GB2184321A

GB2184321A - Confocal scanning microscope

Info

Publication number: GB2184321A
Application number: GB08629923A
Authority: GB
Inventors: John Graham White
Original assignee: Medical Research Council
Current assignee: Medical Research Council
Priority date: 1985-12-17
Filing date: 1986-12-15
Publication date: 1987-06-17
Also published as: GB8629923D0; GB8531011D0; GB2184321B

Abstract

A confocal scanning fluorescence light microscope directs light from a laser source (61) along an optical path which includes beam deflecting devices (64, 66) for causing the beam to raster scan the specimen in the object plane of a microscope. The fluorescent beam emitted by the specimen is returned along the same optical path so as to be descanned, and is separated from the incident beam at a dichroic mirror (62) to be incident on the detector (74). Infinity optics in the form of optical telescopes (65,68) are preferably employed for optical coupling along the optical path of the incident and return beams. <IMAGE>

Description

SPECIFICATION Confocat Scanning Wticr.oscope Field'aftie invention This invention relatesto a confocal scanning microscope.

Background to the invention Fluorescence light microscopy is extensively used in biological research and medical diagnosis. It provides the selectivity necessary to enable specific components of a cell ortissueto bevisualised and the spatial organisation of such components to be determined.

A major problem of the technique is that, especially when thick objects are viewed, light emission from out-of-focus regions seriously degrades the signal to noise ratio ofthe image.

Certain types of scanning system can effect considerable improvements in depth discrimination and in signal to noise ratio and such a system is realisable in theso-called conofocal scanning optical microscope. These advantages are especially significant for fluorescence microscopy.

Previous attempts to remove the out-of-focus signal from fluorescent images have used computational techniques which are intensive and not satisfactory, mainly because underlying assumptions have to be made in relation to optical homogeneity of the specimen which are not generally valid. The potential advantage of the confocal scanning microscope is that the out-of-focus signal is removed at source and imperfect techniques for removing noise from the image do not have to be used.

Making referencetotheaccompanyingdrawings, Figure1 diagrammatically illustrates the optical layout of a known confocal scanning microscope. The specimen at 10is scanned by a focussed light beam usuallyobtainedfrom a laser source 12. Scanning is effected by a mechanical drive which displacesthe specimen in three coordinate directions. Only the pixel which is being observed is illuminated, thus eliminating interference from adjacent regions. The light emanating from the specimen is focussed at a detector 14, the output signals ofwhich are fed to a computer 15, which processes such signals to provide vision signalsfora display device 16.In Figure 1, references 18,20,22,24 denote the lens components of the system, objective 24 being of high numerical aperture, whilst references 26 and 28 denote pinhole apertures and reference 30 denotes a partial (dichroic) reflector. Mechanical scan control 32 is derived from the computer 15. Reference 34 denotes a blocking filter.

Laboratory confocal scanning microscopes constructed hitherto generally in accordance with the arrangement of Figure 1 have shown performances in accordance with theoretical predictions. The disadvantages ofthese known prot-otypeinstruments with fixed beam geometry and scanning m.avem.ent of the speci rRert areola practical nature tn particular, firstly relatively' long scanning times are involved and secondly the layout makes it difficult alternately to employ the microscope in a standard, i.e. nonconfocal, mode. It is probablethatthesedisadvan- tages have prevented commercial realisation of the known confocal scanning microscope.

It is an object ofth is invention to provide a confocal scanning ;Tg; microscope wherein the disadvantages of the known system a re are substantially avoided or at least minimised.

The invention According to the invention, there is provided a confocal scanning fluorescence light microscope which comprises a light source, means defining an optical path whereby a diffraction limited spot (pixel) is illuminated on the object, said optical path means including focussing lensesforfocussing said light spot at the object and means for deflecting the illuminating beam so that the focussed light spot traces a raster (line scan and frame scan) on the object, a detector which receives light emanating from the object, means for producing vision signals from the output signals obtained from the detector, and a display device receiving the vision signals.

The deflecting means is preferably a rotating polygon mirrorfor producing the line scan and a galvanometer-driven mirror assembly for producing the frame scan.

Preferably, the light emanating from the object is returned along the same optical path as the incident light so as effectively to be de-scanned, and is separated from the path of the incident light at a semi-reflecting (dichroic) mirror.

The light source is preferably a laser light source, conveniently an argon ion laser, whilst the detector is preferably a photomultiplier detector, preceded by a pinhole spatial filter.

The main advantage arising from the arrangement is that the object, e.g. specimen, may be stationarily located in the object plane of a microscope objective, typically a standard fluorescence microscope, giving a considerably more preacticable instrument overall.

According to a furtherfeature ofthe invention therefore, the confocal imaging system constituted by the above described optical path means is employed as a peripheral to a standard fluorescence microscope. An additional advantage is that, by scanning at high speed and adding synchronising signals to the video signals provided by the detector, the image may be viewed with standard TV equipment.

In a preferred embodiment ofthe invention, socalled "infinity" optics are employed. Firstly, therefore, an optical telescope is used in the path ofthe raster-tracing light beam brought to a focus at the object. Secondly, where the deflecting means comprises spaced devices for producing the line scan and the frame scan,the two devices are optically coupled by a second optical telescope.

Use of infinity optics enables the normally required pinhole apertures to be dispensed with,thereby increasing sensitivity as light is not lost due to scatter at the aperture edges. Also adjustments prior to use are minimised.

Briefdescription of drawings inthaccompanying drawings: Figure 1 showsthe optical system of a prior art confocal scanning microscope; Figure 2 shows the optical system of a confocal scanning microscope which exemplifies the present invention; and Figure3shows a preferred embodiment of the invention.

Description ofdrawings The known microscope system of Figure 1 has been described heretofore.

Referring nowto Figure 2, the microscope system in accordance with the invention comprises an argon ion laser light source 40, the output beam of which passes through a pinhole aperture 42, through a dichroic mirror44 and a lens 46 (equivalent focal length 40 mm)toa rotating polygon mirror48foreffecting line scanning. The latter, wh lIst shown as a circle in the drawing, mayforconvenience have twenty five facets.

The beam from the polygon mirror 48 passes through a lens 50 (equivalentfocal length 40 mm)to a galvanometer-driven mirror assembly 52 for effecting frame scanning. The beam from the laser is therefore now performing a raster scanning action. This beam is incident via reflectors 53,55 and lens 54 (equivalent focal length 40 mm) onto the objective lens of a conventional fluorescence microscope 56 and is broughtto afocusattheobject plane. Atthe object, the pinhole aperture 42 isfocussedto effect illumination ofthe diffraction limited spot (pixel). The illuminated pixel moves over the object in accordance with the scanning raster.

Fluorescent light emitted by the object passes along the same path as the incident beam, but in the opposite direction, as far as the dick roc mirror 44, where it is separated outto pass via a pinhole spatial filter 58to a photomultipliertype detector 60. The return beam is thus effectively de-scanned, i.e. it is incident on a substantially stationary spot on the detector.

Although not shown, the outputsignals from the detector 60 are processed and,with the addition of synchronising signals, passed to a display device in theform of a conventional TV monitor.

In addition to the advantage of constituting a peripheral to a standard microscope, the invention hastheadvantage of enabling scanning up to the scanning rate oftelevision frame scan frequencies, and it is this that facilitates display in the aforesaid manner.

Figure 3 shows a preferred embodiment in which infinity optics is employed.

In the preferred em bodiment, the beam from an argon ion laser 61 is directed through a dichroic beam splitter 62 on to a galvanometer driven mirror 64. The latter is driven by a sawtooth waveform to cause the laser beamto be deflected in a linear sweep along the y axis. Atelescope 65 couples the thus deflected beam onto a motor driven rotating polygon mirror66which deflects the beam along the x axis. A second telescope 68 couples the deflected beam to the exit pupil of a conventional microscope eyepiece 70. From this eyepiece the beam passes into a conventional fluorescence microscope (not shown) to be broughtto afocus in the objective plane, where the beam traces a raster.

Fluorescent emission from the specimen (object) under examination passes through the optical system in the opposite direction,therebyeffectively being de-scanned.The now stationary return beam is separated from the incident beam atthe dichroic beam splitter 62 and passes through an iris diaphragm 72 to a photomultiplier detector 74. The signal output is electronically processed and synchronising signals are added to enable the image to be viewed with conventional television equipment.

Various modifications ofthe illustrated arrange ments of Figures 2 and 3 are possiblewithin the scope of the invention as defined by the appended claims.

Claims

1. A confocal scanning fluorescence I ight micro- scope which comprises a light source, means defining an optical path whereby a diffraction limited spot (pixel) is illuminatedon the object, said optical path means including focussing lenses for focussing said light spot at the object and means for deflecting the illuminating beam so that the focussed lightspot traces a raster (line scan and frame scan) on the object, a detector which receives light emanating from the object, means for producing vision signalsfromthe output signals obtained from the detector, and a display device receiving the vision signals.

2. A microscope according to claim 1, wherein the deflecting means comprises separated devices for producing the line scan andtheframescan.

3. A microscope according to claim 2, wherein the said devices are a rotating polygon for producing the line scan and a galvanometer-driven mirrorfor producing the frame scan.

4. A microscope according to claim I or claim 2 or claim 3, wherein the fluorescent light emanating from the object is returned along the same path asthe incident beam so as effectivelyto be de-scanned.

5. A microscope according to claim 4, wherein the return beam is separated from the incident beam, after de-scanning,ata beam splitter.

6. A microscope according to any of claims 1 to 5, wherein the light source is a laser.

7. A microscope according to any of claims 1 to 6, wherein the detector is a photomultiplier.

8. A microscope according to any of claims 1 to 7, wherein means are provided for electronically processing the detector output and adding synchronising signals thereto for enabling the imageto be viewed on a TV monitor.

9. A microscope according to any of claims 1 to 8, wherein the optical path means comprises infinity optics.

10. A miscroscope according to claim 9, wherein an optical telescope is employed to couple the raster-scanning incident beam from the deflecting means with the eyepiece of a microscope at which the incident beam is brought to a focus in the object plane.

11. A microscope as claimed in claim 10, when appendantto claim 2 or claim 3, wherein a second optical telescope is employed to couplethetwo beam deflecting devices.

12. Amicroscope according to any of claims 1 to 10, comprising a standard fluorescence microscope having said optical path means provided as a peripheral thereto.

13. A confocal scanning fluorescence lightmicroscope substantially as hereinbefore described with reference to Figure 2 or Figure 3 of the accompanying drawings.