CONFOCAL IMAGING SYSTEM FOR MICROSCOPY

Abstract: A confocal imaging system for use in conjunction with an optical microscope, in which a slit-shaped or bar-shaped beam of light is scanned over the specimen, descanned with a fixed mask and rescanned for viewing or recording, the focussing and scanning system being constituted by wholly reflective optical systems (A1, B1 and A2, B2).

Description: Title Confocal Imaging System for Microscopy Field of the invention This invention relates to a confocal imaging system for microscopy and, in particular, to such a system which utilises slit scanning. Prior art The principle of confocal imaging was first described by Minsky (see U.S. Patent No. 3,013,467). Moving-slit, confocal imaging systems have been described by Baer (U.S. Patent No. 3,547,512 and Lichtman (see Lichtman, J.W., Sunderland, .J., and Wilkinson, R.S. (1989) in The New Biologist 1, 75-82. Also, forms of confocal microscope have been described in which a slit or bar pattern of light is scanned across the specimen and an image of this pattern formed by reflection or emission from the specimen is caused to fall on a stationary slit-shaped aperture. This use of a stationary slit is a feature of the systems described by Koester (Koester, C.J. (1980) Appl. Optics 19, 1749-1757., Burns et al. (Burns, D.H., Hatangadi. R.B. and Spelman, F.A. (1990) Scanning, 12, 156-160) and of the design of Brakenhoff and Visscher (Brakenhoff, G.J. & Visscher, K. (1990) Trans. Roy. microsc. Soc. 1^, 247- 250) . A reflecting autocolli ating system with no primary aberrations was described by Offner (see A. Offner, Optical Engineering (1975), 14*, 131). Background to the invention _ -> — Confocal imaging systems have become established as an effective means of eliminating out-of-focus interference in optical microscopy. In these systems only a very small area in the object plane is illuminated at any given time. The illumination in the object plane may be in the form of a single point, an array of points, a single line or an array of lines. A mask with the same geometrical form as the illuminating pattern is incorporated in a plane conjugate with the object plane, so that only signals from the immediate vicinity of the illuminated regions are admitted through the mask into the viewing system. In this way, interfering signals emanating away from the illuminated regions are rejected. A complete image is built up by scanning the illuminating pattern in such a way as to cover the whole of the area of the object plane, whilst keeping the mask in register with the illumination. There are advantages in using a slit or array of slits rather than a single point as the pattern of illumination. For example, a complete image can be formed more rapidly and the instantaneous intensity of illumination required at any single point in the specimen can be reduced. An increase in the rate of formation of images (framing rate) is of particular value, since it can be used with direct visual observation to create the impression of continuous imaging. If a slit is used, it is desirable to be able to vary the width of the slit in the mask relative to that of the illuminated slit-shaped area. In some prior art, such as that of Lichtman, this is impossible, since one and the same mask serves to define both the illumination and the area of detection. The closest prior art, in this respect, is that of Koester, of Burns et al. and of Brakenhoff and Visscher, where the system is so constructed that the detection mask is stationary, so that the mask is easy to adjust in a continuous fashion during the operation of the microscope system. Koester' s system fails to employ the full aperture of the objective lens of the microscope and is therefore compromised in image quality. The system of Baer depends on chromatic dispersion for its operation and is therefore not suitable for the main field of application of confocal microscopes, which is the examination of fluorescence in specimens. The closest prior art (ie that of Burns et al. and Visscher and Brakenhoff) requires lenses to relay the image within the apparatus. This presents considerable problems since the chromatic performance of lenses is poor outside the restricted range of wavelengths for which they are designed. Also, in order to make the instrument conveniently small, the relay lenses must have high numerical apertures, which makes them difficult to design and expensive to manufacture. The invention According to the present invention, there is provided, in combination with a microscope, a confocal imaging system comprising means for forming a slit-shaped or bar-shaped illuminating beam, or an array of such beams, a beam- splitting means by which the light is directed into an optical microscope having an objective lens, and an optical scanning means means whereby the illuminating beam or beams is or are caused to scan and be directed into the objection lens of the microscope in order to scan the specimen, such optical scanning means consisting of a wholly reflective optical system for both focussing and scanning . Preferably, the same reflective optical system causes the emitted or reflected beam to be descanned (ie brought to a stationary state) and focussed upon a stationary confocal mask such as a slit of variable width, or an array of such slits. The stationary slit image delimited by the mask can then be rescanned by a second wholly reflective optical system and brought to focus within an eyepiece or upon the photosensitive surface of a video camera as a two- dimensional image. In a preferred embodiment the first-mentioned reflective system, and likewise the second reflective system, consists of a modified Offner autocolli ation system, in which the inner element of the Offner system is caused to oscillate. This modification converts the known Offner relay into a, unique reflective scanning system. The use of reflective elements for focussing at appropriate points in the system is also an improvement over prior art, in that such elements are inexpensive to manufacture and perform well over a very wide range of wavelengths. Description of Embodiment The confocal imaging system according to the invention is exemplified in the following description, making reference to the single figure of drawings, in which a preferred system is shown schematically. The system is interposed between the objective (OBJ) and the eyepiece (EYE) of a conventional microscope. It includes two Offner reflecting autocollimation units (A1,B1 and A2,B2) . An Offner autocollimation system consists of a pair of mirrors with radii of curvature in the ratio 1:2. The mirror of smaller radius is convex, the other concave. As described by Offner, this system functions as a 1:1 optical relay if light is reflected through it in the manner shown in the drawing. Thus, the Offner system consisting of mirrors Al and Bl focusses the slit C at the plane shown as a dotted line in the eyepiece (EYE). ' The other Offner system, consisting of mirrors A2 and B2, creates conjugate foci in the plane OP and on the median plane MP at the slit C and within the lens LI. The value of the Offner system in the present context is its ease of fabrication, total achromatism and freedom from primary aberrations. Illumination is injected via a beam splitter (BS) . The light could be provided by an illuminated slit. However, in the preferred embodiment a slit is not used. Instead a parallel laser beam is passed through a cylindrical lens (CL) and then through a conventional spherical lens ( 2) to provide a focussed line of laser light in the plane OP which is conjugate with the object plane. The illuminating beam is directed through the Offner unit proximal to the microscope (B2, A2) . In accordance with the invention, the Offner units are modified in that the convex mirrors Bl and B2 are each caused to oscillate about an axis perpendicular to the plane of the diagram, whereby the beam is caused to scan. Both of the convex mirrors shown in Figure 1 lie in the aperture planes in relation to the microscope rather than in image planes and they are therefore -ideally placed to serve as scaning elements. The light passes from B2 in a scanning state and enters the objective lens (OBJ) via a coupling lens l, which functions to bring the back aperture of the objective into a plane conjugate with that of B2. The signal from the object being illuminated then traverses this path in reverse and is scanned by the mirror B2. It then passes through the beam splitter BS to the masking slit C where it is brought into focus and traverses the slit. Interfering signals emanating from regions away from the illuminated line are rejected by the slit. The slit is adjustable in width to allow stringent confocal conditions to be imposed (slit narrow) or a brighter image of a less confocal nature to be formed (slit wide). The oscillating mirror Bl in the second Offner unit acts to descan the signal, recreating a stationary two-dimensional image at the mid-plane (MP) which can be viewed directly with the eyepiece or be recorded in a camera. The movement of the convex mirrors Bl and B2 may take any form that scans the whole field, but sweeps of uniform angular velocity in alternate directions are preferable as this reduces dead time and gives an image of uniform brightness. In the preferred embodiment the mirror B2 works in synchronism but antiphase to the oscillating mirror Bl . The beam splitter BS could be either a simple device producing any desired ratio of reflected and transmitted intensities, or alternatively a dichromatic reflector (dichroic) as is conventional in fluorescence microscopy.

Claims: Claims 1. In combination with an optical microscope, a confocal imaging system which comprises means for forming a slit- shaped or bar-shaped illuminating beam, or an array of such beams, a beam-splitting means by which the light is directed into an optical microscope having an objective lens, and an optical scanning means whereby the illuminating beam or beams is or are caused to scan and be directed into the objective lens of the microscope in order to scan the specimen, such optical scanning means consisting of a wholly reflective optical system for both focussing and scanning. 2. The combination claimed in claim 1, wherein, the same reflective optical system causes the beam emitted or reflected from the specimen to be descanned and focussed through the beam splitter on a stationary confocal mask. 3. The combination claimed in claim 2, wherein said mask is a slit of variable width. 4. The combination claimed in claim 2 or 3, wherein the stationary image delimited by the mask is rescanned by a second wholly reflective optical system and brought to a focus for viewing or recording. 5. The combination claimed in any of claims 1 to 4, wherein the first-mentioned reflective system is a modified Offner collimation system in which the inner mirror is caused to rotationally oscillate. 6. The combination claimed in claim 5, wherein the second reflective system is a modified Offner collimation system in which the inner mirror is caused to rotationally oscillate. 7. The combination claimed in claim 6, in which the respective inner mirrors of the two reflective systems oscillate in synchronism but in anti-phase. 8. The combination claimed in claim 7, in which the respective inner mirrors oscillate with uniform angular velocity in alternate rotational senses. 9. The combination claimed in any of claims 1 to 9, in which the means for forming the slit-shaped or bar-shaped beam includes a cylindrical lens which shapes the beam by refraction. 10. The combination claimed in claim 9, wherein the illuminating beam is a laser beam. 11. The combination as claimed in any preceding claim, wherein the full aperture of the objective lens is used.

Inventors: AMOS WILLIAM BRADSHAW, FORDHAM JAMES MICHAEL, WHITE JOHN GRAHAM, AMOS, WILLIAM, BRADSHAW, FORDHAM, JAMES, MICHAEL, WHITE, JOHN, GRAHAM
Applicants: MEDICAL RES COUNCIL, MEDICAL RESEARCH COUNCIL
Application year: 1991
Main classification: G02B26
IPC codes: G02B17, G02B21, G02B26