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WO1988007695A1 - Scanning confocal optical microscope - Google Patents

Scanning confocal optical microscope

Info

Publication number
WO1988007695A1
WO1988007695A1 PCT/US1988/000904 US8800904W WO1988007695A1 WO 1988007695 A1 WO1988007695 A1 WO 1988007695A1 US 8800904 W US8800904 W US 8800904W WO 1988007695 A1 WO1988007695 A1 WO 1988007695A1
Authority
WO
Grant status
Application
Patent type
Prior art keywords
light
object
disc
lens
microscope
Prior art date
Application number
PCT/US1988/000904
Other languages
French (fr)
Inventor
Gordon S. Kino
Guoqing Xiao
Original Assignee
The Board Of Trustees Of The Leland Stanford Junio
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

Links

Classifications

    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036Scanning details, e.g. scanning stages
    • G02B21/0044Scanning details, e.g. scanning stages moving apertures, e.g. Nipkow disks, rotating lens arrays
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/06Means for illuminating specimens
    • G02B21/08Condensers
    • G02B21/082Condensers for incident illumination only
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/06Means for illuminating specimens
    • G02B21/08Condensers
    • G02B21/10Condensers affording dark-field illumination

Abstract

A scanning confocal optical microscope using a Nipkow disc (21) having a plurality of pinholes to project light and an objective lens (26) to receive the light and illuminate points on an object to be viewed. Rotation of the disc scans the points across the object. The object is viewed by viewing the reflected light focused on the pinholes by the objective lens (26).

Description

- / -

SCANNING CONFOCAL OPTICAL MICROSCOPE

This invention relates generally to confocal optical microscopes and more particularly to a scanning confocal optical microscope.

Conventional confocal optical microscopes are" known for their extremely short depth of focus and improved trans¬ verse resolution. The main drawback with conventional confocal scanning optical microscopes is that they illumi¬ nate only one point on the object at a time. To scan the object either the sample or beam must be mechanically moved to form a raster image of the object. Mechanical scanning is time consuming.

In a paper appearing in The Journal of the Optical Society of America, Vol.58, No.5, May 1968, there is described a tandem-scanning reflected-light microscope. In the described microscope both the object plane and the image plane are scanned in tandem so that only light reflected from the object plane is included in the image. The object to be viewed is illuminated with light passing through a plurality of holes in a rotating disc which are focused onto the object by an objective lens. Thus, a large number of points on the object, corresponding to the holes which are illuminated, are illuminated at one time. The holes themselves are located along spiral paths. By rotating the disc a raster scan of the object is obtained. Reflected light from the illuminated spots is directed to the opposite side of the same apertured disc and passes through optically congruent holes on diametrically oppo¬ site sides of the rotating disc. The image obtained had better contrast and sharpness than possible with the usual reflected-light microscope. This is due to the fact that reflected light from within the microscope and other stray light coming from other points is intercepted by the opaque portions of the disc and the image only comprised reflection from the illuminated spots. The system of the prior art is extremely complex and difficult to align because of the necessity of assuring that the light reflected from the illuminated spots must pass through congruent holes on the opposite side of the rotating disc; Therefore, despite its obvious advantages, only two or three microscopes of this type have been constructed in the last 20 years.

The present invention employs the principals of a confocal optical microscope such as illustrated schematically in Figure 1. Light input from a laser, arc lamp or other light source impinges upon a plate 11 which includes a pinhole 12. The light travels throughthe pinhole to the objective lens 13 and is focused on the object plane 14. Light reflected from the object plane travels back through the pinhole as shown and can be viewed. Reflection from out of focus planes such as plane 16 does not converge at the pinhole and is therefore blocked by the plate 11. In operation the reflected light passing through the pinhole impinges upon an associated detector. The detector output is maximum when the object is located at the focus of the lens, otherwise the light received at the pinhole is defocused and the amplitude of the signal falls off rapidly as the position of the object is- changed on either side of the object plane. The system has a very short depth of focus in addition to excellent transverse defini- tion. As previously described, such a microscope can be mechani¬ cally scanned to form a raster image by moving the object or pinhole in a raster pattern.

It is a general object of the present invention to provide a scanning confocal microscope which overcomes the mechan¬ ical alignment problems of the prior art Tandem Scanning Microscope and which presents a total image of the object without the necessity of mechanical scanning of the light beam or object.

In accordance with the invention there is provided a scanning microscope for viewing an object which comprises a scanning disc with a plurality of holes arranged in a predetermined pattern and a light source for illuminating a portion of said disc. A polarizer receives the light from the source and polarizes the light prior to the light illuminating the disc. An objective lens focuses light diffracted through said plurality of illuminated holes onto an objective lens which focuses a plurality of spots on the object. The objective lens receives light reflect¬ ed from the object spots and focuses the light on the corresponding pin hole. A quarter wave plate is inter¬ posed between the objective lens and the disc.

A beam splitter receives the reflected light traveling through the holes and directs the light to a polarizer. Means are provided for rotating the disc to scan the spots across the object in a raster scan to provide an image of the region of the object illuminated by the spots.

The foregoing and other objects of the invention will be more clearly understood from the following description taken in connection with the drawings of which: Figure 1 shows a prior art confocal optical microscope.

Figure 2 shows' a scanning confocal optical microscope in accordance with the present invention.

Figure 3 shows another embodiment of the scanning confocal microscope in accordance with the invention.

Referring to Figure 2, the microscope includes a circular disc 21, known as a Nipkow disc which has holes disposed in a pattern of several interleaved spirals of several turns each. The holes are spaced approximately ten hole diameters apart. In one example the average radius of the spiral was five centimeters and the spiral extended over a radial distance of 1.8 centimeters with a total of 200,000 holes of 20 micrometer diameter formed in the periphery of the disc. Preferably the disc is of transparent material with a layer of black emulsion or black chrome in which the holes are formed by photo masking techniques.

A suitable light source, for example, either a mercury .arc lamp or a laser 22, is used to illuminate an area of- 1.8 centimeters by 1.8 centimeters containing about 4,000 holes. The light from the light source is polarized by a polarizer 23 and passes through a beam splitter 24 before striking the disc 21. The holes diffract the impinging light and the incident beam converges on objective lens 26. For example, the incident beam converges to a five millimeter diameter on the back of the objective lens 26 so that 4,000 points on the object 27 are simultaneously illuminated. Because of the spacing of the image points there is negligible interference between the different illuminated points.

The disc is rotated, for instance, at 2,000 rpm and since the holes are arranged along the spiral path the rotatio of the disc forms a raster scan across the object, thus forming 7,000 lines 500 frames per second.

The, light focused on the object is reflected and focused at the same pinholes by the objective lens after traveling through a quarter wave plate 28. The light passing through the holes strikes a beam .splitter 29 and is directed at right angles there from to an analyzer 31 and thence can be viewed by the naked eye through a transfer lens 32 which focuses on the pinholes.

In the embodiment of Figure 3, where like reference numerals are applied to like parts, the image travels through a relay lens to a camera 33 and to a TV monitor 34 which provides a display. Alternately the camera may be a photographic camera which takes a picture of the object. It is to be noted that no precise alignment is needed. The same holes are used for transmission and reception of the light making it relatively simple to align the microscope. The eyepiece, or camera, is focused on the pinholes illuminated by reflected light from behind the pinholes. Centering of the spiral is not critical. Vibration is not a severe problem provided that disc vibration has an amplitude much less than the depth of focus of the lenses at the pinholes, typically a distance greater than 1 millimeter.

The use of the polarizers and a quarterwave plate reduces interference from reflections from the disc as well as as the surfaces of the microscope elements by a factor of the order of lθ"* intensity and provide a clear visual image.

With reference to Figure 3 the apparatus also includes a light stop which is located at the focal point of the incident light reflected from the surface of the Nipkow disc.

The purpose of this stop is to * completely remove all remaining reflected light from the disc. When a laser source is employed, the stop is placed at the focus of the reflected converging beam. With a lamp source, an image of the source is focused on the stop. With an incoherent light source of finite diameter, the stop must be larger than it would need to be with a coherent light source. Nevertheless the stop would only intercept a very small fraction of the area of the beam passing through the pinholes to the observer. The microscope can be constructed with solely a light stop, and this may be advantageous when an object having a low reflectivity is being viewed.

The object or the objective lens can be moved up or down in order to focus on various planes in a translucent object such as a biological material like bone, or at various levels of a sample such as an integrated circuit. A very similar system can be used as a range sensor in robotic applications.

The Nipkow disc is illuminated by a collimated laser beam with, perhaps, as much as several watts in the beam. The pinholes in the disc are viewed as described in Figure 3. Images of the pinhole are focused on a distant plane by a camera lens. The beam, after leaving the pinhole, may be expanded by a telescope to give a larger beam at the lens pupil. This is to make it possible to use a lens of larger aperture so as to obtain the optimum transverse and range definitions. When the' Nipkow disc is rotated a region at the focal plane of the lens is imaged. Thus a direct real tim image of a plane at a fixed distance from the lens is obtained in real time. Images of other cross sections are easily obtained by moving the lens back and, forth. This can be used to control a robot.

Claims

What is claimed is:
1. A scanning microscope for viewing an object com¬ prising- a scanning disc with a plurality of pinholes arranged in a predetermined pattern, a 'light source on one side of said disc for illuminating an area of said disc, a first polarizer for polarizing the light which illuminates the disc, an objective lens on the opposite side of said disc for focusing light diffracted by said pinholes onto the object to illum¬ inate a plurality of points on said object, means for rotating said disc to scan the points across the object, said objective lens serving to receive light reflected from the object and focus the light on corresponding pinholes, a quarterwave plate interposed between said objective lens and disc to intercept the light travelling to and reflected from said object, a beam splitter on said one side of said disc between said first polarized and said quarterwave plate for receiving reflected light travelling through said pinholes and said quarterwave plate and directing it at an angle, and a polarizer for receiving the directed light.
2. A scanning microscope as in Claim 1 including a light stop disposed to stop light reflected from the disc.
3. A scanning microscope as in Claim 1 in which the holes are disposed in a pattern of several interleaved spirals of several turns each.
4. A scanning microscope as in Claim 1 in which the disc includes an apertured dark layer on the surface of a trans¬ parent disc.
PCT/US1988/000904 1987-03-27 1988-03-24 Scanning confocal optical microscope WO1988007695A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US3151687 true 1987-03-27 1987-03-27
US031,516 1987-03-27

Publications (1)

Publication Number Publication Date
WO1988007695A1 true true WO1988007695A1 (en) 1988-10-06

Family

ID=21859900

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1988/000904 WO1988007695A1 (en) 1987-03-27 1988-03-24 Scanning confocal optical microscope

Country Status (2)

Country Link
JP (1) JP2836829B2 (en)
WO (1) WO1988007695A1 (en)

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5127730A (en) * 1990-08-10 1992-07-07 Regents Of The University Of Minnesota Multi-color laser scanning confocal imaging system
EP0646768A2 (en) * 1993-09-29 1995-04-05 Ushiodenki Kabushiki Kaisha Confocal optical microscope and length measuring device using this microscope
DE19511937A1 (en) * 1995-03-31 1996-10-02 Zeiss Carl Jena Gmbh Confocal illuminating microscope with inclined shadow mask
US6134009A (en) * 1997-11-07 2000-10-17 Lucid, Inc. Imaging system using polarization effects to enhance image quality
US6134010A (en) * 1997-11-07 2000-10-17 Lucid, Inc. Imaging system using polarization effects to enhance image quality
EP1617251A1 (en) 2004-07-16 2006-01-18 CARL ZEISS JENA GmbH Zoom lens for a light scanning microscope having a point light source and its use
EP1617249A1 (en) * 2004-07-16 2006-01-18 CARL ZEISS JENA GmbH Light scanning microscope with point-like intensity distribution and use thereof
EP1617252A1 (en) 2004-07-16 2006-01-18 CARL ZEISS JENA GmbH Scanning microscope with punctiform source light distribution and use
EP1617253A1 (en) 2004-07-16 2006-01-18 CARL ZEISS JENA GmbH Scanning microscope with linear source light distribution and use
EP1617263A1 (en) 2004-07-16 2006-01-18 Carl Zeiss Jena Gmbh Scanning optical microscope and method of using it
US7271382B2 (en) 2004-07-16 2007-09-18 Carl Zeiss Jena Gmbh Process for the observation of at least one sample region with a light raster microscope with light distribution in the form of a point
US7270431B2 (en) 2004-07-16 2007-09-18 Carl Zeiss Jena Gmbh Illumination device and its use
US7304280B2 (en) 2004-07-16 2007-12-04 Carl Zeiss Jena Gmbh Laser line scanning microscope and method of use
US7323679B2 (en) 2004-07-16 2008-01-29 Carl Zeiss Jena Gmbh Procedure for the optical acquisition of objects by means of a light raster microscope with line by line scanning
US7369305B2 (en) 2004-07-16 2008-05-06 Carl Zeiss Jena Gmbh Optical zoom system for a light scanning electron microscope
EP1944642A2 (en) 2004-07-16 2008-07-16 Carl-Zeiss Jena GmbH Light raster microscope
US7459698B2 (en) 2004-07-16 2008-12-02 Carl Zeiss Microimaging Gmbh Process for the observation of at least one sample region with a light raster microscope
US7488931B2 (en) 2004-07-16 2009-02-10 Carl Zeiss Jena Gmbh Optical zoom system for a light scanning electron microscope
US7561326B2 (en) 2004-07-16 2009-07-14 Carl Zeiss Microimaging Gmbh Light scanning microscope and use
US7593581B2 (en) 2004-07-16 2009-09-22 Carl Zeiss Micro Imaging Gmbh Process for the acquisition of images from a sample with a microscope
US7649683B2 (en) 2004-07-16 2010-01-19 Carl Zeiss Microimaging Gmbh Process for the observation of at least one sample region with a light raster microscope with linear sampling
USRE43702E1 (en) 2004-07-16 2012-10-02 Carl Zeiss Microimaging Gmbh Microscope with heightened resolution and linear scanning

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2663766B2 (en) * 1991-10-31 1997-10-15 横河電機株式会社 Confocal optical scanner
JP2970223B2 (en) * 1992-06-09 1999-11-02 横河電機株式会社 Confocal optical scanner
JPH09264720A (en) * 1996-03-29 1997-10-07 Komatsu Ltd Cofocal optical device
JPH09274139A (en) * 1996-04-08 1997-10-21 Komatsu Ltd Confocal optical device
JPH09288444A (en) * 1996-04-23 1997-11-04 Komatsu Ltd Confocal optical device and hologram exposure device
JP2005195739A (en) * 2004-01-05 2005-07-21 Nikon Corp Confocal optical system and height measuring apparatus

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US4062623A (en) * 1975-04-07 1977-12-13 Canon Kabushiki Kaisha Device for observing an object
US4251129A (en) * 1977-10-05 1981-02-17 Canon Kabushiki Kaisha Photoelectric detecting device

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JPS638402B2 (en) * 1977-10-05 1988-02-23 Canon Kk
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US3517980A (en) * 1966-12-05 1970-06-30 Ceskoslovenska Akademie Ved Method and arrangement for improving the resolving power and contrast
US3926500A (en) * 1974-12-02 1975-12-16 Ibm Method of increasing the depth of focus and or the resolution of light microscopes by illuminating and imaging through a diaphragm with pinhole apertures
US4062623A (en) * 1975-04-07 1977-12-13 Canon Kabushiki Kaisha Device for observing an object
US4251129A (en) * 1977-10-05 1981-02-17 Canon Kabushiki Kaisha Photoelectric detecting device

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5127730A (en) * 1990-08-10 1992-07-07 Regents Of The University Of Minnesota Multi-color laser scanning confocal imaging system
EP0646768A2 (en) * 1993-09-29 1995-04-05 Ushiodenki Kabushiki Kaisha Confocal optical microscope and length measuring device using this microscope
EP0646768A3 (en) * 1993-09-29 1997-05-14 Ushio Electric Inc Confocal optical microscope and length measuring device using this microscope.
DE19511937A1 (en) * 1995-03-31 1996-10-02 Zeiss Carl Jena Gmbh Confocal illuminating microscope with inclined shadow mask
US6134009A (en) * 1997-11-07 2000-10-17 Lucid, Inc. Imaging system using polarization effects to enhance image quality
US6134010A (en) * 1997-11-07 2000-10-17 Lucid, Inc. Imaging system using polarization effects to enhance image quality
US6577394B1 (en) 1997-11-07 2003-06-10 Lucid, Inc. Imaging system using polarization effects to enhance image quality
US6710875B1 (en) 1997-11-07 2004-03-23 Lucid, Inc. Imaging system using polarization effects to enhance image quality
US7235777B2 (en) 2004-07-16 2007-06-26 Carl Zeiss Jena Gmbh Light scanning microscope and use
EP1617249A1 (en) * 2004-07-16 2006-01-18 CARL ZEISS JENA GmbH Light scanning microscope with point-like intensity distribution and use thereof
EP1617252A1 (en) 2004-07-16 2006-01-18 CARL ZEISS JENA GmbH Scanning microscope with punctiform source light distribution and use
EP1617253A1 (en) 2004-07-16 2006-01-18 CARL ZEISS JENA GmbH Scanning microscope with linear source light distribution and use
EP1617263A1 (en) 2004-07-16 2006-01-18 Carl Zeiss Jena Gmbh Scanning optical microscope and method of using it
EP1617251A1 (en) 2004-07-16 2006-01-18 CARL ZEISS JENA GmbH Zoom lens for a light scanning microscope having a point light source and its use
US7271382B2 (en) 2004-07-16 2007-09-18 Carl Zeiss Jena Gmbh Process for the observation of at least one sample region with a light raster microscope with light distribution in the form of a point
US7270431B2 (en) 2004-07-16 2007-09-18 Carl Zeiss Jena Gmbh Illumination device and its use
US7304280B2 (en) 2004-07-16 2007-12-04 Carl Zeiss Jena Gmbh Laser line scanning microscope and method of use
US7323679B2 (en) 2004-07-16 2008-01-29 Carl Zeiss Jena Gmbh Procedure for the optical acquisition of objects by means of a light raster microscope with line by line scanning
US7369305B2 (en) 2004-07-16 2008-05-06 Carl Zeiss Jena Gmbh Optical zoom system for a light scanning electron microscope
EP1944642A2 (en) 2004-07-16 2008-07-16 Carl-Zeiss Jena GmbH Light raster microscope
US7459698B2 (en) 2004-07-16 2008-12-02 Carl Zeiss Microimaging Gmbh Process for the observation of at least one sample region with a light raster microscope
US7488931B2 (en) 2004-07-16 2009-02-10 Carl Zeiss Jena Gmbh Optical zoom system for a light scanning electron microscope
US7561326B2 (en) 2004-07-16 2009-07-14 Carl Zeiss Microimaging Gmbh Light scanning microscope and use
US7593581B2 (en) 2004-07-16 2009-09-22 Carl Zeiss Micro Imaging Gmbh Process for the acquisition of images from a sample with a microscope
US7649683B2 (en) 2004-07-16 2010-01-19 Carl Zeiss Microimaging Gmbh Process for the observation of at least one sample region with a light raster microscope with linear sampling
USRE43702E1 (en) 2004-07-16 2012-10-02 Carl Zeiss Microimaging Gmbh Microscope with heightened resolution and linear scanning

Also Published As

Publication number Publication date Type
JPH01503493A (en) 1989-11-22 application
JP2836829B2 (en) 1998-12-14 grant

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