WO2002086578A2

WO2002086578A2 - Objective-type dark field scattering microscope

Info

Publication number: WO2002086578A2
Application number: PCT/IL2002/000323
Authority: WO
Inventors: Ido Braslavsky; Joel Stavans; Roee Amit
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2001-04-24
Filing date: 2002-04-24
Publication date: 2002-10-31
Also published as: IL142782A0; WO2002086578A3; AU2002307784A1

Abstract

A dark field microscope comprising an objective (18) lens which both illuminates and images a sample carried by a substrate (21), by means of a beam stop (22) located such that it blocks light back-reflected from the substrate, leaving only the scattered light to be detected. The microscope, in its most convenient embodiment, has an additional optical element (23) for forming a conjugate image of the back focal plane (BFP)) of the objective at an equivalent back focal plane located outside of the objective. A beam stop located in the region of this equivalent back focal plane, being outside of the objective lens, can be conveniently shaped and installed. This use of a spatial beam stop obviates the need for fluorescence techniques thus providing means of tracking particles of only tens of nanometers in size for long periods without the bleaching effects typical of fluorescence microscopy.

Description

OBJECTIVE-TYPE DARK FIELD SCATTERING MICROSCOPE

FIELD OF THE INVENTION

The present invention relates to the field of dark field microscopy, especially as related to methods of tracking sub-wavelength sized samples in solution by means of the light scattered from them.

BACKGROUND OF THE INVENTION

The detection of motion of particles suspended in solution near a surface is a powerful method to monitor a variety of physical, chemical and biological processes. Examples include measurements of colloidal forces, DNA elasticity, DNA-protein interactions at the single molecule level, and the unfolding of proteins. A common feature of such studies hitherto performed is the large size of the particles generally used, which range between 2 and 20 Dm. Within this size range, particles such as polystyrene beads scatter light strongly, and therefore their motion can be detected easily with simple devices such as photodiodes.

In many applications, particularly in biology, there is a need to track the motion of much smaller particles attached, for instance, to a molecule of interest. One such example is the recent study of protein-mediated DNA looping, by L. Finzi, and J. Gelles described in the article "Measurement of Lactose Repressor- mediated Loop Formation and Breakdown in Single DNA Molecules," published in Science, Vol. 267, pp. 378-380 (1995), in which changes were followed in the Brownian motion of 0.22 Dm polystyrene beads tethered to a surface by short DNA molecules. For some studies, however, the ability to track even smaller particles would be desirable. The need to use such small particles stems from the requirement that the particles should interfere as little as possible with the phenomenon under study. One problem posed by the use of such small particles is their detection. In the book "Light Scattering by Small Particles", by H. C. van de Hulst, published by Dover, New York in 1981, it is shown that the scattering cross section decreases as the sixth power of the bead size in the Rayleigh-Gans approximation. This precludes the detection of such small beads with bright-field illumination.

Conventional dark-field microscopy (DFM) techniques are able to enhance the contrast between dim or transparent objects in the field of view, and their background. All dark field methods involve the detection of light scattered by an object illuminated by obliquely incident rays. This is usually achieved by placing a beam block in the illumination path, so that the zero-order illumination rays which pass through the transparent object and would normally cause a high level illumination background, are effectively blocked off. This prevents direct transmission of light not scattered in the sample, thereby enabling the observation of phase objects, and generally enhancing contrast and increasing the image signal to noise ratio.

Conventional DFM techniques, however, suffer from a number of drawbacks. Firstly, a specially constructed dark-field combination condenser/ objective lens is generally required, which provides for the transmission of the illuminating annular beam at its outer periphery, and has a reflection means in the nose of the lens housing for directing the illumination as a hollow cone of light onto the object plane at a range of oblique angles of incidence. Secondly, in order to achieve true dark-field conditions, the numerical aperture of the objective must be smaller than that of the condenser, to avoid collecting the unscattered oblique rays, thus limiting both the collection efficiency and the image resolution.

More sophisticated schemes such as fluorescence detection, or differential interference contrast (DIC), can be used for viewing very small particles. Both of these techniques allow the observation of particles tens of nanometers in size. Fluorescence methods, though, have a major disadvantage, in that they make it difficult to achieve long viewing times, since photobleaching effects place a practical limit of an hour, or even less, for detection. DIC methods, on the other hand, conventionally use visible light illumination and viewing at the same wavelength, and also do not arouse photobleaching effects, but generally involve more complex microscope set-ups, and so may be less convenient to use.

A common method of illuminating the object in microscopy is by means of epi-illumination, whereby the object is illuminated through the objective itself. Epi-illumination through the periphery of the condenser/objective allows oblique illumination to be easily achieved. A.L. Stout and D. Axelrod, in an article entitled "Evanescent Field Excitation of Fluorescence by Epi-illumination Microscopy," published in Applied Optics, Vol. 28, pp. 5237-5242 (1989), hereby incorporated by reference in its entirety, describe the use of epi- illumination to achieve total internal reflection microscopy (TIRM). These authors discuss the advantages of an objective to create evanescent illumination, as opposed to the traditional method in which a prism is employed. In this application, oblique illumination is produced by illuminating the back aperture of a large numerical aperture (NA) objective with annular illumination produced by blocking the central portion of an expanded illuminating laser beam with an opaque disk. This disk is positioned along the illuminating optical path, at a plane conjugate to the back focal plane (BFP) of the objective, which Stout and Axelrod call the equivalent back focal plane (EBFP). The diameter of the disk is chosen to allow through only those rays whose angles of incidence are supercritical, such that they are totally reflected at the glass-water interface.

However, this method, being a total internal reflection method, has the disadvantage that since the illumination rays are totally reflected by the glass- water interface, they enter back into the objective and may flood out the scattering signal of a small object, such as a bead, making it impossible to view these signals. In order to view very small objects, this arrangement is therefore only useful for fluorescence applications, where the use of an emission filter allows the fluorescent signal through, but rejects the reflected rays. As stated above, fluorescence microscopy suffers, however, from other disadvantages, limiting its usefulness for long-term tracking of such small particles.

A further disadvantage arising from such a total internal reflection method, is that evanescent field illumination is used. Since the evanescent mode illumination field decays almost completely within about a half-wavelength of the interface, it can only illuminate specimen detail very close to the glass-water interface. Objects deeper in the sample solution cannot therefore be detected.

A dark field illumination apparatus, which should be capable of deeper illumination within such samples, and without the disadvantages of fluorescence excitation, is described in U.S. Patent No. 4,291,938 to D. Wagner. The annular illumination is produced by means of an array of optical fibers with their end faces arranged annularly in the back focal plane of a microscope objective. Light emitted by the optical fiber ends becomes dark field illumination for the object plane of the objective lens, when viewed through the central aperture inside of the annular region. This method, though, has another disadvantage, in that it requires the use of a dedicated objective lens and microscope tube assembly, such that the widely available range of conventional microscope component elements cannot be easily used or interchanged within the set-up. To the best of the applicants' knowledge, this method never achieved widespread use.

There therefore exists an important need for a simple and efficient method and apparatus for the long-term viewing and tracking of sub-wavelength sized particles, using visible wavelength illumination, and by means of simple adaptations to a standard microscope arrangement.

The disclosures of each of the publications mentioned in this section and in other sections of the specification, are hereby incorporated by reference, each in its entirety.

SUMMARY OF THE LNV

The present invention seeks to provide a new, fluorescence-less method and apparatus capable of the detection and the long-term tracking of particles of sizes down to tens of nanometers in solution near the specimen slide surface. The apparatus need involve only simple modifications to an epi-illumination microscope and can be used as an alternative to DIC microscopy, but with significantly less complexity, and without the need for special-purpose objectives. The method uses epi-illumination over only a part of the available optical aperture. Unlike the prior art oblique ray illumination methods for improving image contrast, such as that of Stout and Axelrod and others, in which the strongly back-reflected rays are removed by means of a wavelength dependent optical filter, according to the present invention the back-reflected light of the illumination is blocked by a suitably located and shaped beam stop, enabling the comparatively weak back scattered light from the sample to be observed. According to different preferred embodiments of the invention, the illumination can be fully or partially annular illumination, whether peripheral or elsewhere across the diameter of the optical aperture, or it can be axially directed through the objective aperture. Though these are the most convenient types to use, the illumination can be in any other convenient location across the objective aperture, and of any convenient shape, on condition that an easily determined equivalent lateral position and shape can be defined for the beam stop. For this reason, the simpler defined types of illumination are generally preferred. It should be noted though that differently positioned illumination may result in different resolution. Thus, for instance, axial or near axial illumination produces a lower resolution image than peripheral illumination.

There is therefore provided, according to a preferred embodiment of the present invention, a microscopy method of viewing very small particles, wherein the back-reflected illumination beam of the object is blocked by inserting a field stop at an appropriate location along the reflected light path, leaving only the light scattered by the particles to proceed unhindered to the viewing camera. Since the scattered light is of the same wavelength as the light reflected back by the glass-water interface into the objective, such that filters cannot be used to separate the two, if the field stop of the present invention were not used, the high intensity reflected light would flood out the low intensity scattered light which is to be detected. A preferred location for the field stop is at a second EBFP formed on the return path of the image by an imaging lens. The total effect is one of dark field illumination, and the method is thus called objective-type, dark-field scattering microscopy (hereinafter ODFSM). Unlike the above-mentioned prior art fluorescence microscopy applications, which use a spectral filter to block the unwanted directly reflected light, and also the scattered light, in ODFSM, a spatial filter is used to block the directly reflected light and to leave the scattered light intact, as far as is possible. An alternative preferred location for the field stop is at or close to the true back focal plane of the objective, though this position may not always be accessible, and is generally not as convenient to use as the EBFP mentioned above.

In conventional objective-type TIRM, the function of the annular illumination is generally to produce evanescent illumination within the substrate. In ODFSM, on the other hand, the function of annular illumination, if such illumination is used, is primarily to facilitate the elimination of reflected rays. This is accomplished, according to two different preferred embodiments of the present invention, either by illuminating with a complete annulus and using an annular field stop to absorb the entire reflected annular beam, or by using a crescent-shaped or partly annular illumination beam, and absorbing the crescent- shaped or partly annular reflected light by means of a semi-circular or partly semi-circular field stop located at the diametrically opposite side of the optical path to that of the incident illuminating crescent beam. If another form of spatially partial illumination is used, other than peripheral illumination, the elimination of directly reflected rays is preferably achieved by the use of a suitably shaped and positioned beam stop. Thus, for example, a narrow circular axial illumination beam would require a centrally located axial stop, preferably at the EBFP, to absorb the generally circularly shaped, axially positioned, reflected light.

The use of oblique illumination has been implemented in the past in order to enhance image contrast, and to observe phase objects without using special- purpose objectives. Many such applications are described in a review article entitled "Diffracted-Light Contrast Enhancement: A Re-Examination of Oblique Illumination", by W. B. Piekos, published in Microscopy Research and Technology, Vol. 46, pp. 334-337 (1999), and in the numerous references therein. In the article by D. Axelrod, entitled "Zero-Cost Modification of Bright Field Microscopes for Imaging Phase Gradient on Cells: Schlieren Optics", published in Cell Biophysics, Vol. 3, ρp.167-173 (1981), there is described a method of modifying a bright field transmission microscope in order to enable phase contrast to become visible. In this method, beam stops are inserted at the front focal plane of the condenser, and the back focal plane of the objective, and the phase gradient of the sample becomes visible by the schlieren effect. However, as is mentioned in that article, "the focal planes of the objective and condenser are not always easily accessible", and in particular, "high magnification objectives in which rear access to the back focal plane is blocked by a lens cannot be modified easily for schlieren optics." For those objectives where it is possible, the back focal plane stop can be mounted on a thin-walled hollow tube inserted from the back of the objective. This then has the disadvantage that it takes up part of the objective aperture. Furthermore, it is not clear from the Axelrod article how the schlieren method could be adapted for application in reflection microscopy.

According to another preferred embodiment of the present invention, by illuminating through the objective from less than half of the circular cross section of the objective and by blocking the reflected light at the opposite half of the objective's circular cross section, either at the BFP or in its vicinity immediately at the out port of the objective, an alternative embodiment of ODFSM can be achieved. . Similar results can also be obtained with the beam block at or as close as possible to the BFP, by illuminating through any other selected part of the objective's aperture, and blocking at the corresponding region where the reflected beam outputs the objective, so long as less than half of the effective cross section of the objective is illuminated.

In those of the preferred embodiments of the present invention in which use is made of a beam block at an additional EBFP created outside of, and at a distance from the objective, the ODFSM technique has the additional flexibility of blocking unwanted light simply and without needing to change the mechanical or optical structure of the objective, as for instance, is necessary in the Wagner method, and in the Axelrod schlieren transmission method mentioned hereinabove, and in the preferred embodiments mentioned above where the beam stop is at the BFP, where this falls within the objective. Furthermore, by illuminating through the objective, which then also plays the role of a condenser, in most ODFSM configurations, use can be made of almost the whole of the objective's high numerical aperture, thus benefiting from all the known advantages of objective-type illumination. The technique of illuminating and blocking only a small portion of the EBFP, as performed in the ODFSM technology according to the preferred embodiments of the present invention, ealmost the entire numerical aperture of the objective lens to be utilized for its light collection function. Though the numerical aperture of the illumination function could thereby be compromised, this is more than overcome by the gain in sensitivity for collection of the scattered signal, which is usually, very small. For applications in tracking small individual particles, it is important to collect as much as possible of the weak optical scattered signal and resolution is a less important factor.

According to another preferred embodiment of the present invention, the illumination can be generated by means of a diffractive optical element. Use of such an element enables the majority of the incident source light to be directed into the annulus, or into the other preferred illuminating beam shape used, unlike prior art annular illumination systems using beam blocks, which consequently waste that part of the illumination not transmitted in the annulus. Using an annular beam shape as an example for illustrating the advantages of such a diffractive optical element, the diameter and aspect ratio of the annular illuminating beam can be varied, by means of axial motion of an imaging lens down-beam from the diffractive element. Variation in size and rim thickness of the annular illuminating beam varies the range of angles of incidence of the illumination on the sample substrate interface, thus allowing the microscope of the present invention to be used, according to another preferred embodiment, either in a pure non-evanescent mode, or with a partially or fully evanescent field illumination mode, depending on the diameter of the annular illumination beam selected.

The sensitivity of the method is such that, using only a simple CCD camera, it is possible to detect polystyrene beads as small as 60 nm, and gold particles as small as 20 nm in diameter, with a signal-to-background ratio of the order of 5 to 6.

There is thus provided in accordance with a preferred embodiment of the present invention, a dark field microscope comprising an objective lens for illuminating a sample carried by a substrate and also for imaging the sample, and a beam stop located such that it blocks light back-reflected from the substrate.

In the dark field microscope as described above, the objective lens preferably has a back focal plane, and the microscope also preferably comprises an optical element for forming a conjugate image of the back focal plane at an equivalent back focal plane located outside of the objective, and the beam stop is preferably located in the region of the equivalent back focal plane. Alternatively and preferably, the beam stop may be located either at or in the region of the back focal plane itself. Furthermore, the sample may be preferably illuminated by a beam incident on part of the objective lens, and the beam stop preferably has a shape such that it blocks illumination back-reflected from the substrate. According to more preferred embodiments, the part of the objective lens may be an annular part and the beam stop annular such that it blocks the illumination back-reflected from the substrate, or it may be at least a part of an annulus of the objective lens and the beam stop partly annular such that it blocks the illumination back-reflected from the substrate, or it may be an axial part, and the beam stop axial such that it blocks the illumination back-reflected from the substrate.

There is further provided in accordance with yet another preferred embodiment of the present invention, a dark field microscope, comprising an optical assembly having an object plane and a back focal plane, illumination incident on part of the optical assembly, an optical element for forming a conjugate image of the back focal plane at an equivalent back focal plane located outside of the optical assembly, and a beam stop located in the region of the equivalent back focal plane to block light back-reflected from the object plane. Additionally, the part of the of the optical assembly may preferably be at least a part of the periphery of the optical assembly, or an axial part of the optical assembly. Furthermore, the beam stop may preferably be located at the equivalent back focal plane. Additionally, the optical assembly may preferably be an objective lens, and the object plane may consist of a substrate carrying a sample to be viewed.

In accordance with still more preferred embodiments of the present invention, in the previously described dark field microscope, the illumination may preferably be in the shape of an annulus and the beam stop in the shape of an aperture. Alternatively and preferably, the illumination may be in the shape of at least part of an annulus and the beam stop may likewise be in the shape of at least part of an annulus. Furthermore, the illumination may preferably consist of off- axis rays incident on part of the periphery of the optical assembly, and more preferably, on no more than one half of the periphery of the optical assembly. Additionally, the illumination may preferably be a narrow axial beam and the beam stop may be in the shape of a small axial plate.

There is further provided in accordance with still another preferred embodiment of the present invention, a dark field microscope as described above, and wherein the illumination is in the shape of a first arced part of an annulus, and the beam stop has a shape which covers at least part of a circle, and is oriented such that the at least part of a circle is located essentially diametrically opposite to the first arced part of an annulus with respect to the optical axis of the optical assembly. Preferably, the shape of the above-mentioned first arced part of an annulus, is a crescent. Alternatively and preferably, the above-mentioned at least part of a circle is a second arced part of an annulus.

In accordance with a further preferred embodiment of the present invention, there is also provided a dark field microscope as described above, and wherein the illumination enters the periphery of the objective lens on one side relative to the optical axis of the objective lens, and exits the periphery of the objective lens after reflection from the substrate at a diametrically opposite side of the objective lens with respect to the optical axis of the objective lens.

In accordance with yet further preferred embodiments of the present invention, in any of the dark field microscopes described above, the beam stop is operative to prevent the reflected light from flooding out back-scattered light from the sample. Preferably, the back-scattered light from the sample may have the same wavelength as the illumination beam.

There is even further provided in accordance with a preferred embodiment of the present invention, a dark field microscope as described above, and wherein the objective lens illuminates the sample through its periphery, and collects light from the sample through the majority of its numerical aperture.

Furthermore, in accordance with yet another preferred embodiment of the present invention, there is provided a dark field microscope as described above, and also comprising a beam steering element which laterally moves the illumination across the optical assembly such that the range of angles of incidence of illumination on the object plane is varied with lateral movement of the beam steering element.

There is also provided in accordance with a further preferred embodiment of the present invention, a dark field microscope as described above, and also comprising a beam expanding element which varies the width of the illumination incident on the optical assembly such that the range of angles of incidence of illumination on the object plane is varied with lateral movement of the beam steering element. In either of the two previously mentioned preferred embodiments, the microscope may be such as to be variable from a pure non- evanescent mode to at least a partially evanescent mode of illumination. In accordance with yet another preferred embodiment of the present invention, there is provided a dark field microscope comprising a source of light and a diffractive optelement which generates an essentially annular illuminating beam from the light emitted by the source. The dark field microscope may preferably also incorporate an axially movable imaging lens operative to image the annular illuminating beam, such that the size of the annular illuminating beam may be varied with axial movement of the imaging lens. Furthermore, in this microscope, the range of angles of incidence of illumination on the sample substrate interface may preferably be varied with axial movement of the imaging lens. Additionally, the microscope may thus preferably be varied from a pure non-evanescent mode to at least a partially evanescent mode of illumination.

There is further provided in accordance with yet another preferred embodiment of the present invention, a method of viewing microscopically a sample on a substrate, comprising the steps of providing an optical assembly having an object plane and a back focal plane, illuminating at least part of the periphery of the optical assembly by means of an incident beam, forming by means of an optical element, a conjugate image of the back focal plane at an equivalent back focal plane located outside of the optical assembly, and blocking light back-reflected from the object plane by positioning a beam stop in the region of the equivalent back focal plane. The optical assembly may preferably be an objective lens. In addition, the object plane may preferably consist of a substrate carrying a sample to be viewed.

In accordance with still another preferred embodiment of the present invention, in the method described above, the incident beam may in the shape of at least part of an annulus and the beam stop likewise in the shape of at least part of an annulus.

There is further provided in accordance with still another preferred embodiment of the present invention, a method of viewing the motion of sub- wavelength sized particles near a surface, comprising the step of mounting the particles in a dark field microscope as described above, and viewing the motion of the particles.

In accordance with a further preferred embodiment of the present invention, in the above-mentioned method, the sample may be viewed for effectively unlimited periods of time.

There is provided in accordance with yet a further preferred embodiment of the present invention, a method of three dimensional tracking of sub- wavelength particles close to a surface, comprising the steps of mounting the particles in a dark field microscope as described above, axially moving the imaging lens such that the sub-wavelength particles are illuminated with partially evanescent illumination, and tracking the sub-wavelength particles three dimensionally.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be appreciated more fully from the following detailed description, taken in conjunction with the drawings, in which:

Fig.l is a schematic illustration of an objective-type, dark-field, scattering inverted microscope, constructed and operative according to a preferred embodiment of the present invention, with annular illumination and an annular reflected beam stop at the EBFP;

Fig.2 A is a schematic illustration of an objective-type, dark-field, scattering inverted microscope, similar to that shown in Fig. 1, but using partially annular illumination and an off-axis beam stop located at the EBFP;

Fig.2B is a schematic illustration of an objective-type, dark-field, scattering inverted microscope, similar to that shown in Fig. 2A, but with the beam stop located at or near the BFP;

Fig.3 is a schematic illustration of an objective-type, dark-field, scattering inverted microscope, similar to that shown in Fig. 1, but using near axial illumination and a near axial beam stop;

Fig. 4 is a schematic drawing of the optical arrangement for implementing the scheme shown in Fig. 1 for achieving ODFSM conditions in a commercial epi-illumination microscope, according to a preferred embodiment of the present invention; and

Fig. 5 is a graph of the measured signal to noise of the images of small beads of various sizes, comparing prior art bright field illumination with ODFSM according to a preferred embodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Reference is now made to Fig. 1, which illustrates schematically an objective-type dark-field scattering inverted microscope, constructed and operative according to a preferred embodiment of the present invention. In order to better illustrate the operation of the ODFSM method according to the present invention, the light paths in the preferred embodiment of Fig. 1 (and in those of Figs. 2 and 3) are drawn differently according to their functions. Thus the incident illuminating rays are drawn as full lines, the directly reflected rays are shown as fine dotted lines, and the scattered light from the object is shown by dashed lines. A diffractive element 10 made of a binary surface with a radial phase grating is preferably used to convert the cross-section of the illuminating laser beam 12 into a thin expanding annulus 14. About 80% of the laser beam is typically concentrated into the diverging annulus. This method of production of the annular illuminating beam is in contrast to the conventional circular block illuminator used both in the Stout and Axelrod article, and in many dark-field microscopy methods, where the blocked part of the illumination is effectively lost, and the illumination on the sample, for a source of the same luminance, is significantly lower. Alternatively and preferably, a waxicon type of aspheric reflective element may be used to provide the annular illuminating beam, this too providing an illumination efficiency level significantly higher than the prior art beam blocks. Either of these methods are considerably simpler to implement than the annular optical fiber ends described in the Wagner patent document. It is appreciated, though, that other methods of producing an annular beam can also be used according to other embodiments of the present invention.

Lens LI forms an image of the annular diffraction pattern at its focal plane. Residual traces of zeroth-order illumination from the diffractive element are eliminated by an opaque disk 16 at this plane, whose diameter is slightly smaller than the inner diameter of the annulus. However, unlike prior art illumination beam blocks, this opaque disk does not determine the size of the annulus, this being determined by the optical properties and the location of the optical components as will be explained hereinbelow.

A second lens, L2, images the annulus onto the back focal plane (BFP) of the objective 18, in such a way that rays reach the BFP essentially parallel. Therefore, the focal plane of LI where the image of the annulus is first formed, is conjugate to the back focal plane of the objective, and defines an equivalent back focal plane, marked as EBFP1. For the sake of clarity in following the various beam paths through the objective, the incident illumination is shown impinging on the BFP on one side only of the objective, and the associated reflected outgoing rays are shown exiting the objective only on the opposite side. It is to be understood though that the actual incoming and outgoing beams are preferably both complete annuli.

The relative distances between the four optical components LI, L2, the diffractive element 10, and the objective 18 are arranged such that the outer diameter of the annular illumination impinging on the objective is of the same size, or slightly larger than the back aperture of the objective 18. Parallel illumination at the BFP of the objective is achieved when the distance between the diffractive element 10 and LI is adjusted so that a spot image of the beam impinging on the diffractive element is formed at the focus of L2. By varying the positions of the optical elements, the size of annulus formed at the BFP can be varied accordingly. This feature of the present invention has novel applications for ODFSM microscopy, as will be further expounded below.

The beam is steered into the objective by means of a beam splitter 20. In this co, the beam is focused into a spot of limited size on the sample 19, under nearly evanescent illumination conditions. The size of the illumination spot, about 30 μm in the embodiment shown in Fig. 1, is proportional to the size of the spot at the focus of lens L2, as will be explained below.

In order to block that part of the rays transmitted through the beam splitter 20, which were directly reflected at the glass/water interface of the sample substrate 21, a field stop 22 is preferably placed at a plane EBFP2, conjugate to the objective BFP, formed by lens L3. This field stop 22, preferably in the form of a hole in a screen, blocks the annular-shaped image produced by lens L3, of the rays emerging from the objective 18, thus creating a dark field effect. The scattered light from the object specimen is, however, transmitted paraxially through the hole in the field stop, and an image of the scattering objects is formed on the CCD camera 24 by means of lens L4. The need for L4 arises from the fact that the location of the EBFP2 lies beyond the focal plane of L3, in which an image of the sample plane is formed when an infinity-corrected objective is used. If the diameter and width of the annular illumination are chosen appropriately, total internal reflection conditions can be attained. Pure evanescent illumination is, however, hard to achieve by objective-type methods, as explained hereinbelow. The use of higher NA objectives, may make this easier to achieve, though, using such objectives require special high refractive index glass slides and special oil.

The creation of an additional EBFP2 plane outside of the body of the objective, thus enables any amendments to the optical performance of the standard microscope to be implemented outside of the microscope structure, thus greatly simplifying the execution of the method.

The size of the illuminating spot, 30 μm, as mentioned above, is proportional to the cross section of the laser beam as it impinges on the diffractive element 10. This is because the cross-section determines the size of the spot at the focus of lens L2, since the location of the diffractive element 10 and the focus of L2 are conjugated by LI. Since the focus of L2 and the sample plane are conjugate, the illuminated spot is circular and proportional to the impinging beam cross section at the diffraction element. The size of the illuminating spot can therefore be controlled by expanding the spot size on the diffractive element itself. This differs from the way in which the illumination spot size is controlled in the Stout and Axelrod article mentioned above, in which use is made of a diffusive element.

Reference is now made to Figs. 2A and 2B, which are schematic illustrations of an objective-type dark-field scattering microscope, constructed and operative according to alternative preferred embodiments of the present invention. Instead of the full annulus illumination used in Fig. 1, in these embodiments, the BFP of the objective is illuminated with a beam having a crescent-shaped cross' section 30. This beam is created by blocking the illuminating laser beam at the EBFP of the objective formed by the lens L3, with a slightly off-axis disk 32. Such a crescent-shaped beam enters the objective from one side 36, while the rays directly reflected at the glass/water interface of the sample substrate 33, emerge from the side 38 diametrically opposite with respect to the optical axis, to that of the incident beam.

In the embodiment shown in Fig. 2A, in order to block these directly reflected rays, a field stop 40 is placed preferably at a plane EBFP2, conjugate to the objective BFP, formed by lens L4. This field stop 40, which generally covers less than half of the objective's back aperture, blocks the crescent-shaped image produced by lens L4, of the rays emerging from the objective 34, thus creating a dark field effect.

Since the field stop is located at the EBFP, and there is therefore no interference between the illuminating beam and the reflected beam block, it is possible to make the illuminating beam larger than half of the periphery and the beam block correspondingly larger than half of the aperture of the objective. If the stop were, alternatively and preferably, to be located at or close to the real BFP, this would not be possible, and only up to half of the respective sides of the aperture could be used. However, even for the situation according to this preferred embodiment of the present invention, with the stop at the EBFP, in general no more than half of the aperture would practically be used, in order not to limit the useful numerical aperture unnecessarily.

The scattered light from the object specimen is, however, transmitted paraxially past the field stop, and an image of the scattering objects is formed on the CCD camera 46 by means of lens L5. The need for L5 arises from the fact that the preferred location of the EBFP2 lies beyond the focal plane of L4, in which an image of the sample plane is formed when an infinity-corrected objective is used. Although the preferred embodiment shown in Fig. 2 uses a crescent shaped illuminating beam, simply formed by the use of a disk 32 disposed slightly off-axis in the illuminating beam, it is to be understood that a similar effect is obtained with an illuminating beam in the form of any arc of part-annular shape, or which covers a lateral segment of the cross section of the aperture.

The preferred embodiment shown in Fig. 2 also illustrates the use of an extended source for illuminating a wide area on the sample. Lenses LI and L2 constitute a conventional illumination beam telescope. The focal plane of L2 is conjugate with the sample plane. If the lens LI images point-like illumination on this focal plane, the result will consequently also be point-like illumination on the sample plane. On the other hand, if a diffusing screen 44 is disposed at this plane, the laser beam is converted into an effectively extended source, and illuminates at a range of oblique angles of incidence, such that a wider area on the sample is illuminated. The diffusing screen is preferably rotated in order to reduce the speckle effect arising from the use of a coherent source. Alternatively and preferably, removal of the diffusing screen enables the configuration of Fig. 2A to be also used for spot-like illumination of the sample, as described in the previously described embodiment.

Reference is now made to Fig. 2B which is a schematic illustration of an objective-type dark-field scattering microscope, constructed and operative according to an alternative preferred embodiment of the present invention. Instead of locating the beam stop at the EBFP2, as used in Fig. 2A, in this embodiment, the beam stop is located near or at the BFP of the objective. The ideal location for the beam stop in this configuration would be at the BFP. It is in this position that the size of the beam stop can be minimal for effective blocking of the reflected illumination. However, this position is not generally easily accessible. A more accessible position is at the out-port of the objective, though a larger beam stop is needed there to block the reflected illumination, thus reducing the effective numerical aperture of the system. As in the embodiment of Fig. 2A the BFP is illuminated with a beam having a crescent-shaped cross section 30, or up to a semi-circular shaped cross section. Such a beam enters the objective from one side 36, while the rays directly reflected at the glass/water interface of the sample substrate 33, emerge from the side 38 diametrically opposite, to that of the incident beam, with respect to the optical axis. In order to block these directly reflected rays, a beam stop 35 is placed preferably at the objective BFP. Alternatively and preferably, the beam stop 37 is placed close to the BFP at the out-port of the objective. This beam stop 35 or 37, which covers less than half of the objective's back aperture, blocks the crescent-shaped, or the up to semicircular shaped reflected illumination, thus creating a dark field effect. Although the position of the field stop 37 does not coincwith the BFP, if it is located close to the out port, complete blocking of the reflected beam is generally achieved.

The scattered light from the object specimen is, however, transmitted paraxially past the field stop, and an image of the scattering objects is formed on the CCD camera 46 by means of lens L4. Although the preferred embodiments shown in Figs. 2A and 2B use a crescent shaped illuminating beam, simply formed by the use of a disk 32 disposed slightly off-axis in the illuminating beam, it is to be understood that a similar effect is obtained with an illuminating beam in the form of any arc of part-annular shape, or which covers a less then a half of the lateral segment of the cross section of the aperture.

Although the preferred embodiment of Fig. 2B illustrates the use of a beam block located at or close to the BFP with a crescent shaped, or a part arc segment shaped, or up to a semicircular shaped illuminating beam, it is to be understood that the use of a block at or close to the BFP is equally applicable when other types of beam shape are used, on condition that the beam stop does not block the illumination itself. This limitation does not exist when the beam stop is located at the EBFP.

Reference is now made to Fig. 3, which is a schematic illustration of an objective-type dark-field scattering microscope, constructed and operative according to an another preferred embodiment of the present invention. Instead of the full annulus illumination used in Fig. 1, and the off-axis partial annulus illumination used in Figs. 2A and 2B, in the embodiment of Fig. 3, the BFP of the objective is illuminated with a narrow axial beam 43. This beam is created, like that shown in the embodiment of Fig. 2, by focusing the illuminating laser beam preferably at the EBFP of the objective formed by the lens L3. Alternatively and preferably, the beam can be blocked down by means of a point-like apertured field stop 41 preferably located at the EBFP of the objective formed by the lens L3. The illumination size at the sample plane is determined by the range of illumination angles impinging on the BFP. This can be controlled by the amount of expansion of the laser beam prior to entry into lens LI.

The narrow axial shaped beam 43 enters the objective 34 axially near its center, while the rays directly reflected at the glass/water interface of the sample substrate, also emerge axially from the objective. In Fig. 3, for the sake of clarity in separating the various types of rays, the input illumination is shown slightly off-axis in a near axial configuration, such that the directly reflected rays also exit the objective slightly off-axis. In order to block these directly reflected rays, a field stop 45 is placed preferably at a plane EBFP2, conjugate to the objective BFP, formed by lens L4. This field stop 45 is preferably in the form of a small axial plate, which blocks the small axial circular image produced by lens L4, of the rays emerging from the objective, thus creating a dark field effect. The scattered light from the object specimen, on the other hand, is transmitted paraxially past the small field stop, and an image of the scattering objects is formed on the CCD camera 46 imaging plane by means of lens L5, as previously described.

The configurations shown in Figs. 1 to 3 may preferably be implemented around an inverted microscope, using a standard x63 objective with a nominal numerical aperture of 1.4, such as that manufactured by Carl Zeiss GmbH, of Oberkochen, Germany. For illumination, light from a lO W HeNe laser at 633nm or a 30mW argon laser at 488nm may preferably be used, but these sources can also be substituted by any well-collimated monochromatic coherent or non-coherent source. Images are preferably captured with a CCD camera, preferably a Model 4710, manufactured by the Cohu Company of San Diego, California, and may preferably be recorded on video for further analysis.

Reference is now made to Fig. 4, which is a schematic drawing of the optical arrangement for achieving ODFSM conditions in a commercial epi- illumination microscope, according to a preferred embodiment of the present invention, and using the scheme of Fig. 1 with an annular beam stop. Fig. 4 shows the illumination path of a Zeiss Axiovert 135 microscope, converted for ODFSM use according to another preferred embodiment of the present invention. The standard microscope body is schematically shown enclosed within the lines 50. The front port is preferably used for the detection path. In order to find an appropriate equivalent back focal plane in which preferably to place the field stop 52 to block the reflected rays, lens L2 is added. L2 forms a new equivalent back focal plane EBFP* outside the microscope body 50, conjugate to the EBFP formed by the standard lenses inside the microscope body. L2 is needed to enable the placement of the field stop at the EBFP*, since the first EBFP is inaccessible, being situated inside of the microscope body. In the Zeiss Axiovert 135 microscope, there are three lenses 54, 56, 58, after the objective 60. The first lens 54 images the sample at its focus and, by means of the transfer lenses 56, 58, again further down. The other two lenses 56, 58, are located between the sample image and the EBFP. These lenses form a new image of the sample outside the microscope body. Lens L2 is added at the outport, in the vicinity of the image formed by lens 58, thus reproducing the internal EBFP at the position EBFP* outside the microscope body, while changing the position of the image only slightly.

To determine the exact position of EBFP* after L2, the objective is preferably illuminated with parallel illumination by introducing a pinhole in the BFP of the condenser, and adjusted the objective to its working position by focusing on a sample using the eye-piece. The plane where light emerging from the front port is focused by L2 into a point, is the EBFP*, and the field stop is placed at this position. Finally, another lens, L3, is placed beyond the EBFP*, to form an image for detection with a CCD camera 62. The image plane is determined by matching the features observed beyond L3 with those observed simultaneously by the eye-piece. For the Zeiss Axiovert 135, lenses with a focal length of 50 mm are suitable for L2 and L3. The distance between L2 and the field block is preferably 100 mm. The overall length between the microscope outport and the CCD imaging plane is preferably 225 mm.

Reference is now made to Fig. 5, which is a graph of the measured signal- to-noise ratio of the images of small polystyrene beads of various sizes, as a function of bead diameter, comparing normal bright field illumination with ODFSM according to the preferred embodiment of the present invention shown in Fig. 1. The bright field illumination results are shown as open circles, while the ODFSM results are shown as full circles.

The signal-to-noise ratio (S/N) in these images is measured by taking the average intensity of the bead image, I_bead, minus the average intensity of the background, ac_kp-_ound , and dividing by the standard deviation of the background

^background-

C* - /* head - ^Λ / background

N ^~ σ background

This definition of S/N is robust with respect to linear transformations such as the autogain facility of the camera. As is clearly observed in Fig. 5, the S/N of the images obtained using ODFSM, according to preferred embodiments of the present invention, is significantly higher than that obtained with normal bright field illumination. Fig. 5 shows that using ODFSM, it is possible to detect beads with sizes of below 300nm, with a high S/N. In contrast, using bright field illumination, the same sized beads are not even detectable. Finally, it is observed that using ODFSM according to preferred methods of the present invention, beads with a diameter of only 60nm are clearly observed, with a S/N of the order of 6. In contrast to this, for bright field illumination, the S/N did not exceed 6 even for beads as large as 800nm.

Using the preferred ODFSM apparatus described in Fig. 2A, and an argon laser for illumination, it has been found possible to detect gold particles stuck to a glass s, of dimensions as small as 20nm. The image of such particles appears as a bright spot on the dark field background, with a S/N of 5.

It is to be understood that the applications given hereinabove are only examples of typical results achievable with ODFSM, using the optical elements and detection techniques described in the above mentioned preferred embodiments. It is thus understood that the method and apparatus described in this application are not limited to the examples described hereinabove but include any other applications utilizing the methods and apparatus as claimed.

In the prior art methods of TIRM, using evanescent mode illumination, a very narrow range of angles of incidence of the illuminating beam is used, limited on the one hand by the need to have supercritical incidence at the glass-water sample interface, and on the other hand by the geometrical limitation of the numerical aperture of available objective lenses. Purely evanescent illumination is hard to achieve with objectives with a nominal numerical aperture of 1.4, since in practice, the measured numerical apertures are even slightly less than this, though new objectives with higher numerical aperture are becoming available. Furthermore, the presence of macromolecules and other solutes may slightly increase the index of refraction of the sample, restricting further the condition for total internal reflection. To satisfy the condition of purely evanescent illumination, the beam at the objective's back aperture must be very thin, as explained above, and is therefore difficult to collimate precisely with the objective rim. On the other hand, in ODFSM applications, where only the transverse bead displacement is monitored, a small portion of non-evanescent light can be tolerated, by means of also allowing some illumination with a less oblique angle of incidence than the critical angle, such that some light penetrates the sample solution, with the additional advantage that objects which are located further from the interface surface can be detected.

The ability to use both evanescent and non-evanescent illumination with ODFSM, enables the achievement of a further preferred embodiment of the present invention, whereby the annular illumination beam is varied in size and aspect ratio, thereby switching between evanescent and non-evanescent conditions, or a mixture of both. For an annulus of fixed rim thickness, when the annulus has a smaller diameter, pure non-evanescent conditions exist. As the diameter of the annulus is allowed to grow, a level of evanescent illumination is added, until the point is reached when all of the illumination is incident at supercritical angles, at which point the illumination is purely evanescent. An annulus with a broader rim allows the microscope to function in both non- evanescent and evanescent modes.

As previously mentioned, the size of the illuminating annulus, meaning in this specification, either its diameter or its rim thickness, or both, are determined by the optical properties and the location of the above-mentioned elements used to generate the annulus. For any given diffractive optical element, motion of the lenses LI and L2 along the optical axis, while maintaining a well-collimated illumination beam, causes the diameter of the annulus to change. According to yet another preferred embodiment of the present invention, there is therefore provided an ODFSM, capable of switching between evanescent and non- evanescent illumination, and mixtures thereof, by means of the adjustment of the annular illuminating beam. This adjustment can preferably be performed as described above. Alternatively and preferably, this effect can also be achieved in the embodiments shown in Figs. 2A and 2B, by means of a beam steering element disposed such as to laterally move the arc of annular illumination across the objective aperture, such that the incident angle on the glass/water interface switches between the subcritical and supercritical angle. According to a further preferred embodiment of the present invention, ODFSM with completely evanescent illumination may allow for three-dimensional tracking by using the sensitive exponential drop in intensity as a function of height, as is characteristic of total internal reflection techniques.

It is appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove. Rather the scope of the present invention includes both combinations and subcombinations of various features described hereinabove as well variations and modifications thereto which would occur to a person of skill in the art upon reading the above description and which are not in the prior art.

Claims

CLAIMSWe claim:

1. A dark field microscope comprising: an objective lens having a back focal plane, which illuminates a sample carried by a substrate, and also images said sample; and a beam stop located such that it blocks light back-reflected from said substrate.

2. A dark field microscope according to claim 1 and also comprising an optical element for forming a conjugate image of said back focal plane at an equivalent back focal plane located outside of said objective, and wherein said beam stop is located in the region of said equivalent back focal plane.

3. A dark field microscope according to claim 1 and wherein said beam stop is located in the region of said back focal plane.

4. A dark field microscope according to any of claims 1 to 3, and wherein said sample is illuminated by a beam incident on part of said objective lens, and said beam stop has a shape such that it blocks illumination back-reflected from said substrate.

5. A dark field microscope according to claim 4, and wherein said part of said objective lens is an annular part and said beam stop is annular such that it blocks said illumination back-reflected from said substrate.

6. A dark field microscope according to claim 4, and wherein said part of said objective lens is at least a part of an annulus of said objective lens and said beam stop is partly annular such that it blocks said illumination back-reflected from said substrate.

7. A dark field microscope according to claim 4, and wherein said part of said objective lens is an axial part, and said beam stop is axial such that it blocks said illumination back-reflected from said substrate.

8. A dark field microscope, comprising: an optical assembly having an object plane and a back focal plane; illumination incident on part of said optical assembly; an optical element for forming a conjugate image of said back focal plane at an equivalent back focal plane located outside of said optical assembly; and a beam stop located in the region of said equivalent back focal plane, and being shaped to block light back-reflected from said object plane.

9. A dark field microscope according to claim 8, and wherein said part of said optical assembly is at least a part of the periphery of said optical assembly.

10. A dark field microscope according to claim 8, and wherein said part of said optical assembly is an axial part of said optical assembly.

11. A dark field microscope according to any of claims 8 to 10, and wherein said beam stop is located at said equivalent back focal plane.

12. A dark field microscope according to any of claims 8 to 11, and wherein said optical assembly is an objective lens.

13. A dark field microscope according to any of claims 8 to 12, and wherein said object plane comprises a substrate carrying a sample to be viewed.

14. A dark field microscope according to either of claim 8 and 11, and wherein said illumination is in the shape of an annulus and said beam stop is in the shape of an aperture.

15. A dark field microscope according to either of claim 8 and 11, and wherein said illumination is in the shape of a narrow axial beam and said beam stop is in the shape of an axial plate.

16. A dark field microscope according to either of claim 8 and 11, and wherein said illumination is in the shape of at least part of an annulus and said beam stop is in the shape of at least part of an annulus.

17. A dark fiemicroscope according to either of claim 8 and 11, and wherein said illumination comprises off-axis rays incident on part of said periphery of said optical assembly.

18. A dark field microscope according to either of claim 8 and 11, and wherein said illumination is in the shape of a first arced part of an annulus, and said beam stop has a shape which covers at least part of a circle, and is oriented such that said at least part of a circle is located essentially diametrically opposite to said first arced part of an annulus with respect to the optical axis of said optical assembly.

19. A dark field microscope according to claim 18, and wherein said shape of a first arced part of an annulus is a crescent.

20. A dark field microscope according to either of claims 18 and 19, and wherein said at least part of a circle is a second arced part of an annulus.

21. A dark field microscope according to any of claims 18 to 20, and wherein said illumination enters the periphery of said objective lens on one side relative to the optical axis of said objective lens, and exits the periphery of said objective lens after reflection from said substrate at a diametrically opposite side of said objective lens with respect to the optical axis of said objective lens.

22. A dark field microscope according to any of claims 8 to 21, and wherein said beam stop is operative to prevent said reflected light from flooding out back- scattered light from said sample.

23. A dark field microscope according to claim 22, and wherein said back- scattered light from said sample has the same wavelength as said illumination beam.

24. A dark field microscope according to claim 12, and wherein said objective lens illuminates said sample through its periphery, and collects light from said sample through the majority of its numerical aperture.

25. A dark field microscope according to any of claims 14 to 21, and also comprising a beam steering element which laterally moves said illumination across said optical assembly such that the range of angles of incidence of illumination on said object plane is varied with lateral movement of said beam steering element.

26. A dark field microscope according to any of claims 14 to 21, and also comprising a beam expanding element which varies the width of said illumination incident on said optical assembly such that the range of angles of incidence of illumination on said object plane is varied with lateral movement of said beam steering element.

27. A dark field microscope according to either of claims 25 and 26, and wherein said microscope is variable from a pure non-evanescent mode to at least a partially evanescent mode of illumination.

28. A dark field microscope comprising a source of light and a diffractive optical element adapted to generate an essentially annular illuminating beam from the light emitted by said source.

29. A dark field microscope according to claim 28, and also comprising an axially movable imaging lens operative to image said annular illuminating beam, such that the size of said annular illuminating beam may be varied with axial movement of said imaging lens.

30. A dark field microscope according to claim 29, and wherein the range of angles of incidence of illumination on the sample substrate interface is varied with axial movement of said imaging lens.

31. A dark field microscope according to claim 30, and wherein said microscope is variable from a pure non-evanescent mode to at least a partially evanescent mode of illumination.

32. A method of microscopically viewing a sample on a substrate, comprising the steps of: providing an optical assembly having an object plane and a back focal plane; illuminating at least part of said optical assembly by means of an incident beam; forming by means of an optical element, a conjugate image of said back focal plane at an equivalent back focal plane located outside of said optical assembly; and blocking light back-reflected from said object plane by positioning a beam stop in the region of said equivalent back focal plane.

33. The method of claim 32, and wherein said optical assembly is an objective lens.

34. The method of claim 32, and wherein said object plane comprises a substrate carrying a sample to be viewed.

35. The method of claim 32, and wherein said incident beam is in the shape of at least part of an annulus and said beam stop is in the shape of at least part of an annulus.

36. The method of claim 32, and wherein said samples are sub-wavelength sized particles.

37. The method of claim 32, and wherein the motion of said sample is viewed for effectively unlimited periods of time without effecting the sample.

38. A method of three dimensional tracking of sub-wavelength particles close to a surface, comprising the steps of: mounting said particles in an object plane of an objective lens having a back focal plane; illuminating at least part of the periphery of said optical assembly by means of at least a partially annular beam; forming by means of an optical element, a conjugate image of said back focal plane at an equivalent back focal plane located outside of said optical assembly; positioning an annular beam stop in the region of said equivalent back focal plane such that it blocks said annular illuminating beam back-reflected from said substrate; changing the diameter of said annular beam such that said sub- wavelength particles are illuminated with at least partially evanescent illumination; and tracking said sub-wavelength particles three dimensionally.