Background Art Phase contrast imaging is a technique used to obtain images of objects, such as biological objects.

Phase imaging has particular application where the object is transparent or exhibits minimal absorption and cannot readily be imaged with conventional intensity imaging techniques such as usual photography. One technique for obtaining phase contrast images is disclosed in International patent application no. PCT/AU99/00949 in the name of The University of Melbourne.

In order to obtain a phase image it is necessary for images to be obtained at a minimum of two different planes and most preferably at three planes with one of the planes being the actual focal plane and the other two planes are defocused planes one on each side of the focal plane. By obtaining intensity information from these three planes the transport-of-intensity equation CM. R. Teague, J.

Opt. Soc. Am 73,1434-1441 (1993, can be solved in order to obtain phase information and enable a quantitative phase image to be constructed.

Traditionally, optical instruments have relied upon basic, mechanical methods to adjust the location of their focal planes. This has quite simply involved moving one or more of the instrument's optical components relative to a fixed image plane or, alternatively, moving the entire optical system relative to the object it is imaging.

Obvious examples of each are the focusing ring on a camera lens and the focus control knob of a microscope.

Where single images, or multiple images at a fixed focus, are required, such an approach is entirely

acceptable. If, however, a reproducible focal position or precise amounts of defocus are required, such methods are quite inadequate without major mechanical modification.

More importantly, if such changes in image focus also require a non-detectable change in the image magnification, or should mechanical construction or sample thickness prevent the aforementioned adjustments, even these mechanical modifications can prove worthless.

Typical imaging instruments also frequently incorporate dual imaging methods. For example, an eyepiece for operator viewing plus a recording medium for image storage, both of which must be in focus at the same time.

There is, therefore, the need for separate focal adjustments.

The need to obtain images at three different focal planes introduces some complications into lens systems which are used in instruments of this type. This is because of the need to move lenses in order to obtain the required images and this normally results in a different magnification of the image at each of the required planes.

Furthermore, typically in Z-stacker configurations in which it is desired to obtain images through different slices of a sample, the sample is moved by a small amount in the direction of propagation of the radiation towards a detector so that radiation from different planes within the sample are imaged at a common location at which a recording device such as a camera can be located. Generally, in order to obtain images at planes which are spaced a very small distance apart, very delicate and expensive equipment is required in order to move the sample the small distance required. In some situations, the ability to obtain required samples is limited by the precision of the movement which can be achieved.

Summary of the Invention The object of a first aspect of the present

invention is to provide a lens system which will enable radiation to be focused at different planes whilst obtaining a required power factor or magnification of the image at those planes.

The invention in a first aspect provides a lens system, including : a first lens array for receiving electromagnetic radiation from a source and for collimating the radiation into a collimated beam of radiation; a second lens array for receiving the radiation from the first lens array and for focusing the radiation; and moving means for moving the second lens array which receives the collimated radiation so as to move the focal plane of the radiation whilst maintaining a predetermined magnification or power factor.

Thus, by moving the second lens array the focal plane can be readily adjusted to thereby enable radiation to be focused at different planes. In applications of the invention applicable to phase imaging, a first of the focal planes can coincide with an imaging device for recording the radiation to provide an in focus image, and other focal planes can be adjusted by movement of the second lens array to be on either side of the imaging device so as to provide two defocused images to provide information to enable the transport-of-intensity equation to be solved.

In one embodiment of the invention the lens assembly provides zero power, that is unitary or unchanged magnification, at each focal plane, which is achieved by movement of the second lens array by providing the first lens array with the same focal length in magnitude as the second lens array.

However, if other than unitary magnification is required at each of the different focal planes then the focal length of each of the lens arrays can be different.

In one embodiment of the invention the first lens array is formed by a single negative lens and the second

lens array is formed by a single positive lens. The negative lens and positive lens each having the same focal length in magnitude.

In one embodiment of the invention the moving means comprises a microprocessor controlled translator for moving the second lens array. This provides precise control of the movement of the second lens array to thereby provide defocused images at planes which are shifted from the focal plane by a precise distance. This aspect also enables the focal plane to be readjusted with extreme accuracy so that the same focal plane can be reproduced as required.

In the preferred embodiment of the invention the translator comprises a motorised microminiature translator sold by National Apertures Inc, of Salem New Hampshire U. S. A., Module No. MM-3M-F.

Preferably the translator is contained within a housing coupled to an imaging device and also to a source of radiation, the housing including a carrier, a mounting plate connected in the carrier, the translator being connected to the mounting plate, the mounting plate having a support portion for supporting the first lens array in a stationary position, the translator having a support plate for mounting the second lens array so that the second lens array can be moved by the translator relative to the first lens array, and a closure plate for closing the carrier.

Preferably the housing is coupled to a microscope mount by a mounting bracket including a platform and a hollow tubular sleeve, and the housing is connected to the imaging device by a mounting bracket including a platform and a hollow tubular sleeve, the said platforms being connected to the carrier so as to form end walls of the carrier and enclose the first and second lens arrays within the carrier.

In another embodiment the housing is cylindrical and includes a cut out or window in which the translator is located. Preferably in this embodiment the housing is a

mounting assembly including a stem which carries the first lens array at an end of the stem remote from the translator, and a mounting plate opposite the mounting assembly, the mounting plate being for coupling the housing to a camera.

In the preferred embodiment of the invention, the radiation which is received by the first lens array is radiation converging towards the first lens array, the first lens array forming a negative lens for collimating the diverging radiation received by the first lens array.

An object of a second aspect of the invention is to provide a system in which images of different planes through a sample can be obtained, different planes being spaced apart by only a very small distance.

This aspect of the invention provides a lens assembly for imaging different locations of a sample at a common image plane, which locations of the sample are separated one from the other by a very small distance, the image of the sample being provided by an objective lens, the lens assembly including : a first lens array for receiving electromagnetic radiation from a source and for collimating the radiation into a collimated beam of radiation ; a second lens array for receiving the radiation from the first lens array and for focusing the radiation at the common plane ; and moving means for moving the second lens array which receives the collimated radiation so as to produce focused images from the different locations of the sample at the common focal plane whilst maintaining a predetermined magnification or power factor.

This aspect of the invention has particular application in situations where it is desired to obtain images of different locations in a sample, such as to provide a Z-stacker configuration of the sample. Because the first lens assembly is moved rather than the sample, the amount of movement of the lens assembly can be much

greater than what would be required if the sample is moved in order to provide images of planes within the sample which are spaced apart by a very small distance. In other words, a very small spacing or distance between the planes within the sample can be achieved by a much greater displacement of the second lens array. Thus, a small movement of the second lens array produces a much smaller separation of the planes within the sample which are being imaged. Thus, this aspect of the invention provides a much more economical and easy way of providing Z-stacker images of a sample than situations where the sample itself is moved to provide those images.

In one embodiment of the invention the first lens array is formed by a single negative lens and the second lens array is formed by a single positive lens. The negative lens and positive lens each having the same focal length in magnitude.

In one embodiment of the invention the moving means comprises a microprocessor controlled translator for moving the second lens array. This provides precise control of the movement of the second lens array to thereby provide defocused images at planes which are shifted from the focal plane by a precise distance. This aspect also enables the focal plane to be readjusted with extreme accuracy so that the same focal plane can be reproduced as required.

In the preferred embodiment of the invention the translator comprises a motorised microminiature translator sold by National Apertures Inc, of Salem New Hampshire U. S. A., Module No. MM-3M-F.

Preferably the translator is contained within a housing coupled to an imaging device and also to a source of radiation, the housing including a carrier, a mounting plate connected in the carrier, the translator being connected to the mounting plate, the mounting plate having a support portion for supporting the first lens array in a stationary position, the translator having a support plate

for mounting the second lens array so that the second lens array can be moved by the translator relative to the first lens array, and a closure plate for closing the carrier.

Preferably the housing is coupled to a microscope mount by a mounting bracket including a platform and a hollow tubular sleeve, and the housing is connected to the imaging device by a mounting bracket including a platform and a hollow tubular sleeve, the said platforms being connected to the carrier so as to form end walls of the carrier and enclose the first and second lens arrays within the carrier.

In another embodiment the housing is cylindrical and includes a cut out or window in which the translator is located. Preferably in this embodiment the housing is a mounting assembly including a stem which carries the first lens array at an end of the stem remote from the translator, and a mounting plate opposite the mounting assembly, the mounting plate being for coupling the housing to a camera.

In the preferred embodiment of the invention, the radiation which is received by the first lens array is radiation converging towards the first lens array, the first lens array forming a negative lens for collimating the diverging radiation received by the first lens array.

Brief Description of the Drawings Preferred embodiments of the invention will be described, by way of example, with reference to the accompanying drawings in which: Figure 1 is a diagram illustrating the concept of the present invention ; Figure 2 is an exploded view according to one embodiment of the invention ; Figures 3,4 and 5 are ray diagrams showing the formation of different focal planes according to the preferred embodiment of the invention ; Figure 6 is a view of a second embodiment of the

invention ; and Figure 7 is a diagram illustrating imaging according to embodiments of the invention.

Description of the Preferred Embodiments With reference to Figure 1 an embodiment of the lens assembly according to the invention is shown for use with an imaging device 10 (i. e. camera) which is intended to record images of an object. The device, as will be made clear with reference to Figure 2, may be embodied in a microscope or other optical instrument for inspecting objects. The radiation used in the instrument may be visible light.

The system according to the preferred embodiment of the present invention comprises a negative lens 12 and a positive lens 14 which form a lens doublet. In the preferred embodiment of the invention the lens doublet has zero power by providing the lens 12 and 14 with a focal length of the same magnitude but the negative lens 12 obviously having a focal length effectively in the opposite direction to that of the positive lens 14. Radiation received from an object (not shown) and shown by rays 16 is collimated into a parallel and collimated beam 18 by the negative lens 12. The collimated radiation 18 is then focused by the lens 14 as shown by rays 20 onto a focal plane coincident with the imaging device 10. In order to shift the focal plane relative to the imaging device 10 so as to provide two defocused images, the lens 14 is moved in the direction of double headed arrow X in Figure 1. Since the lenses 12 and 14 have the same focal length in magnitude, and the lens 14 is provided with collimated or parallel radiation, the magnification or power factor at each of the focal planes will be the same. Thus, according to this embodiment of the invention, the lens doublet formed by the lenses 12 and 14 has zero power thereby providing the same amount of magnification at each of the focal planes.

Because the positive lens 14 is provided with the collimated or parallel beam 18, the positive lens 14 will always focus the radiation at the focal point of the lens 14 at a distance y from the lens 14 regardless of where the lens 14 is located relative to the lens 12. Thus, by shifting the lens 14 towards the imaging device 10 the focal plane can be moved beyond the imaging device 10 or by moving the lens 14 towards the lens 12 the focal point will be moved to a position in front of the imaging device.

Figures 3,4 and 5 show this arrangement with three different positions of the lens 14 relative to the lens 12.

Figure 4 shows a situation where the lens 14 is positioned so that the focal plane may coincide with the plane of the imaging device represented by line C in Figures 3,4 and 5. By shifting the lens 14 towards the plane C as shown in Figure 3 then the focal plane of the lens 14 is also shifted in the same direction and focuses at a point passed the plane C as clearly shown in Figure 3.

If the lens 14 is moved towards the lens 12 as shown in Figure 5 then the focal plane of the lens 14 is also shifted towards the lens 12 and is in front of the plane C as clearly shown in Figure 5.

The lens assembly according to the preferred embodiment of the invention is shown in detail in Figure 2.

As shown in Figure 2 a mount 22 which may form part of a microscope (not shown) is provided. The mount 22 has an opening 24 having an internal flange 26. The flange 26 defines an aperture through which light collected by the microscope can pass. A mounting in bracket 30 has an upper platform 34 with a circular hole 35 and a tubular portion 36 which is hollow and which extends downwardly from the hole 35. The tubular section 36 has an inwardly projecting flange (not shown) which sits on the flange 26 so that the bracket 30 can be connected to flange 26 by screws (not shown) which pass through the flange 26 and the inwardly projecting flange (not shown) in the tube 36. The platform

34 has holes 38.

A U-shaped carrier 40 sits on the platform 34 and screws 42 pass upwardly through the apertures 38 and screw into holes (not shown) in the bottom wall of the carrier 40. The top wall of the carrier 40 is also provided with apertures 44 and a support plate 46 having a opening 47 is coupled to the carrier 40 by screws 48. A tube 50 projects through the opening 47 and screws onto a screw threaded collar 52 which supports a camera 60 including the imaging device 10 shown in Figure 1.

A mounting plate 62 is attached by screws (not shown) to wall 65 of the mount 40. The plate 62 carries a translator 70 which, in the preferred embodiment of the invention is a MM-3M-F translator made by National Apertures Inc, of Salem, New Hampshire, U. S. A. The translator 70 has a support plate 74 to which is bolted a lens mount 78. The lens mount 78 has a base 79 which has an aperture 80 in which is mounted lens 14. The lens 14 is retained in place by a collar 82. The lens mount 78 also has side walls 83 and 84 and rear wall 85. The rear wall 85 has apertures 86 and the mount 78 is connected to the plate 74 by screws 81 which pass through the holes 86 and screw into holes 87 provided on the plate 74. A lens mount 90 is also provided which bolts onto the plate 62 by screws 91 which pass through holes 93 in the plate 62 and screw into holes (not shown) in rear wall 94 of the mount 90.

The mount 90 has the central hole 95 which receives lens 12. The lens 12 is retained in place by collar 96.

Thus, according to the preferred embodiment of the invention the lens 12 is mounted in a fixed position on the plate 62 and the lens 14 is mounted on the translator 70 for movement by the translator when the translator 70 is actuated.

A cover plate 98 screws onto front surfaces 63 of the mount 40 so as to close the mount 40.

In order to move the lens 14 relative to the lens 12 the translator 70 is actuated from, for example, a

processor or personal computer so as to provide control signals to the translator 70 to cause the translator 70.

Actuation of the translator 70 causes the plate 74 to move in the direction of double headed arrow M in Figure 2 (which coincides with the direction X in Figure 1) thereby moving the lens 14 in the direction of double headed arrow M relative to the lens 12. This enables the focal plane of the lens 14 to be shifted relative to the plane of the charged couple device 10 within the camera 60 so that a focused image can be obtained which is focused at the plane of the imaging device 10 and two defocus images also obtained as previously explained.

Data recorded by the imaging device 10, can be forwarded to a processor for processing in order to obtain a phase image.

Figure 6 shows a second embodiment of the invention. This embodiment operates in the same manner as the previous embodiment, except the configuration of the embodiment is different. In this embodiment, primed reference numerals are intended to indicate similar parts to that described with reference to Figure 2.

A mount assembly 30'includes a tube 101 which carries fixed lens 12'. The mount assembly 30'connects with the photo-port (not shown) of a microscope (not shown). The assembly 30'includes an adaptor fitting 31' which can be used to couple the mount assembly 30'to different brands and models of microscope. An adaptor fitting 31'is screwed into position onto mount assembly 30'. Such adaptors are well known and therefore will not be described in any further detail.

Carrier body 40'is cylindrical in configuration and connects on to the mount assembly 30'. The body 40' also connects to camera mounting plate 50'. The plate 50' couples with a CCD camera (not shown) via the standard C- mount of the CCD camera.

A translation stage assembly 70'is mounted within the cylindrical carrier body 40'and operates in the

manner previously described. A lens carrier 78'is mounted on platform 102 of translation stage 71'and the moveable lens 14'is located in the carrier 78'for movement with the carrier 78'and translation stage 71'. The position of the moveable lens 14'is therefore accurately controlled in the same manner as previously described.

The translation stage 70'mounts in the carrier body 40 by locating in a cut out or window 105 in the carrier body 40', so that the lens 14'is coaxial with the lens 12'and the opening 106 in the mounting plate 50'.

The translation stage 70'may include openings 107 on front peripheral edge 109 which receive screws or like fasteners which couple the assembly 70'to the body 40'so that the translation stage 71'and lens carrier 78'are sealed within the body 40'.

Whilst the preferred embodiment of the invention has been described with reference to a system intended for obtaining phase contrast images, it should be understood that the invention has application in other environments and may be used in any environment which requires critical control of image focus and defocus.

The preferred embodiment of the invention can provide focusing distances of less than 1 Mm. In the case of magnifying instruments such as microscopes, the longitudinal magnification of the camera is actually the square of the instruments transverse magnification. This means that for a microscope having X10 magnification, a 1 Mm defocus at the camera amounts to a change at the sample at just 1 x 1/100 Mm or 10 nm-clearly beyond the resolution of even the most precise optical instrument.

Figure 7 is a diagram illustrating imaging of a sample in accordance with embodiments of the invention.

As depicted in Figure 7, a sample S is imaged by a microscope (illustrated by objective lens 200) into the lens assembly according to the preferred embodiment of the invention, which is comprised of the lens 12 and lens 14 as previously described. The lens 14 is moved in the manner

previously described with reference to Figures 2 and 7. At locations A', B'and C'of the lens 14, an in focus image of the sample plane b of sample S is obtained at corresponding points A, B and C at image location 210. For phase imaging applications, where the CCD array is located at the image location 210 and, in particular, with the CCD array being at point B, lens position B'provides an in focus image, while lens positions A'and C'provide out of focus images, all of plane b of the sample, as is described with reference to Figures 3,4 and 5.

Conversely, for Z-stacker applications where it is desired to obtain in focus images at different planes or locations through the sample S, an in focus image at plane B of the image location 210 is provided at sample locations a, b, c, corresponding to the position of lens 14 at locations A', B', The image of the sample is magnified asymmetrically-the width w of the image is the width of the sample multiplied by the microscope's objective lens magnification M (ie. W = W x M). The depth H of the image is the depth of the sample multiplied by the square of the magnification (ie. H = h x M2).

For any application, a displacement of the moving lens produces an identical displacement of the image plane, ie. distance A'B'at the lens equals distance AB within the image. Within the sample itself, however, the actual distance between planes imaged is smaller by the square of the magnification, ie. distance A'B'at the lens equals AB multiplied by M2. That is, at the magnification of X20 and for a shift within the sample of 100 nanometres, the lens would shift by 100 nanometres times 202 equals 40 micrometres.

Thus, by providing movement of the lens 14, an effective much smaller change in the plane at the sample S which is actually focused at plane B in the image location 210 can be obtained. Thus, rather than attempting to move the sample S a minute distance to enable Z-stacker

applications in which different planes of the sample S are imaged at the focal plane B and which is normally achieved by movement of the sample S, according to the preferred embodiments of this invention, the lens 14 is moved.

In order to move the sample S, the very small amounts such as the 100 nanometre distance referred to above in order to provide images at different planes for the sample S, extremely expensive and delicate equipment would be needed and, even then, the degree of accuracy and the precision of such a small movement would be extremely difficult. However, with the arrangement of the present invention, the effective small displacement of the plane in the sample S which is being focused is obtained by a much larger movement of the lens 14, which can be achieved much more easily and with less expensive apparatus as per the embodiments of Figures 2 and 6 previously described.

By carefully selecting lens surface curvatures of the lens 12 and 14 and also using achromatic optical components, distortions can be minimised to the point where they are much less than the size of individual camera picture or pixels which therefore cannot be detected and thus can largely be ignored.

The device according to the embodiment of Figure 2 or Figure 6 can also be used as a parfocalising device to ensure that an image of the sample viewed through the eyepiece of a microscope (not shown) to which the device is coupled and the image as detected by the camera 10 are parfocal. That is, the image seen through the eyepiece and the image detected by the camera are both in focus. This is important in many applications apart from the phase imaging and Z-stacker applications previously described.

The reason for this is that when a camera is used to take images of a sample, the light received by the camera and the light which passes through the eyepiece of the microscope are not one and the same and effectively travel through different optical systems. Thus, by looking through the eyepiece of the microscope, there is no

guarantee that the camera image is in focus. Movement of the moveable lens 14 in the manner previously described, can locate the moveable lens in a particular position which will provide a parfocal image at the eyepiece of the microscope and also at the camera 10. Thus, at one particular location of the moveable lens 14, parfocal images can be obtained and if this is necessary, the lens 14 can be adjusted to that position so as to provide those parfocal images at the camera and the eyepiece of the microscope. In order to obtain the parfocal images, the image at the eyepiece can be observed through the eyepiece of the microscope and the image detected by the camera observed on a display to which the camera is connected.

Since modifications within the spirit and scope of the invention may readily be effected by persons skilled within the art, it is to be understood that this invention is not limited to the particular embodiment described by way of example hereinabove.