GB2152697A - Improvements in or relating to scanning optical microscopes - Google Patents

Abstract

A scanning optical microscope has a laser L1 passing a parallel beam through beam splitter O3 to be focussed by lens L2 on specimen S1. Radiation reflected from S1 is directed by splitter O3 to photodiode detector d1 whose output passes to a display unit having a visible element (e.g. CRT spot) whose intensity varies with the signal. The lens mount O5 is moved rapidly to give a line scan and the specimen support moved slowly to produce a frame scan; transducers supply signals to drive the visible element to correspond to the scan movement. The parallel beam enables specimen and scanner to be remote from the laser, allowing examination of highly radioactive specimens. A phase retarder can be inserted to enable examination of the specimen using polarised light. An automatic focussing arrangement is described. <IMAGE>

Description

SPECIFICATION Improvernents in or relating to scanning microscopes This invention relates to scanning microscopes, particularly, but not exclusively, to scanning microscopes for use in handling radioactive specimens or material.

According to one aspect of the invention a scanning microscope comprises a source of coherent electromagnetic radiation, means for supporting an object to be viewed, means for receiving a parallel beam of radiation from the source and adapted to focus radiation on the object, means responsive to radiation from the object for forming an image of the object, and means for effecting relative movement between the focussing means and the support for scanning the object.

The radiation may be reflected from or transmitted through the object.

In one arrangement the support is moved to obtain a frame scan and the means for focussing is moved to obtain a line scan.

The radiation may be polarized before being focussed, and means for rotating the plane of polarization, for example to obtain different imaging modes.

There may be a visible display element moved in response to said relative movement, and the intensity of the display may be dependent on the radiation from the object.

In this way a magnified image of the object is mapped out element by element as the movement takes place.

The sensing means may have a capacitance which changes in response to said line scan.

The responsive means may comprise a beam splitter through which said parallel beam passes, a photodiode for receiving from said beam splitter radiation reflected from the object, and visual display apparatus connected to the photodiode for displaying an image of the object.

The invention may be performed in various ways and one specific embodiment with possible modifications will now be described by way of example with reference to the accompanying drawings. in which: Figure 1 is a showing of a scanning optical microscope according to the invention; Figure 2 is a schematic showing of a system for handling radioactive specimens; Figure 3 shows part of Figure 2 on an enlarged scale; Figure 4 shows an imaging system: Figure 5 shows a modification; and Figure 6 shows another modification.

In Figure 1, L1 is low power laser from which the output beam 10 is expanded through a beam expander 01 to produce a parallel beam 11 of monochromatic light. The The beam is focussed to a small spot (eg 1calm) by a lens L2 mounted at the end of a scanning arm 05. The spot is on the surface of sample S1. As the lens L2 is scanned to and fro across the sample, different areas of the beam 11, are focussed to produce the scanning spot. The full line and dotted line positions of L2 show the limits of the scanning movement. The rays reflected from the sample retrace the same path back through the lens L2 and are reflected by a beam splitter 03 to produce beam 11 a through a lens 04 to impinge on a photodiode d1 and via amplifier 69 to display unit 70.The diode d1 produces an analog electric output signal which goes to a visual display unit which has a visible display element moved in response to relative movement between the lens and a support for the sample, the intensity of the element being dependent on the signal from the diode.

One form of scanning arrangement is shown in Figure 3. The lens L2 is mounted on an arm 05 fixed to torsion bar 51 fixed in structure 52. The other end of arm 05 is between two electromagnets 53, 54 which can be selectively energised by power amplifier 55 to oscillate or vibrate arm 51 between limits at its natural frequency, for example 200 cycles per second. Thus the lens L2 describes an arc in a plane parallel to the samole surface (for clarity, the sample is shown spaced from its mounting 61).

The specimen or sample stage or support 61 is reciprocated (period of say 7 seconds) in the Y direction by motor 62 and a transducer 63 provides an output signal responsive to the Y movement. The movement (X) of the lens L2 and the movement in the Y direction of the sample are both at right angles to optical axis 64. Because the sample is not moved at the high frequency for a line scan the corresponding expensive and powerful line scan drives are not needed.

The position of the arm 05 is measured by position sensors 56, 57 in the X direction and transducer 63 for the Y direction and these sensors may have an analogue or digital output as desired. For example, transducer 63 may inciude a grating gauge; and sensors 56, 57 may be conducting plates with a centre plate 58 may sense changes in capacitance between 56 and 58, and between 57 and 58, and in association with a charge amplifier provide an output signal representing movement in the X direction. The imaging system can be arranged to compensate for non-orthogonal X, Y directions. The output signals are fed to a display unit 70 and move the display spot correspondingly in the X and Y directions.The intensity of the spot is modified in accordance with reflected light from the specimen S1 detected by photodiode d1 and the display on the screen of unit 70 is for example photographed to produce a permanent image. The duration of the exposure is set to correspond with a single frame scan (Y) so the image is built up and stored on the negative. The signal may pass to a video store where the accumulated display can be seen.

By careful choice of the laser focussing lens L2 it is to be expected that a spot size as amall as 0.8,um can be achieved with a helium neon laser. A nickel-cadmium laser, which emits light in the UV region, or an ray source to obtain even higher resolution, might be used.

A 'harmonic scanning microscope' might be used. This adaptation of the scanning optical microscope may yield resolution above the classical limit set by the illuminating light. Its operation is based on the properties of certain crystals, which lack a centre of symmetry, to generate harmonic light frequencies, at high light intensities. Light reflected from the sample at the fundamental frequency is filtered out and an image of the sample built up in the normal way using the harmonic light.

The purpose of lens L2 is simply to focus parallel light and as such it need not be highly corrected. It may be replaced by a diffraction grating and the scanning arm reduced in size or replaced by a piezo bi-morph strip. The resulting system would be capable of oscillating at much higher frequencies and would therefore produce an image at a higher frame rate than the arrangement of Figure 1.

Means can be provided, for example, by use of an electromagnet 72, mounted above or below the scanner, to alter the position of the lens L2 in a direction parallel to the optical axis 64 to effect focussing by applying a bending movement to the end of the scanner arm 05. The same mechanism can be used to maintain the focus during the period of one scan.

Referring to Figures 1 and 3, an electromagnet 71 is mounted to the optical base 61 in close promimity to a light weight soft iron pole piece 72 mounted on the end of the scanner arm 05. When current is passed through the coil 71, the scanner is pulled upwards in the Z direction. Automatic focussing is derived from a quadrant diode detector d2 (Figure 5) as follows: The already-split return beam is split at beam splitter 06 and a weak cylinder lens 07 interposed between splitter 06 and photodiode d2. The gross astigmatism so introduced gives the return beam an asymmetrical envelope which is used to give the control signal for the coil a direction.

A scanning type of microscope is inherently remotely operated and is particularly suitable for examining radioactive samples in shielded cells. The only connection between the control panel 20 Figure 2 and the parts located in cell 21 is a bundle of control cables 22. The optical extension tubes used to bring the image from a conventional microscope out of the shielded cell are not required. The output is in the form of an electronic signal which can easily be interfaced with a computer for automatic measurement, image analysis or processing. The equipment is much smaller, more compact and not bound to the cell wall.

Unlike conventional 'in cell' microscopes it can be unplugged from its cables, moved around in the cell, replaced or removed as required. This has a large impact on maintenance costs, and simplifies health physics procedures for preparation for maintenance.

Figure 2 depicts how the microscope might look in cell. The operator only stands in front of the cell for a short time while the sample is positioned on the microscope.

There is a low radiation area 80, intermediate radiation area 81, high radiation area 82 and an optics cabinet 83 having laser source Li.

The scanning optical microscope uses a laser light source. The power level can be chosen from a wide range and can be orders of magnitude brighter than conventional microscope lamps. (High power lasers are not used to illuminate conventional microscopes because of the risk of damage to the eye). All the light is focussed down to a spot of the order of 1 ,am in diameter so that the reflected signal even from a poor reflector can be large compared to other types of microscope.

This leads to two advantages of the laser scanning optical microscope.

(a) Less gain needs to be applied to the detector amplifier and hence the signal-tonoise ratio of the image is higher.

(b) The usual blackening of the objective lens of a microscope on exposure to radiation eventually reduces image intensity down to such a level that the objective lens has to be replaced. This is not a problem with the scanning optical microscope since sufficient light is available to enable it to be automatically controlled to provide constant intensity throughout the working life of the microscope.

The properties of the scanning optical microscope mentioned above are particularly important when the sample is being observed in polarised light with "crossed polars" (dark field).

In this situation it is not unusual for image intensities to be many orders of magnitude less than the normal and quite often the performance of conventional equipment is severely limited.

For radioactive work the microscope 30 has no requirement, unlike a conventional microscope, for lens-changing controls. The parts of the microscope 30 located in cell 21 are of low cost and small size and these features appiy for non-nuclear use. It is therefore envisaged that a spare microscope 31 would be kept on stand-by in the cell. This would mean that a microscope would always be available for use.

When using a conventional microscope the operator has only limited control over the image in the eyepiece. To change the magnification he has to change either the eyepiece or the objective. On a microscope located behind shielding this is a time consuming operation if it is required to go up and down in magnification frequently. However, in the scanning optical microscope, the magnification is not a function of the lens focal length but is controlled quickly and easily from the control panel 20 by altering the sensitivity of the position measuring transducers. This causes the video signal to be displayed over a larger or smaller area of the screen.

The arrangement is particularly suitable for polarised light operation. Laser L1 emits polarized light and is positioned so that the plane of polarization corresponds with the plane of maximum transmission of the polarized light beam splitter 03. A variable phase retarder (Soleil Babinet compensator) 02 enables the plane of polarization to be rotated through an angle S. The beam makes a second pass through retarder 02 and is again rotated through an angie S. When 20 = 90 90, all the reflected light from the sample is reflected to the photodiode d, by beam splitter 03.When 20 = 180 only light rotated 90 by the sample structure is collected by the diode (the greater part of the light is reflected by the sample through the beam splitter back to the laser and is attentuated if necessary for safety). These conditions are used to produce an image of samples which will rotate the plane of polarization so as to show their surface structure. The sample can now be analysed by rotating 02 allowing light which has been rotated by the sample through to the detectors d1.

In certain cases, for example, transparent biological samples, an image of the internal structure may be obtained by sensing radiation which has been transmitted through the sample rather than reflected with the photodiode positioned to receive the transmitted radiation.

Figure 4 shows the details of a computer imaging system for the microscope. The X scan position and video signal 91 is stored in a buffer line store 90 in order to scan convert from a sinusoidal scan input to a raster-scan.

The Y ramp signal 92 is also not input directly but is forced to follow a raster ramp by feedback control. The contents of the store are continuously read out to a 1000 line monitor 94 via a high speed digital to analogue converter 93. Simultaneously read/write operation allows the image to be viewed while it is being formed in the memory. This is useful for checkjng focus and allows the sample to be moved part way through a scan. The original slow scan image can be displayed on the screen 70 using photorecord electronic apparatus 95 which allows synchronisation of the frame scan with the line scan of an oscillator (400 Hz); this allows the same video monitor to be used as the photocord module by photographing the screen. A keyboard 96 allows control of the system through computer 99.

Images can be stored (97) on magnetic disc, moved to and from the frame store 98 and copies obtained from the video photorecord 94 as required.

Lens Aberrations Many scanning optical systems are based along conventional lines of imaging the object using a lens and then scanning this two dimensional image. The system described above is different in that the intermediate step is omitted and an image is not formed until the data reaches the digital memory. This is achieved by using a parallel beam and scanning perpendicular to the optical axis. The objective lens L2 then is focussing a parallel beam to a diffraction limited spot and collecting the reflected light. The lens does not therefore show its aberrations of astigmatism or coma which would be present if the lens were imaging a two-dimensional object. In addition, chromatic aberration inaccuracies are not present because of the single line spectrum of the laser.The result is that the only aberration of importance is spherical aberration which can be corrected to a large degree.

By the same token the lens can be computed for maximum focal length whilst still retaining diffraction limited performance. In practice the lens used has a sample S1 to lens L2 clearance of 3mm compared to a normal high power objective of 1 mm. A further consequence of this is that the scanning microscope is suitable for imaging fracture surfaces when protruding parts might not allow sufficient clearance using a conventional microscope.

A photographic record has been mentioned above. Any attempt to take measurements from the photographic film would suffer errors due to the cathode ray tube, camera and film characteristics, for example amounting to a few percent. An advantage of the scanning microscope system is achieved when these measurements or manipulations are done on the image data, when stored to digital memory such as a computer store. Such a computer system may be designed to achieve 0.1% accuracy. The limit to imaging accuracy is then determined by the following parameters: a. Spatial linearity and resolution b. Greyscale linearity and resolution.

In the present arrangement many optical image aberrations are avoided by not producing an optical image and by using a parallel beam. Linearity errors associated with one and two dimensional detectors such as video or television cameras are avoided by using a single point detector. Thus spatial linearity depends solely on the characteristics of the position measuring system. A number of posi tion measuring instruments are applicable to the microscope. The following requirements should be considered: a. Maximum linearity.

b. Maximum resolution c. High speed (200 kHz in case of X scan) d. Non contact or light weight so as not to affect performance of the scanner.

The measuring devices can be either analogue or digital, depending on interfacing requirements. Additionally, an alternative system can infer X scanner position and assume it to be a sine wave and Y position to be a smooth ramp. In this way high signal to noise ratios can be obtained perhaps with compromise on linearity. The following types of instruments would meet the above requirements for position measurement: Optical - interferometer - grating - mirror deflection Magnetic - inductance - eddy current - hall effect Electronic - accelerometer - strain gauge - capacitance Spatial resolution on the other hand is ultimately limited by the size of the scanning spot. In the present system this may be a 1 micrometre.The scanning range, hence the minimum magnification, achievable is limited by the dynamic range of the position measuring devices, although if reduced resolution is accepted then greater scan amplitude can be achieved, up to the limit of the scanner. The greyscale characteristics are determined by the photo-detector/video amplifier/digitiser noise characteristics. Modern photodetectors have good performance with signal to noise ratio better than 60 dB and dynamic range over three orders of magnitude. The arrangement described produces a visible reflected signal which allows the photodiode to operate in room light conditions and being a point detector much of the strong light from points other than the laser beam focus is rejected.

The bandwidth of the video signal, being slow scan, is only 200 kHz, so carries proportionally less noise than for example a television camera with a 4 mHz bandwidth.

The scanning optical microscope produces an output in the form of an electronic signal so not only can the signal be displayed on a cathode ray tube to be viewed by any number of observers but also the signal can be interfaced to a computer for functions such as: 1. Archive storage in digital memory.

2. Production of hard copy in less time than photographs.

3. Electronic processing such as image enhancement.

4. Automatic operations such as particle counting.

(a) with the device there can be a remote viewing of the image for example in order to reduce radiation dose to the operator when the sample is radioactive and allowing centralising of operations in one control room.

(b) Interfacing of the microscope to other hardware to facilitate; (1) image enhancement by computer; (2) large archive storage in computer memory; (3) production of 'hard copy' from video printer rather than photographs; (4) measurement of image parameters, automatic particle counting etc using software programs.

The resolution may be approximately 2 jum, very close to the resolution limit set by the wavelength of the illuminating light. This is equal if not better than that produced by existing equipment.

The scanning optical microscope image shows details of metallurgical features in a way different to other microscopes. This may be useful in imaging objects which have hitherto been difficult to observe using existing equipment (eg oxide layers).

The system does not suffer from degradation of the image due to radiation effects on the lens. The high intensity obtainable from the laser allows any lens blackening to be compensated for.

The high intensity of light available from the laser results in a higher quality image than other systems with less electrical noise on the displayed image, particularly when image intensity is low, such as when viewing in dark field using polarised light.

Lower maintenance costs will result when the microscope is used in the difficult environment of the shielded cell. The 'in-cell' components of the microscope can represent no more than 10% of the total cost of the instrument.

Possible modifications are: a tricolour system and extension of the scanning to larger areas. An increase in scanning frequency to produce the image in shorter time allowing the operator to move around the sample faster. Other imaging modes; for example, by using a microphone detector the equipment becomes an acoustic microscope, possibly detecting sub-surface structure. Detection of the infra-red reflection would lead to an image mapping of conductivity and specific heat; detection of Raman scattering using a Spectrum analyser in beam 11 UaU would give insight into the molecular species present. The accuracy of the image depends on the position-measuring equipment. There are a wide range of possibilities of which the interferometer type is probably the most accurate and capable of better than 1 ,tm resolution.

The system is suitable for automated, high accuracy measurements such as particle counting using a computer.

There has been described a microscope which comprises a laser source, a support for an object to be viewed, means for focussing radiation from the source on the object, means for effecting relative movement between the support and the focussing means, a visible display movable in orthogonal directions, and means responsive to said relative movement for effecting movement of the dis play in the orthogonal directions. There has also been described a microscope comprising a source of electromagnetic radiation; a sup port for an object to be viewed, means for focussing radiation from the source on the object, means for effecting relative movement between the radiation and the support, and a visual display means for providing a display responsive to the radiation from the object.

Claims (1)

1. A scanning microscope comprising a source of coherent electromagnetic radiation, means for supporting an object to be viewed, means for receiving a parallel beam of radiation from the source and adapted to focus the radiation on the object, means responsive to radiation from the object for forming an image of the object, and means for effecting relative movement between the focussing means and the support means for scanning the object.

2. A microscope as claimed in claim 1, in which the radiation is reflected from the object.

3. A microscope as claimed in claim 1, in which the radiation is transmitted through the object.

4. A microscope as claimed in claim 1 or claim 2, in which the radiation is polarized, and means for rotating the plane of polarization.

5. A microscope as claimed in any preceding claim, including a visible display element moved in response to the relative movement.

6. A microscope as claimed in claim 5, including means correlating the intensity of the display and the radiation from the object.

7. A microscope as claimed in any preceding claim, in which the focussing means is moved to obtain a line scan and the support means is moved to obtain a frame scan.

9. A microscope as claimed in claim 2. said responsive means comprising a beam splitter through which said parallel beam passes, a photodiode for receiving from said beam splitter radiation reflected from the object, and visual display apparatus connected to the photodiode for displaying an image of the object.

8. A microscope as claimed in claim 7, comprising sensing means having a capacitance which changes in response to said line scan.

9. A scanning microscope substantially as hereinbefore described with reference to the accompanying drawings.