CONFOCAL LASER SCANNING MICROSCOPE

Abstract: Confocal laser scanning microscope comprising a laser (1) as point light source, a deflection system for the line and frame scanning and a lens system, at least one objective (9) near the object, an object stage, a spatial filter (14) and a detector (15), and an electronic control and imaging-processing system. The object is scanned point by point by the light beam and measure­ ments are made with the detector only where the point light source is focused so that out-of-focus light is not detected. As a result resolution and contrast in three dimensions, in particular, axially to the image plane can be improved considerably, and 3D information can be derived from the object. For the more rapid line scanning, the deflection system of the present invention comprises an acousto-optical deflector (5) and comprises for the slower frame scanning, another deflector (7) constructed in a manner such that at least the frame-scanning movement of the return light beam is completely eliminated, as a result of which the return light is focused on the spatial filter.

Description: The invention relates to a laser scanning microscope as indicated in the heading of the main claim. The operation of laser scanning microscopes is described in the book entitled "Theory and Practice of Scanning Optical Microscopy" by T. Wilson and C. Sheppard, Academic Press, 1984. In such a microscope, the focused laser spot is scanned over the stationary object or preparation. Use is generally made of galvanometric deflection by means of mirror galvanometers in said scanning movement of the focused laser spot both for the line scanning and for the frame scanning. The object is also often scanned by means of the moving object stage with respect to a stationary focus laser spot. The disadvantage of such methods of scanning is that they have a mechanical nature and are therefore inherently slow. This also produces long frame times. The object of the invention is to provide a laser scanning microscope for confocal imaging, with a deflection which can operate with high speed and flexibility, i.e. with variable scanning amplitude and for various types of confocal microscopy, and which has an optically relatively simple embodiment. As a result of rapid line and frame scanning, it is then possible, in a very short time, to combine electronically a number of thin image sections to form an image with an increased depth of focus. In a laser scanning microscope of the type mentioned in the introduction, this is achieved according to the invention with the measures as indicated in the chracterizing clause of the main claim. The use of an acousto-optical deflector, having an inherently small deflection angle and thus a small field of view, is known per se in the case of non-confocal laserscanning microscopy e.g. from AT&T Technical Journal, vol. 65, no. 1, pages 68-77. The microscope described in this AT&T Technical Journal is used for the inspection of surfaces of patterned semiconductor wafers for particle contamination. It is a non-confocal optical system, in which a beam splitter is provided between the first deflector, i.e. acousto-optical deflector, and the second deflector in order to split off the return light beam. In order to make this optical system suitable for confocal purposes, in which the return light beam follows the same optical path as the laser light beam through the deflector, one has to meet the demand of entering at the Bragg-angle into the acousto-optical deflector for both illuminating and reflected light. This severe requirement has to be fulfilled for achieving a high efficiency and even illumination over the field of view and for descanning the laser light for confocal detection. The expert in confocal microscopy would expect numerous problems entailed by an acousto-optical deflector having inherently small deflection angle and therefore a small field of view which problems affect the resolution and the confocality of the system. Zooming in with such a system is difficult due to the cylindrical lens effect. Also upon variation of the scan speed the cylindrical lens effect has to be corrected or for instance mechanically displaced. If this is used in confocal microscopy the known disadvantages of the dispersive nature and the necessity of using several lenses then occur, as a result of which, respectively, confocal microscopy using light to be observed of a wavelength other than that of the light incident on the object (fluorescence etc.) is not readily possible and the disturbing effect of reflections at surfaces of the lenses used is considerable. In addition, the latter have to be of good quality to avoid aberrations. In the confocal microscope of the invention the above problems are abviated. In an advantageous embodiment, the second deflector in the deflection system consists of a mirror galvanometer. In a further advantageous embodiment, in which a beam splitter is incorporated in the light path in order to split off the return light beam and to direct it to the detector, said beam splitter is mounted between the acousto-optical deflector and the point light source, and the deflection system with the acousto-optical deflector and the lens system are constructed such that the return light beam follows the same optical path as the laser light beam up to the beam splitter whereby the line-scanning movement introduced by the acousto-optical deflector is eliminated, and the spatial filter is mounted, after a further objective, as a pin-hole filter which forms a point detector with the subsequent detector. Advantageously, in the confocal laser scanning microscope according to the invention a quarter-wave plate may be incorporated in the outward path in front of the objective in order to suppress the disturbing effect of optical reflections in the outward path which are produced by the use of the acousto-optical deflector with the necessity, associated therewith, of using several lenses. A polarizing filter, whose polarization direction is perpendicular to the polarization direction of the outward light should then be incorporated in the path of the reflected light beam. The outward linearly polarized light is converted into circularly polarized light by the quarter-wave plate. After reflection by the object, said circularly polarized light again passes through the quarter-wave plate so that that light is linearly polarized with a polarization direction perpendicular to that of the incident beam. The polarization filter incorporated in the path of the reflected light transmits only light having a polarization direction perpendicular to that of the outward beam, and this can then be detected by the point detector. Advantageously the confocal laser scanning microscope according to the invention can be used in fluorescence microscopy or other forms of microscopy in which the wavelength of the return light beam differs from that of the laser light beam, a dichromatic mirror being incorporated in the light path between the acousto-optical deflector and the second deflector in order to deflect the return light beam with differing wavelengths behind the second deflector and to direct it via yet a further objective and the subsequent spatial filter to the subsequent detector, the subsequent spatial filter being a slit filter which forms a line detector with the subsequent detector. US 4634880 describes a confocal microscope in which a quarter-wave plate is used for eliminating disturbing reflections. The invention will now be explained in more detail on the basis of an exemplary embodiment with reference to the drawings, in which: Figure 1 gives a diagrammatic view of a first embodiment of the confocal laser scanning microscope according to the invention; Figure 2 gives a diagrammatic view of the manner in which the light beam is incident on the objective; Figure 3 gives a detailed view of the spatial filter in the microscope of Figure 1; and Figure 4 gives a diagrammatic view of a second embodiment of the confocal laser scanning microscope according to the invention. The first embodiment is explained with reference to Figure 1. The laser light beam 1 first passes the lenses 2 and 3 forming together a beam expansion optical system, followed by a beam splitter 4, an acousto-optical deflector 5 having a planocylindrical lens 5.1 and a planoconvex lens 5.2 both at the entrance and at the exit side, a lens 6, a deflector 7, which may be a mirror galvanometer, a lens 8, a quarter-wave plate 16, and an objective 9. In the object plane 10, an object, not shown, is further placed on a stationary object stage. The reflected light traverses a return path identical to the outward path up to the beam splitter 4 after which it is split off to a polarizing filter 11, a further objective 13, a spatial filter 14, a lens 17, a band pass or cut-off filter 12, and finally a detector 15. The expansion optical system mentioned, which has an expansion factor of three, ensures, in combination with the other optical elements, that the light completely fills the entrance pupil of the objective 9. The beam splitter 4 ensures that light reflected by the object is separated from the outward laser light. The acousto-optical deflector 5 ensures more rapid line scanning over the object, and the mirror galvanometer 7 ensures a relatively slower frame scanning across the object. In this connection, the acousto-optical deflector can achieve a deflection of the laser beam with such a high frequency that the latter takes place with video speed or even higher speed. The lens 6 increases the angle through which the laser beam is deflected by the acousto-optical deflector. The focused spot of the laser beam ends up in a position such that the objective is used in the correct manner. The mirror galvanometer 7 is at the focal point of said lens and in the centre point of the lens 8. As a result of this, the laser beam is stationary both at the mirror galvanometer and at the rear side of the objective so that, at these positions, only the angle of incidence, but not the position of incidence, of the laser beam changes. In this connection, compare also Figure 2. The objective 9 focuses the outward laser beam 18 on the object, which may be, for example, a biological preparation or any other object. The laser light 19 reflected or scattered by the object follows the same optical path backwards up to the beam splitter 4. After this, the reflected light follows the path already specified through the elements 11, 13, 14, 17 and 12 up to the detector 15. The scanning X-Y movement of the laser beam introduced by the acousto-optical detector and the mirror galvanometer is eliminated again on the return path so that the reflected light is focused on the stationary spatial filter 14, which is a pin-hole filter (2-micron hole). This filter, with the detector immediately behind it, forms a point detector. As a result of this the microscope has confocal characteristics. In this first embodiment according to the invention, the disturbing reflections introduced in the outward path by the acousto-optical deflector are eliminated by means of a quarter-wave plate 16 incorporated in front of the objective 9 and a polarizing filter 11 incorporated after the beam splitter. The outward linearly polarized light is converted into circularly polarized light by the plate 16, after which said light, after reflection, again passes through said quarter-wave plate and is converted again into linearly polarized light with a polarization direction perpendicular to the incident beam. The polarization filter 11 incorporated after the beam splitter is also adjusted to this polarization direction so that only reflections from the object and the objective 9 are detected. Further disadvantageous effects may possibly be caused by the dispersive nature of the acousto-optical deflector, as a result of which return light of a wavelength (for example, fluorescence) other than the laser light no longer passes through the spatial filter, are taken care of by displacing the spatial filter 14 in those cases. Such a spatial filter may advantageously be displaced by three piezoelectric crystals, each for one of the three axes of the XYZ coordinate system, as indicated in Figure 3. Advantageously, such a microscope can be used for examining fluorescent preparations which have this property inherently or have been marked for this purpose. The light emitted as a result when light is incident on the object has a wavelength other than that of the outward laser light. By using a band filter or cut-off filter 12 in the return path which is matched to the expected wavelength of the return light, the latter can be selectively transmitted so that no disturbing effect is experienced from the reflected laser light. Since the light of differing wavelengths also undergoes a different deflection in the acousto-optical deflector 5, the spatial filter 12 must be arranged in another position corresponding to the angular dispersion of the acousto-optical deflector. Figure 4 indicates a second embodiment. A diachromatic mirror 20 has been incorporated in the light path between the planocylindrical lens 5.1 and the lens 6. Said mirror transmits the (short-wave) laser light and deflects the long-wave return light originating, for example, from fluorescence. This light is passed through a correction lens 21 and focused with the objective 22 on a special spatial filter 23 which is a slit filter (330 m x 1 m), as a result of which this system has confocal characteristics. In this manner, a line detector is formed with the subsequent lens 24 and detector 26. Between the lens 24 and the detector 26 one more band pass or cut-off filter 25 has been incorporated which has the same function as that of the band pass or cut-off filter 12. With this embodiment, return light which has a wavelength other than that of the outward light can be advantageously examined if the acousto-optical deflector has too low an efficiency for said light, i.e. brings about too large an attenuation. With this microscope it is possible to assemble, for example, 20 images per second at a line frequency of 20 kHz. That is to say, each image contains 1000 lines. With such a number of 20,000 lines per second and if there are to be 1000 image points or pixels per line, the detector and the subsequent electronics which measure the half tone of a pixel which have to have at least a response of not more than 50 nsec. Advantageously, with such a fast image assembly, an image with increased depth of focus can be built up by combining a number of thin image sections. According to the above example, 20 sections situated underneath each other can be combined in one second so that a object which is 20 sections thick can be reproduced with complete sharpness. In addition to being used in the field of application of biology, such a microscope according to the invention having a line-scanning frequency of, for example, 20-30 kHz and a frame-scanning frequency of, for example, 90 Hz can also be used in forensic examination and in the microelectronics industry. In all cases the fact that it is not necessary with this microscope to work with the object under vacuum is of great advantage. In contrast to the scanning electron microscope, for which the preparations have to be covered with a thin conductive metal layer, this method is also non-destructive. The fact that, in the microscope according to the invention, no mechanical forces are exerted on the preparation is also an advantage over systems in which the preparation is scanned. In the microelectronics industry, this microscope can be used for production control of LSI and VLSI chips, the production of "custom design" chips and also the functional control of chips by means of the so-called "optical beam induced current" method. The present microscope can also be used advantageously for examining optical memories.

Claims: Laser scanning microscope provided with a laser as light source (1), a deflection system for line and frame scanning including an acousto-optical deflector (5) for the line scanning, a first lens system (5.1; 5.2), an object stage, at least one objective (9) near the object stage for focusing the laser beam (18) onto an object, a second deflector (7) positioned in the beam path between the acousto-optical deflector (5) and the objective (9) for the slower frame scanning, the second deflector and the objective being positioned so that the return light beam (19) from the object follows the same beam path as the laser light focused onto the object up to and including the second deflector, a detector (15; 26) positioned in the return light beam path behind the second deflector (7) for detecting the return light from the object, and an electronic control and image processing system, the object being scanned point by point by the laser light beam and measurements being made with the detector, characterized in that a spatial filter (14; 23) is positioned in front of the detector (15; 26) between the second deflector and the detector in the beam path from the object to the detector in order to realize confocal imaging; and in that a lens (8) is positioned in the beam path between the objective (9) and the second deflector (7) to direct the light from the objective onto the second deflector, whereby the frame-scanning movement introduced by the second deflector is eliminated as a result of which the return light can be focused on the spatial filter. Laser scanning microscope according to claim 1, characterized in that the second deflector (7) in the deflection system consists of a mirror galvanometer. Laser scanning microscope according to claim 1 or 2, in which a beam splitter (4) is incorporated in the optical path in order to split off the return light beam and to direct it to the detector (15), characterized in that the beam splitter (4) is mounted between the acousto-optical deflector (5) and the point light source (1), and that the deflection system with the acousto-optical deflector (5) and the first lens system (5.1, 5.2) are constructed such that the return light beam follows the same optical path as the laser light beam up to the beam splitter (4) whereby the line-scanning movement introduced by the acousto-optical deflector (5) is eliminated, and in that the spatial filter (14) is mounted, after a further objective (13), as a pin-hole filter which forms a point detector with the subsequent detector (15). Laser scanning microscope according to claim 1 or 2 for use in fluorescense microscopy or other forms of microscopy in which the wavelength of the return light beam (19) differs from that of the laser light beam (18), wherein the spatial filter (14) is mounted on three piezoelectric crystals and can accordingly be moved in a 3D coordinate system, as a result of which the effect of the dispersive nature of the acousto-optical deflector (5) on the return light of a different wavelength, which is deflected through an angle other than the reflected laser light, is eliminated and wherein a correspondingly matched bandpass or cut-off filter (12) is incorporated in the return light path to filter out the reflected laser light. Laser scanning microscope according to claim 1 or 2 for use in fluorescence microscopy or other forms of microscopy in which the wavelength of the return light beam (19) differs from that of the laser light beam (18), wherein a dichromatic mirror (20) is incorporated in the light path between the acousto-optical deflector (5) and the second deflector (7) in order to deflect the return light beam with differing wavelengths behind the second deflector and to direct it via yet a further objective (22) and the subsequent spatial filter (23) to the subsequent detector (26), the subsequent spatial filter being a slit filter which forms a line detector with the subsequent detector (26).

Inventors: DRAAIJER, ARIE, HOUPT, PIETER MARCUS
Applicants: NEDERLANDSE ORGANISATIE VOOR TOEGEPAST-NATUURWETENSCHAPPELIJK ONDERZOEK TNO
Application year: 1988
Main classification: G02B21
IPC codes: G01N21, G02B27, G02B21, G02B26, G01Q90, G01Q30, G01Q20