CONFOCAL MICROSCOPE WITH PLURAL SCANNING BEAMS

Abstract: A confocal scanning microscope comprises a variable wavelength light source and means for generating, from the light source, plural scanning beams of light and illuminating a sample, in use with the beams of light. Further means receive, in use, light reflected from the sample and generate an output image therefrom.

Description: CONFOCALMICROSCOPE WITHPLURAL SCANNING BEAMS This invention relates to confocal microscopy. Traditional confocal microscopes operate by scanning a beam of light from a single wavelength light source (usually a laser) across a sample and collecting light reflected from the sample or emitted by fluorescence with a photo ultiplier to determine the intensity of the reflected light. Whilst such confocal microscopes are of considerable value, they have a number of problems associated with them. Firstly, it is difficult to scan the illuminating light beam at a speed which is high enough to provide for rapid generation of images from the photomultiplier. Furthermore, photomultiplier devices are expensive and inconvenient and require considerable associated circuitry in order to generate an image. A further problem is that the use of laser light restricts the apparatus to a single operating wavelength or to a small number of wavelengths that match poorly the wide range of fluorphores available for microscopy applications. This generally means that an image cannot be created from reflections or emissions of light at more than one wavelength unless expensive provision of different wavelength lasers is provided. The present invention seeks to overcome the above and other problems. According to the present invention there is provided a confocal scanning microscope comprising: a variable wavelength light source; means for generating, from the light source, plural scanning beams of light and illuminating a sample, in use with the beams of light; and means for receiving, in use, light reflected from the sample and generating an output image therefrom. By generating plural scanning beams from a single light source it is possible to increase considerably the scanning speed of the microscope compared to prior art arrangements. Furthermore, the provision of multiple beams means that the means for receiving reflected light and generating an image may be provided by a CCD camera of the like or by any type of two dimensional imaging system using light sensitive elements to detect light, and thus supplying directly the image generating and acquisition circuitry. Furthermore, the provision of a variable wavelength light source means that images at different wavelengths can be generated, increasing the microscopes and flexibility and allowing its use in wider range of applications than are possible with the prior art. The means for generating plural light beams may be a perforate spinning disk illuminated by the light source which may or may not have additional focusing elements such as lenses or mirrors incorporated on the disk, or may comprise plural optical fibre elements with ancillary optical components, all of which are illuminated at one end from the light source. Alternatively, a beam-splitting grating which generates plural light beams from a single beam may be provided. The variable wavelength light source may be provided by a white light source and appropriate diffraction grating, reflecting and light filtering optical components, or by a filter wheel or filter changer making use of optical interference filters or barrier filters or other similar light filtration devices. The variable wavelength light source may or may not also include a means for affecting synchronisation of the light with the confocal scanner and/or with the imaging device referred to above. One example of the present invention will now be described with reference to the accompanying drawings, in which: Figure 1 is a schematic optical diagram for a conventional confocal microscope; Figure 2 is a schematic optical diagram for an example of the present invention; and Figure 3 is a schematic optical diagram for a variable wavelength light source for employment in the present invention. Figure 1 shows the basic configuration of a standard confocal microscope. A laser light source 1 generates a monochromatic beam which is directed onto a sample 2 via a half mirror 3 and objective lens 4. Light reflected from the sample 2 passes back through the objective lens 4 and through the half mirror 3. The reflected light is then screened by a pin hole arrangement 5 and passes onto a light detector (usually a photomultiplier tube) 6. Such an arrangement has an advantage over a more traditional microscope in that the provision of point illumination and point detection provides a high resolution and the arrangement has the ability to resolve 3-dimensional images in view of the fact that only light reflected from a single plane is picked up in a single scan. Figure 2 shows an example of the present invention. Components corresponding to those in figure 1 are numbered identically. With the device shown in figure 2, a light source 1 of variable wavelength which may be a scanning monochromator or a filter wheel or filter changer or another device as described above is provided and illuminates an optional first rotating disk 10 which has formed on it a series of microlenses arranged in a series of involute curves. Light from the light source 1 passes through the apertures which may or may not have microlenses on the disk 10 and through a half mirror prism 3. It will be appreciated that the disk 10 does not have to be present, but provides a far more efficient arrangement in which light from the light source 1 is collected and focused to the required position, reducing the intensity requirement of the light source 1. After light has passed through the half mirror prism 3, it passes through a series of pinholes in a second disk 5. The pin holes in the second disk 5 are placed in positions corresponding to the microlenses in the first disk 10 and, in use, the two disks are rotated in unison to produce a scanning effect. As more than a single microlens is illuminated at any one time, light passes through more than one pinhole at any one time, and plural beams of light are provided to the surface of a sample 2. Light reflected from the sample 2 is reflected by the half mirror 3 via a lens 11 onto the surface of a CCD camera 6. Because the disks 10, 5 can be rotated at high speed, and because plural light beams are transmitted to the surface of the sample 2 at any one time, a scanning speed which is high enough for a CCD camera 6 to be employed is possible. It will be appreciated that alternatives to the spinning disk arrangement can be provided. For example, the light source 1 could be provided to plural optical fibres, whose outputs are then provided via the half-mirror 3 to the sample 2. It may further be possible to provide a beam splitting grating to provide plural beams from a single light source. Figure 3 shows the internal construction of a light source 1 that may be employed with the present invention. The light source 1 has a high intensity white light source 20 which transmits light onto a moveable mirror 21. The position of the mirror 21 can be controlled accurately by a user by either automated means such as a galvanometer, motor, acousto-optical deflector or other device which can effect movement and accurate positioning. Light reflected from the mirror 21 is transmitted to the surface of a fixed diffraction grating and mirror arrangement which reflects only light of a wavelength dependent upon the relative positions of the mirror 21 and diffraction grating 22 onto the surface of a second mirror 23. As only light of a selected wavelength is then reflected from the second mirror 23 out of the light source arrangement, it is possible to provide a single wavelength source of light of sufficient intensity for the present invention. Because the wavelength is chosen by movement of the mirror 21, this can be controlled either by a user or electronically so that scanning at the appropriate wavelength or wavelengths can be provided. The same principle of operation can be obtained by interchanging the diffraction grating 22 with the mirror 21, so that the diffraction grating moves and the mirror is static. The confocal microscope construction may instead of a single detector or camera also include multiple light detectors in the form of CCD cameras or other electronic 2D imaging device. In this example, light emitted from the sample may be passed through an additional beam splitter or dichroic filter in order to separate the distinct wavelengths of light present in the sample into their component wavelengths or into wavelength groupings which may be further passed through optical filter arrangements prior to such light being used to form an image on the detector or detectors. This light may be separated using a further wavelength changer such as a filter wheel or filter changer, or it may be separated by a series of one or more fixed filters, or by filters which may be interchanged manually. In another variant of the above example, the confocal microscope can include manual or motorised means for collecting data at different optical sections of the sample. This may be either manual or automated movement of the microscope focusing mechanism such that the relationship between the sample and the viewing objective or the confocal imaging plane is changed spatially such that confocal images are acquired at different optical sections or planes of the sample being viewed. It will be appreciated that automated movement of focus or optical collection plane may be achieved by a number of different means, including motorised or mechanical movement of any relevant optical component including the objective lens, the focusing mechanism of the microscope, or the mechanical stage on which the specimen rests. In this way, a series of optical sections may be acquired by progressively moving the focal plane, and capturing the required optical section using the confocal microscope described here with an imaging device so that the plurality of beams being used to expose the optical section at each depth results in an image at each optical section. These sections can then be used to produce a three dimensional representation of the sample by using appropriate volume rendering or volume projection software. A further variation of this can be effected so as to produce four dimensional imaging capability by using time lapse imaging procedures to collect stacks of optical sections at defined or random time intervals. In this example, the four dimensions are defined as X, Y and Z spatial axis, and time. A further variation of four dimensional imaging is the capture of images at X,Y and Z spatial axis and with the additional capture of multiple wavelengths of emitted light in each spatial dimension. It will be appreciated that the capturing of multiple wavelengths of optical data allows the capture of multiple optical probes which may be used to visualise features of the sample. In this way, three dimensional image stacks of multiple optical probes may be acquired and reconstructed to provide a representation of each probe in a 3D context viewed as a volume rendered or optically reconstructed imag . A further variation of this can be effected so as to produce five dimensional imaging by collecting not only a series of optical sections at defined or random time intervals, but also introducing the capture of multiple wavelengths of optical data at each level of optical section and each time interval. It will be appreciated that the capturing of multiple wavelengths of optical data allows the capture of multiple optical probes which may be used to visualise features of the sample. In this way, five dimensions of data including X, Y and Z spatial axis, time and colour or wavelength are captured. It will be appreciated that each variation of this confocal microscope will benefit from software and hardware for automating the capture of, and analysing, and viewing the image data. Software and hardware is also required for controlling various mechanical aspects of the system including focusing control, multiple wavelength control unit which may be a filter wheel or monochromator or multiple control units on a single microscope.

Claims: CLAIMS 1. A confocal scanning microscope comprising: a variable wavelength light source; means for generating, from the light source, plural scanning beams of light and illuminating a sample, in use with the beams of light; and means for receiving, in use, light reflected from the sample and generating an output image therefrom. 2. A microscope according to claim 1, wherein the means for generating plural light beams is a perforate spinning disk illuminated by the light source. 3. A microscope according to claim 1, wherein the means for generating plural light beams comprises plural optical fibre elements, which are illuminated at one end from the light source. 4. A microscope according to claim 1, wherein the means for generating plural light beams is a beam-splitting grating which generates plural light beams from a single beam. 5. A microscope according to any of claims 1 to 4, wherein the variable wavelength light source is provided by a white light source, diffraction grating, and reflecting and light filtering optical components. 6. A microscope according to any preceding claim, further comprising means for varying the plane from which light is received by the light receiving means. 7. A microscope according to claim 6, wherein the output image generating means provides an image of the sample from light received from plural planes. 8. A microscope according to any of the preceding claims, further comprising means for controlling the variable wavelength light source and scanning light beam generator to enable polychromatic images to be generated from the light received by the light receiving means. 9. A microscope according to any of the preceding claims, further comprising means for receiving light reflected from the sample at different times and generating images based on the received light. 10. A microscope according to any of the preceding claims, wherein the generating means, variable wavelength light source and light receiving and output generating image generating means are all controlled by a central control unit. 11. A microscope according to claim 10, wherein the central control unit can receive inputs from a user to vary the wavelength of the light source, the times at which light is received, the plane from which light is received, or a combination thereof.

Inventors: MASON WILLIAM, MASON, WILLIAM
Applicants: MASON WILLIAM, WALLAC OY, MASON, WILLIAM
Application year: 1999
Main classification: G02B21
IPC codes: G02B21