP0951778A1 (fr) * 1997-01-09 1999-10-27 Interface Multigrad Technology (IMT) Ltd. Methode et dispositif pour surveiller un echantillon biologique
EP1240535A1 (fr) * 1999-11-16 2002-09-18 Ikonisys, Inc. Microscope de composition
US6548796B1 (en) 1999-06-23 2003-04-15 Regents Of The University Of Minnesota Confocal macroscope

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2152697A (en) * 1983-10-25 1985-08-07 Atomic Energy Authority Uk Improvements in or relating to scanning optical microscopes
AU3960889A (en) * 1988-07-13 1990-02-05 Optiscan Pty Limited Scanning confocal microscope
JPH03125108A (ja) * 1989-10-09 1991-05-28 Fuji Photo Film Co Ltd 共焦点走査型顕微鏡
AU7653191A (en) * 1990-04-06 1991-10-30 Optiscan Pty Limited Confocal microscope

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2152697A (en) * 1983-10-25 1985-08-07 Atomic Energy Authority Uk Improvements in or relating to scanning optical microscopes
AU3960889A (en) * 1988-07-13 1990-02-05 Optiscan Pty Limited Scanning confocal microscope
JPH03125108A (ja) * 1989-10-09 1991-05-28 Fuji Photo Film Co Ltd 共焦点走査型顕微鏡
AU7653191A (en) * 1990-04-06 1991-10-30 Optiscan Pty Limited Confocal microscope

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN, P-1242, page 157; & JP,A,3 125 108 (FUJI PHOTO FILM CO LTD), 28 May 1991. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0951778A1 (fr) * 1997-01-09 1999-10-27 Interface Multigrad Technology (IMT) Ltd. Methode et dispositif pour surveiller un echantillon biologique
EP0951778B1 (fr) * 1997-01-09 2005-10-19 Interface Multigrad Technology (IMT) Ltd. Methode et dispositif pour surveiller un echantillon biologique
WO1998038542A1 (fr) * 1997-02-24 1998-09-03 Bodenseewerk Perkin-Elmer Gmbh Dispositif de balayage lumineux
EP0862055A2 (fr) * 1997-02-27 1998-09-02 Basf Aktiengesellschaft Procédé et dispositif pour déterminer le degré de réticulation de couches d'adhésif à contact réticulé
EP0862055A3 (fr) * 1997-02-27 1999-03-24 Basf Aktiengesellschaft Procédé et dispositif pour déterminer le degré de réticulation de couches d'adhésif à contact réticulé
US6548796B1 (en) 1999-06-23 2003-04-15 Regents Of The University Of Minnesota Confocal macroscope
EP1240535A1 (fr) * 1999-11-16 2002-09-18 Ikonisys, Inc. Microscope de composition
EP1240535A4 (fr) * 1999-11-16 2005-07-20 Ikonisys Inc Microscope de composition

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