AU672876B2 - Confocal microscope - Google Patents

Description

CONFOCAL MICROSCOPE Technical Field

This invention relates to the field of microscopy and more particularly to confocal microscopes. Background of the Invention

The principles of a scanning confocal microscope are disclosed in United States Patent No 3,013,467 of Marvin Minsky. The basic principle is that illumination of the specimen or object to be observed is confined to a single point observational field and observation or detection is confined to that illuminated point field. A complete image is derived by scanning the specimen or object under observation point by point through a complete field of view of the microscope. In early confocal microscopes, including that proposed in the Minsky patent, the optical system remained fixed and scanning was achieved by moving the specimen or object to be observed in a scanning patte.rn across the focal point of illumination. More recent high speed scanning microscopes have employed beam scanning techniques rather than movement of the specimen. Commonly, 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.

International Patent Applications PCT/AU89/00298 and PCT/AU91/00129 describe the design of confocal microscopes which in place of a pinhole (or equivalent) of a convention confocal microscope use an optical fibre or fibre bundle, thus allowing the imaging end to be more freely mobile, transportable and capable of miniaturisation. The present invention provides a further development whereby 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. This enables confocal microscopic examination of objects which are inconvenient for any current microscopic techniques due to limitations of distance, safety or motion of the object to be examined. The use of 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.

However, 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

According to the invention there is provided 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.

Alternatively, 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. Brief Description of Drawings

In order that the invention may be more fully explained, two particular embodiments will be described in some detail with reference to the accompanying drawings in which:

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; and

Figure 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. It is to be understood that 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. In this way 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.

There are a mimber of situations in which it may be necessary to carry out microscopic examinations of objects which are extremely inconvenient for any current microscopic techniques due to limitations of distance, object motion etc. Examples are the need for microscopic examination of sheet metal travelling in rolling mills, electroplating, enamelling, sheet paper and silicon wafer fabrication. The apparatus can be particularly useful in examining objects which have slight vibration or random movements, such as in the tissue of living laboratory animals. If the objective lens is physically attached to the specimen X, Y, Z movement of the specimen produces much less motion of the image than, if the objective lens was stationary and the object moved beneath it. The movement is effectively de-magnified. However, there are applications such as in the examination of moving sheet material in which the objective lens would not be physically attached to the specimen but merely set at a fixed position adjacent to the specimen. The apparatus illustrated in Figure 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. However 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. Such a system is illustrated in Figure 3 which also illustrates a development by which a series of objective lenses may be scanned and interrogated by a single transmitter and scanning system.

In the microscope illustrated in Figure 3 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.

As also illustrated in Figure 3 the objective lens may be one of a series of objective lenses to be scanned and interrogated by the one scanning and detector system. Thus 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.

Although in the illustrated systems 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. For example in the examination of a moving sheet material 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.

In such a case it would be necessary to have 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.

Such a system would most conveniently be applied with the bulk optical embodiment.

Claims (10)

CLAIMS:

1. 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.

2. A microscope as claimed in claim 1, wherein the objective lens is one of a 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 is operable to aim the light beam transmitted from the transmitter alternatively onto each of the objective lenses.

3. A microscope as claimed in claim 1 or claim 2, wherein the aiming means is operable adjustably to move the transmitter thereby to vary the aim of the light beam through said free space.

4. A microscope as claimed in claim 1 or claim 2, wherein the aiming means comprises moveable reflector means associated with the transmitter so as to reflect the light beam and moveable to change the direction of reflection thereby to vary the aim of the light beam through said free space.

5. A microscope as claimed in any one of the preceding claims, wherein there is optical transmission means for transmitting the light beam from the light source to the transmitter and for transmitting the return beam of object emanated light from the transmitter to said detector.

6. A microscope as claimed in claim 5, wherein the optical transmission means comprises a beam splitter for separating from the source light beam the return beam of object emanated light for application to the detector.

7. A microscope as claimed in claim 6, wherein the optical transmission means comprises a first optical fibre means extending from the light source to the transmitter via the beam splitter and a second optical fibre means extending from the beam splitter to said detector.

8. A microscope as claimed in any one of the preceding claims, wherein the light beam transmitter comprises scanning means operable to move the light beam in a scanning pattern.

9. A microscope as claimed in claim 8, wherein the scanning means comprises moving mirror means for reflection of the light beam and means to move the mirror means so as to move the light beam in said scanning pattern.

10. A microscope as claimed in any one of claims 1 to 7 and intended for examination of a moving object, wherein the confined observational field is linear and transverse to the direction of movement of the object to be examined and the detector is adapted to detect light emanating from individual points along the linear observational field.