WO1994018593A1 - Detecting means for a scanning optical microscope - Google Patents

Detecting means for a scanning optical microscope Download PDF

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Publication number
WO1994018593A1
WO1994018593A1 PCT/GB1994/000249 GB9400249W WO9418593A1 WO 1994018593 A1 WO1994018593 A1 WO 1994018593A1 GB 9400249 W GB9400249 W GB 9400249W WO 9418593 A1 WO9418593 A1 WO 9418593A1
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WO
WIPO (PCT)
Prior art keywords
detecting means
microscope
light
optical
sensors
Prior art date
Application number
PCT/GB1994/000249
Other languages
French (fr)
Inventor
William Bradshaw Amos
Original Assignee
Medical Research Council
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 Medical Research Council filed Critical Medical Research Council
Publication of WO1994018593A1 publication Critical patent/WO1994018593A1/en

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/1006—Beam splitting or combining systems for splitting or combining different wavelengths
    • G02B27/1013—Beam splitting or combining systems for splitting or combining different wavelengths for colour or multispectral image sensors, e.g. splitting an image into monochromatic image components on respective sensors
    • 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
    • G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/145—Beam splitting or combining systems operating by reflection only having sequential partially reflecting surfaces

Definitions

  • This invention relates to a detecting means for a scanning optical microscope.
  • the scanning optical microscope has been developed in a confocal epi-illumination form (White, GB Patent Specification 2184321A and US Patent 5032720) in which a laser is used as the source of illumination.
  • a laser is used as the source of illumination.
  • a particularly effective laser for this purpose is the argon-krypton mixed gas laser, which provides blue, yellow and red wavelengths, simultaneously.
  • the beam is frequently scanned by means of mirrors and the various wavelengths are therefore not appreciably dispersed: they fall upon the same spot in the specimen at any one time.
  • a detecting means for detecting light from a polychromatic source such as a lamp or multiline laser or combination of lasers, the detecting means comprising a plurality of optical sensors for responding to different wavelengths of light, the sensors being placed in such a position that they can receive the light transmitted through a specimen by a scanning optical microscope.
  • the detecting means may comprise three optical sensors, each responding to a different component of the light, corresponding to a different range of wavelengths.
  • the detecting means preferably comprise beam splitter plates for splitting light from the specimen into the different components and directing the light components onto the respective sensors, conveniently photodiodes.
  • These sensors are used to detect signals, preferably simultaneously, from which a colour transmission image may then be generated by electronic means.
  • the invention also provides a scanning optical microscope assembly having a polychromatic source, a scanning head, a microscope having an eyepiece, an optical objective, and a condenser lens for receiving light transmitted through a specimen scanned with light of different wavelengths through the objective, and a detecting means comprising a plurality of optical sensors for responding to different wavelengths of light received from the condenser lens in the microscope.
  • the drawing shows a scanning, confocal, optical microscope assembly comprising a laser light source 10, a scan head 12, a microscope 14, a detecting means 16, and a display unit 18.
  • Polychromatic light from a laser system constituting the source 10 which is preferably a multiline laser or a number of lasers, enters the scan head 12, which incorporates a dichroic mirror 20, from which light passes via fixed mirror 22 and scanning mirror 24 into the microscope 14.
  • the latter has an eyepiece 26, optical objective 28, and specimen site 30.
  • scanning mirror 24 is for simplicity shown as a single mirror, in practice a more complex arrangement of rotating or oscillating mirrors will be employed to produce scanning in two dimensions.
  • Eyepiece 26 may also be a more complex lens system.
  • the optical objective 28 focusses a scanning beam of multi-wavelength light on the specimen site 30.
  • the complete apparatus as thus far described comprises a substantially conventional scanning optical microscope, preferably having confocal detection.
  • the microscope assembly can also detect light transmitted through a specimen at site 30.
  • transmitted light passes through a condenser lens 38 in the microscope to a mirror 40, and thence through lens or lens system 42 to the detecting means 16.
  • Mirror 40 can be moved aside, as indicated by arrow 44, when the detecting means 16 is not required for use, for example to allow transmitted light to fall on a conventional detector analogous to the detector 36.
  • the polychromatic light entering the detecting means 16 is divided into three components, each covering a different wavelength, by beam splitting plates 46, 48, and each light component is focussed by optics 50, 52, 54 on to an optical sensor 56, 58, 60 responsive to light in the wavelength range of the corresponding component.
  • the outputs of the sensors 56, 58, 60 can be utilised in other ways, in the illustrated embodiment these outputs are fed to the display unit 18, which comprises substantially conventional electronic processing circuitry 62 producing signals fed to a colour monitor 64, on which is displayed a colour transmission image characteristic of light absorption in the specimen.
  • the lens 42 at the point of beam entry to the detecting means 16, is used to make the telecentric points in the microscope 14 confocal with the optical sensors 56, 58, 60. Accordingly, the lens 42 may comprise more complex optics than is illustrated. The lens 42 thus ensures that, when no specimen is present, each sensor 56, 58, 60 receives approximately the same intensity of light throughout the scanning cycle.
  • the preferred source of illumination 10 is an Argon/Krypton mixed gas multiline laser, used in conjunction with chromatic splitters which achieve separation of the red, yellow and blue components of the light from this laser.
  • chromatic splitters which achieve separation of the red, yellow and blue components of the light from this laser.
  • electronic means which activate red, red plus green and blue phosphors respectively for the three channels.
  • Lasers, or combinations of lasers, providing red, green and blue light lend themselves to display even more simply by using the appropriae phosphor for each channel.
  • This invention is not restricted to three wavelength- ranges within the visible spectrum. It can be modified to cover wavelengths outside the visible spectrum, for instance in the ultra-violet and infra-red.
  • the resulting images may be displayed in conventional red, green and blue colours, forming false-colour transmission images.
  • an absorption image in the ultra-violet can be displayed using one visible colour, while others are used to display the " simultaneous absorption images formed by light of a different wavelength.
  • the invention is also applied to coloured transmission imaging in cases where the colour is not due to absorption, but to other effects, such as scattering, birefringence, dichroism, dispersion staining, Rheinberg illumination or the use of interference optics.
  • analogue electronic means particularly the use of offset and gain controls in amplifiers and signal subtraction and differential methods, used in the handling of the signal, or by digital methods, the colour images formed are readily made more sensitive than those formed by the unaided human eye, both in respect of the detection of th'e degree of colour saturation and the distinction of hues. This provides assistance to histological observation and demonstration.
  • the invention allows the colour transmission image to benefit from valuable properties such as zooming of magnification and panning of position, which result from manipulation of the size and position of the scanned raster on the specimen.

Abstract

A scanning, confocal, optical microscope assembly has a detecting means (16) for receiving multi-wavelength light transmitted through a specimen located in the microscope (14). This detecting means (16) comprises beam splitters (46, 48) for splitting the multi-wavelength transmitted light into three components corresponding to different wavelength ranges, and three optical sensors (56, 58, 60) for receiving the respective components. The outputs of the sensors may be processed and a full transmission colour image displayed on a television monitor (64).

Description

Title: Detecting Means for a Scanning Optical Microscope
Field of the Invention
This invention relates to a detecting means for a scanning optical microscope.
Background to the Invention
Young and Roberts described in 1951 (A Flying Spot Microscope, Nature 167, p231) a scanning optical microscope in which a spot of light was caused to move in a raster fashion over a specimen and the light transmitted by the specimen was collected in a non-imaging detector (a photocell). At the same time, a spot was moved in a raster on a cathode ray tube and the brightness of this displayed spot was modulated by the photocell output in such a way as to provide an image of the specimen, showing regions of absorption or scattering of the light. The use of a single detector to generate a transmission image in this way is a well established piece of optical technology, which has also been used in non-microscopic scanning in connection with television signals. Thus Evans described (Television optics, Chapter 7, pp. 79-309 in Applied Optics and Optical Engineering 2., ed. R. Kingslake Academic Press Inc. New York & London, 1965) a microscopic scanner in which a spot of light is scanned over a photographic transparency and the transmitted light is focussed on to a detector, the output of which is used to modulate a television signal. Of particular relevance in the present connection is the form of this apparatus described by Evans for colour television, in which white light is used to scan the specimen and the transmitted light is then passed through chromatic beam splitters in order to divide it into red, green and blue components which are directed separately to three detectors. The electronic signals are ultimately combined to form a television image in full colour.
The scanning optical microscope has been developed in a confocal epi-illumination form (White, GB Patent Specification 2184321A and US Patent 5032720) in which a laser is used as the source of illumination. For biological applications such systems are normally used with a multiline laser, providing illumination at a number of wavelengths. A particularly effective laser for this purpose is the argon-krypton mixed gas laser, which provides blue, yellow and red wavelengths, simultaneously. In such scanning microscopes the beam is frequently scanned by means of mirrors and the various wavelengths are therefore not appreciably dispersed: they fall upon the same spot in the specimen at any one time.
It is already accepted practice to form multiple confocal images in different colours of fluorescence or reflection simultaneously. However, present apparatus does not provide for the production of a full-colour absorption image. This is a useful addition to the capabilities of a scanning optical microscope, since it allows visualisation of conventionally stained specimens in familiar transmission colours. It is then possible to switch quickly to epi-illumination modes of microscopy such as confocal fluorescence and reflection. The Invention
According to the present invention, there is provided a detecting means for detecting light from a polychromatic source such as a lamp or multiline laser or combination of lasers, the detecting means comprising a plurality of optical sensors for responding to different wavelengths of light, the sensors being placed in such a position that they can receive the light transmitted through a specimen by a scanning optical microscope.
The detecting means may comprise three optical sensors, each responding to a different component of the light, corresponding to a different range of wavelengths. The detecting means preferably comprise beam splitter plates for splitting light from the specimen into the different components and directing the light components onto the respective sensors, conveniently photodiodes.
These sensors are used to detect signals, preferably simultaneously, from which a colour transmission image may then be generated by electronic means.
The invention also provides a scanning optical microscope assembly having a polychromatic source, a scanning head, a microscope having an eyepiece, an optical objective, and a condenser lens for receiving light transmitted through a specimen scanned with light of different wavelengths through the objective, and a detecting means comprising a plurality of optical sensors for responding to different wavelengths of light received from the condenser lens in the microscope. Description of Drawing
A preferred embodiment of the invention is shown in the accompanying drawing.
The drawing shows a scanning, confocal, optical microscope assembly comprising a laser light source 10, a scan head 12, a microscope 14, a detecting means 16, and a display unit 18.
Polychromatic light from a laser system constituting the source 10, which is preferably a multiline laser or a number of lasers, enters the scan head 12, which incorporates a dichroic mirror 20, from which light passes via fixed mirror 22 and scanning mirror 24 into the microscope 14. The latter has an eyepiece 26, optical objective 28, and specimen site 30. Although scanning mirror 24 is for simplicity shown as a single mirror, in practice a more complex arrangement of rotating or oscillating mirrors will be employed to produce scanning in two dimensions. Eyepiece 26 may also be a more complex lens system. The optical objective 28 focusses a scanning beam of multi-wavelength light on the specimen site 30.
When a specimen is in place, light emitted by reflection, fluorescence or luminescence passes back along the path of the incident light, is descanned and falls on the dichroic mirror 20. Some of the return light passes through this beam splitter 20 and via ancillary optics such as mirror 32 and iris diaphragm 34 reaches a detector 36.
The complete apparatus as thus far described comprises a substantially conventional scanning optical microscope, preferably having confocal detection.
In accordance with the present invention, the microscope assembly can also detect light transmitted through a specimen at site 30. For this purpose, such transmitted light passes through a condenser lens 38 in the microscope to a mirror 40, and thence through lens or lens system 42 to the detecting means 16.
Mirror 40 can be moved aside, as indicated by arrow 44, when the detecting means 16 is not required for use, for example to allow transmitted light to fall on a conventional detector analogous to the detector 36.
The polychromatic light entering the detecting means 16 is divided into three components, each covering a different wavelength, by beam splitting plates 46, 48, and each light component is focussed by optics 50, 52, 54 on to an optical sensor 56, 58, 60 responsive to light in the wavelength range of the corresponding component.
Although the outputs of the sensors 56, 58, 60 can be utilised in other ways, in the illustrated embodiment these outputs are fed to the display unit 18, which comprises substantially conventional electronic processing circuitry 62 producing signals fed to a colour monitor 64, on which is displayed a colour transmission image characteristic of light absorption in the specimen.
The lens 42, at the point of beam entry to the detecting means 16, is used to make the telecentric points in the microscope 14 confocal with the optical sensors 56, 58, 60. Accordingly, the lens 42 may comprise more complex optics than is illustrated. The lens 42 thus ensures that, when no specimen is present, each sensor 56, 58, 60 receives approximately the same intensity of light throughout the scanning cycle.
The preferred source of illumination 10 is an Argon/Krypton mixed gas multiline laser, used in conjunction with chromatic splitters which achieve separation of the red, yellow and blue components of the light from this laser. In displaying the colour image from such a system on a television monitor more accurate rendition of colours is achieved by electronic means which activate red, red plus green and blue phosphors respectively for the three channels. Lasers, or combinations of lasers, providing red, green and blue light lend themselves to display even more simply by using the appropriae phosphor for each channel.
This invention is not restricted to three wavelength- ranges within the visible spectrum. It can be modified to cover wavelengths outside the visible spectrum, for instance in the ultra-violet and infra-red. The resulting images may be displayed in conventional red, green and blue colours, forming false-colour transmission images. For example, an absorption image in the ultra-violet can be displayed using one visible colour, while others are used to display the "simultaneous absorption images formed by light of a different wavelength.
The invention is also applied to coloured transmission imaging in cases where the colour is not due to absorption, but to other effects, such as scattering, birefringence, dichroism, dispersion staining, Rheinberg illumination or the use of interference optics. By analogue electronic means, particularly the use of offset and gain controls in amplifiers and signal subtraction and differential methods, used in the handling of the signal, or by digital methods, the colour images formed are readily made more sensitive than those formed by the unaided human eye, both in respect of the detection of th'e degree of colour saturation and the distinction of hues. This provides assistance to histological observation and demonstration.
The invention allows the colour transmission image to benefit from valuable properties such as zooming of magnification and panning of position, which result from manipulation of the size and position of the scanned raster on the specimen. These advantages were hitherto available only for monochrome transmission images.

Claims

C l a ims
1. A detecting means for a scanning optical microscope having a polychromatic source, wherein the detecting means is positioned to receive light from the source transmitted through a specimen placed in the microscope, and said detecting means comprises a plurality of optical sensors for responding to different wavelengths of light.
2. A detecting means according to claim 1, wherein the detecting means comprises three optical sensors each responding to a different component of the transmitted light, each component corresponding to a different range of wavelengths.
3. A detecting means according to claim 1 or claim 2, wherein the detecting means includes beam splitters for splitting the transmitted light into different components and directing the light components on to the respective sensors.
4. A detecting means according to claim 3, wherein optical means is provided between the microscope and the beam splitters to cause telecentric points in the microscope to be confocal with the optical sensors.
5. A detecting means according to any of claims 1 to 4, in combination with electronic means for processing the outputs of the sensors and a display means for receiving the processed signals and generating a colour transmission image.
6. A scanning optical microscope assembly having a polychromatic source, a scanning head, a microscope having an eyepiece, an- optical objective, and a condenser lens for receiving light transmitted through a specimen scanned with light of different wavelengths through the objective, and a detecting means comprising a plurality of optical sensors for responding to different wavelengths of light received from the condenser lens in the microscope.
7. A microscope assembly according to claim 6, wherein the source comprises a multiline laser.
8. A microscope assembly according to claim 6 or claim
7, wherein a reflector is provided for directing light to the detecting means from the microscope, and said reflector is movable out of the path of the light transmitted through the condenser lens when the detecting means is not required for use.
9. A microscope assembly according to any of claims 6 to
8, wherein the detecting means includes beam splitters for splitting the light received from the condenser lens in the microscope into components corresponding to different ranges of wavelengths and for directing the components to the respective optical sensors, which produce outputs appropriate for generation of a colour transmission image.
10. A microscope assembly according to claim 9, including an electronic means for processing the output signals from the sensors and a display means for receiving the processed signals and generating a colour transmission image.
PCT/GB1994/000249 1993-02-12 1994-02-09 Detecting means for a scanning optical microscope WO1994018593A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB939302799A GB9302799D0 (en) 1993-02-12 1993-02-12 Detecting means for a scanning optical microscope
GB9302799.3 1993-02-12

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WO1994018593A1 true WO1994018593A1 (en) 1994-08-18

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0841557A2 (en) * 1996-10-11 1998-05-13 Bio-Rad Laboratories, Inc. Tunable excitation and/or tunable detection microplate reader
WO1999052005A1 (en) * 1998-04-03 1999-10-14 Bio-Rad Laboratories, Inc. Apparatus and methods for fourier spectral analysis in a scanning spot microscope
EP1128200A2 (en) * 2000-01-27 2001-08-29 Leica Microsystems Heidelberg GmbH Microscope structure
DE102008062650A1 (en) * 2008-12-17 2010-07-15 Carl Zeiss Surgical Gmbh Surgical microscope for observing infrared fluorescence and method therefor

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EP0176358A2 (en) * 1984-09-28 1986-04-02 Lasertec Corporation Image pick-up apparatus
EP0327425A1 (en) * 1988-01-27 1989-08-09 Commissariat A L'energie Atomique Method for optical scanning microscopy in confocal arrangement with large depth of field and apparatus to perform this method
EP0418928A2 (en) * 1989-09-22 1991-03-27 Fuji Photo Film Co., Ltd. Scanning microscope and scanning mechanism for the same
WO1992002839A1 (en) * 1990-08-10 1992-02-20 Regents Of The University Of Minnesota Laser for confocal microscope
EP0548699A1 (en) * 1991-12-12 1993-06-30 Matsushita Electric Industrial Co., Ltd. Scanning laser microscope with photo coupling and detecting unit

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0176358A2 (en) * 1984-09-28 1986-04-02 Lasertec Corporation Image pick-up apparatus
EP0327425A1 (en) * 1988-01-27 1989-08-09 Commissariat A L'energie Atomique Method for optical scanning microscopy in confocal arrangement with large depth of field and apparatus to perform this method
EP0418928A2 (en) * 1989-09-22 1991-03-27 Fuji Photo Film Co., Ltd. Scanning microscope and scanning mechanism for the same
WO1992002839A1 (en) * 1990-08-10 1992-02-20 Regents Of The University Of Minnesota Laser for confocal microscope
EP0548699A1 (en) * 1991-12-12 1993-06-30 Matsushita Electric Industrial Co., Ltd. Scanning laser microscope with photo coupling and detecting unit

Non-Patent Citations (1)

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Title
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0841557A2 (en) * 1996-10-11 1998-05-13 Bio-Rad Laboratories, Inc. Tunable excitation and/or tunable detection microplate reader
WO1999052005A1 (en) * 1998-04-03 1999-10-14 Bio-Rad Laboratories, Inc. Apparatus and methods for fourier spectral analysis in a scanning spot microscope
US6853455B1 (en) 1998-04-03 2005-02-08 Bio-Rad Laboratories, Inc. Apparatus and methods for fourier spectral analysis in a scanning spot microscope
EP1128200A2 (en) * 2000-01-27 2001-08-29 Leica Microsystems Heidelberg GmbH Microscope structure
JP2001235683A (en) * 2000-01-27 2001-08-31 Leica Microsystems Heidelberg Gmbh Microscope assembly
EP1128200A3 (en) * 2000-01-27 2002-05-15 Leica Microsystems Heidelberg GmbH Microscope construction
DE102008062650A1 (en) * 2008-12-17 2010-07-15 Carl Zeiss Surgical Gmbh Surgical microscope for observing infrared fluorescence and method therefor
US8659651B2 (en) 2008-12-17 2014-02-25 Carl Zeiss Meditec Ag Surgical microscope for observing an infrared fluorescence and corresponding method
DE102008062650B4 (en) 2008-12-17 2021-08-12 Carl Zeiss Meditec Ag Surgical microscope for observing infrared fluorescence and methods for this
DE102008062650B9 (en) 2008-12-17 2021-10-28 Carl Zeiss Meditec Ag Surgical microscope for observing infrared fluorescence and methods for this

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