WO2003060589A1

WO2003060589A1 - Auto focussing device and method

Info

Publication number: WO2003060589A1
Application number: PCT/GB2003/000151
Authority: WO
Inventors: Mark Osborne; Harold Swerdlow
Original assignee: Solexa Ltd.
Priority date: 2002-01-15
Filing date: 2003-01-15
Publication date: 2003-07-24
Also published as: GB0200844D0; AU2003202023A1

Abstract

There is disclosed an apparatus for detecting the distance of a surface from a lens, for example for use in a microscope autofocus arrangement. A light beam is directed into the lens, displaced sideways from the lens axis. The displacement of the reflected beam emerging from the lens is used to determine the required distance or to operate a focussing actuator.

Description

AUTOFOCUSING DEVICE AND METHOD AUTO FOCUSSING DEVICE AND METHOD

The present invention relates to a device and method for determining the distance of a plane surface from a reference plane, and particularly the position of a surface with respect to the focal plane of a lens. Such a device can be used to implement an auto focussing arrangement for a microscope.

Auto-focussing is desirable in applications where the rapid and accurate determination of the displacement of an object plane from the focal plane of an imaging lens is required, such that the object can be brought into focus by translating the lens or object by a corresponding amount. Microscopy applications in which auto-focussing may be particularly desirable are cell screening using bright field microscopy, cell profiling using confocal microscopy, surface imaging using total internal reflection microscopy and high resolution imaging using scanning probe microscopies.

US 4342905, US 5483055 and US 4844617 describe known auto focussing devices for microscopes. These devices employ image contrast methods or intensity measurements to determine when a specimen on a stage of a microscope is in focus. Such methods generally require multiple image acquisition in order to determine the maximum contrast position, and require high power lasers and high cost detectors and signal processing means.

The invention provides apparatus for detecting the distance of a surface from a lens having a central axis, the apparatus being arranged: to project an incident light beam into the lens such that the incident beam is incident on and reflects from said surface to form a reflected beam collected by said lens; such that the incident beam enters the lens displaced from the central axis; and to detect a displacement of said reflected beam in a detection plane.

The incident beam will typically be generated using laser source, the lens may be the objective lens of a microscope, and the surface may be a sample surface which it is desired to keep in or close to the focal plane of the objective lens. Because the incident beam enters the lens off axis, the reflected beam emerges from the lens laterally displaced from the incident beam. The magnitude of this displacement depends on the distance of the surface from the lens. The reflected beam may also emerge from the lens slightly aparallel to the incident beam, with the difference in beam axis direction also depending on the distance of the surface from the lens.

The surface needs to be at least partially reflective, so as to allow specular reflection of the incident beam and may be, for example, the polished surface of a glass microscope slide. The reflected beam emerging from the lens is transmitted by suitable optics to the detection plane where lateral displacement of the beam is directly related to corresponding relative movement of the lens and the surface .

Preferably, the incident beam entering the lens is substantially parallel to the central axis, so that the emerging reflected beam is approximately parallel to, and indeed may be overlapping with the incident beam. In this way, common optics may be used to process both the incident and reflected beams.

Preferably, the incident beam entering the lens is either slightly convergent or slightly divergent so that the intensity of the incident beam on the surface is reduced by being slightly out of focus. This reduces effects such as breakthough which may be undesirable in various applications, such as fluorescence microscopy applications. Preferably, the apparatus further comprises a detection element located in the detection plane, such as a CCD array, a light sensitive diode array, a light sensitive quadrant detector, or any pair of closely spaced light sensitive elements, to detect movement of the reflected beam in the detection plane.

The apparatus may further comprise a detection lens arranged to focus the reflected beam to a point near to the detection plane. The lens may thereby be used to improve the detection resolution of the apparatus. It may be desirable to focus the reflected beam to a point near to but not in the detection plane, to increase the magnitude of the detected beam displacement. If the reflected beam is directed to the boundary between a pair of detectors, slight defocus may improve the usefulness of the detected signal .

The apparatus may further comprise electronic circuitry coupled to the detection element and adapted to control the position of the lens in response to the detected displacement of the reflected beam, thereby effecting an autofocus arrangement.

Advantageously, the apparatus may comprise a polarisation dependent optic arranged to separate the reflected beam from the incident beam. This is particularly useful if the incident and reflected beams overlap significantly.

The apparatus may also comprise a dichroic beam splitter adapted to direct the incident beam into said lens and to direct the reflected beam towards said detection plane. If a different wavelength of light is being used for imaging a sample at the surface, then the dichroic beam splitter is conveniently used to separate the light beams used to detect distance from the light being used for imaging.

Preferably, the central axis of the lens is arranged to be approximately normal to the surface. The present invention also provides an apparatus for determining the distance of a plane surface from a first reference plane comprising: means for providing a light beam incident on the surface at an angle with respect to the surface normal, the light beam being at least partially reflected from the surface; and means for measuring the position of the point of incidence of the reflected beam on a second reference plane.

The invention also provides a method for determining the distance of a plane surface from a first reference plane comprising the steps of: providing a light beam incident on the surface at an angle with respect to the surface normal, the light beam being at least partially reflected from the surface; and measuring the position of the point of incidence of the reflected beam on a second reference plane.

Preferably, the surface and the first reference planes are substantially parallel. The second reference plane may also be parallel to the surface.

In embodiments of the invention, the displacement of a reflected beam of light incident at an angle with respect to a surface normal is measured to determine the position of the surface with respect to a first reference plane. If the surface, the first reference plane and the second reference plane in which the displacement is measured are parallel, a simple linear relationship may exist between the magnitude of the beam in the x, y directions of the second reference plane and movement of the surface along the z axis for the entire surface area. By exploiting this linearity or other functional relationship the surface can be moved in the z direction to minimise the beam displacement away from some reference position which represents a desired distance of the surface from the first reference plane. The reference position may be obtained in a one off set up step. Preferably, the apparatus includes means of moving either the surface or the first reference plane in a direction and by an amount dependent on the measured displacement of the reflected beam.

Preferably, the device is employed as an auto focussing device for a microscope wherein the surface is a sample plane and the first reference plane is a focal plane of an objective lens of the microscope. The reference position may be obtained in a one off set up step by focussing on an object at the surface and recording the position of the reflected light beam in the second reference plane. Unlike methods based on image contrast, in the present invention only a single measurement is required. In addition, a single low power laser and a low cost detector can be used compared to more complex techniques.

Preferably, the light beam is directed through the objective lens of the microscope towards the surface with the axis of propagation spaced some distance from the optical axis of the objective lens. Light propagating approximately parallel to but not along the optical axis of the objective lens is redirected towards the optical axis at an angle to the reflective surface. In this case, the reference position may be calculated from the focal length of the lens and the displacement of the reference beam from the optical axis, if these are known accurately. Preferably, the reflected beam is also collected by the objective lens and emerges parallel to the beam incident on the lens. The displacement of the emerging beam from the optical axis of the objective lens can be measured and is dependent on the position of the sample plane in the z direction.

Preferably, the means for measuring the displacement of the point of incidence of the reflected beam on the reference plane comprises a CCD camera, linear diode array or a quadrant detector. A preferred embodiment of the present invention will now be described with reference to the accompanying drawings in which :-

Figure 1 illustrates an autofocus arrangement for a microscope, embodying the invention;

Figure 2 illustrates the operation of a range finding apparatus according to the invention in which a light beam reflected from a surface is incident on a detector;

Figure 3 shows displacement of the light beam of figure 2 on the detector as the reflective surface is moved; and

Figure 4 shows a total internal reflection microscope incorporating an autofocussing device using range finding apparatus according to the invention.

Referring to figure 1 there is illustrated a microscope incorporating an autofocus device embodying the invention. A microscope objective lens 100 is arranged such that its focal point is close to a sample surface 102, for example the surface of a microscope slide or similar body. A laser 104 generates an incident laser beam 106 having a width of about 1 to 2 mm, which propogates towards an uncoated pellicle beamsplitter 108. The pellicle beamsplitter 108 reflects about 10% of the incident beam 106 and transmits about 90% irrespective of polarization. The transmitted light is not used.

The portion of the incident beam 106 reflected from the pellicle beamsplitter 108 is directed to a half wave plate 110 which allows the polarization of the beam to be adjusted. The half wave plate 110 must be selected as appropriate for the wavelength of the beam produced by the laser 104. The incident beam 10 emerging from the half wave plate 110 is directed to the objective lens 100 by a dichroic filter 112. The dichroic filter 112 couples the incident beam 106, having a first wavelength, into the objective lens 100, while allowing fluorescence or other image light, having a second wavelength, from the vicinity of the sample surface 102 to be transmitted to an imaging camera (not shown) . In one embodiment, which is arranged as a total internal reflection microscope, the objective lens is a Nikon Plan fluor, lOOx magnification, NA 1.3, working distance 370 microns. This compound lens has a rear aperture of about 6mm and a sample side aperture of about 1mm. In such an embodiment, the incident beam 106 may have a power of about 20 μ and a diameter of about 1 to 2mm as it enters the objective lens 100.

The incident beam is directed non-centrally or off-axis into the objective 100, but preferably approximately parallel to the central axis of the lens. The beam may be only slightly off center so that the central axis of the lens lies within the beam or close to the center of the beam. Alternatively, the incident beam may be more significantly off center, for example such that the beam lies outside the central axis of the lens.

The incident beam 106 is directed to the sample surface 102 by the objective lens 100, and is at least partially reflected to form a reflected beam 114 which is collected by the objective lens. Because the incident beam enters the objective lens 100 off center, the reflected beam 114 emerges from the objective lens 114 displaced laterally from the incident beam 106 by a distance which depends on the distance of the sample surface 102 from the objective lens 100. The reflected beam 114 emerging from the objective is reflected by the the dichroic filter 112, passes through the halfwave plate 110 and is incident upon the pellicle beam splitter 108. About 90% of this beam is transmitted through the pellicle beam splitter 108 and is focused using detector lens 116 onto a light sensitive element or array of elements 118. In the total internal reflection micropscope embodiment mentioned above the light sensitive element 118 is a quad cell located about lm from sample surface 102, and the detector lens 116 has a focal length of about 30cm. The quadcell is aligned such that two of its elements are spaced apart in a direction parallel to the direction of movement of reflected beam 114 as the sample surface 102 moves towards or away from the objective lens 100. As a result, a change in the distance from the sample to the objective leads to a change in the relative powers of the beam portions incident on each of these two elements. The reflected beam is preferably slightly out of focus at the detector so that it impinges on a part of both elements at the same time. The remaining two elements of the the quadcell are unused. Electronic circuitry coupled to the quadcell drives an activator which adjusts the distance between the objective lens 100 and the sample 102 thus forming a feedback loop which seeks to keep the sample surface 102 at the focal point of the lens 100.

Reflections of the incident and reflected beams 106,114 from the dichroic filter 112 and from the sample surface 102 are polarisation dependent, governed by the Fresnel equations, so that the apparatus can be adjusted to yield a reflection beam 114 which has an optimum or maximum intensity at the light sensitive element. In particular, this can be achieved by rotating the half wave plate.

In the total internal reflection microscope embodiment discussed above, a change in the separation of the objective lens 100 and sample surface 102 of 1 micron results in a movement of the focused reflected beam 114 at the light sensitive element 118 of about 200 to 500 microns, depending on how far from the light sensitive element 118 is from the focal point of the reflected beam 114. Preferably, the light sensitive element 118 is not placed exactly at the focal point of the reflected beam 114.

Preferably, the incident beam 106 is slightly convergent or divergent on entering the objective lens 100, so that during normal operation the incident beam forms a slightly out of focus spot on the sample surface 102. This reduces the intensity of the incident beam at the sample surface, reducing the likelihood of breakthrough or other effects which are undesirable in microscope arrangements such as a total internal reflection microscope.

The pellicle beam splitter 108 may be replaced by a polarisation dependent optic such as a polarising beam splitter which acts such that light of a first polarisation is transmitted and light of a second polarization orthogonal to the first polarization is reflected sideways. Such a component my be used to improve the separation of the incident and reflected beams .

Figures 2 and 3 illustrates how an apparatus such as that claimed or as illustrated in figure 1 or figure 4 may operate. In the arrangement of figure 2, there is provided a lens 2 to image a reference beam of light 1 which is incident at an angle θ to the normal of a reflective or transmitting surface, the position of which is indicated as z, a drive or actuator to move the lens or reflective surface, which may correspond to a sample stage along the optical axis (the z direction) and a detector 3 to measure the position r of the reflected reference beam in a second reference plane.

The reference beam 1 is directed through the lens 2 with the axis of propagation displaced from the optical axis of the lens. Light propagating approximately parallel to but not along the optical axis of the lens is re-directed towards the optical axis at an angle θ to the reflecting surface. The displacement r on the detector 3 at which the reflected light beam intersects the second reference plane then has an r = 2z tan θ dependency on the position z of the surface with respect to the lens 2. The instrument response may be made more sensitive by increasing the angle θ or by increasing the displacement of the beam 1 from the optical axis of the lens 2.

An initial reference position z₀ corresponding to displacement r₀ of the reflected beam is obtained in a one off set-up step by focussing on an object at the surface and recording the position r₀ of the reflected light beam. By measuring the displacement r with respect to the predetermined position r₀, the movement z - z₀ by which the lens must be moved to return the reference beam to the origin r₀ may be determined from a linear relation of the form r - r₀ = m(z - z₀) where m = 2 tan θ and r₀ are constants over any surface. In practice the values of m and r₀ are determined in an initial one off set up from the best fit of the displacement r as a function of the z axis focus position through the focal plane. To do this,. the sample surface is initially located visually by bringing into focus an object at the surface. The reference beam is then introduced and the position of the reflected spot recorded as the origin r₀ at the plane of focus z₀. The surface or lens is then moved to a new position z, along the optical axis, either side of the plane of focus and the new position r of the reflected reference beam recorded as a function of the z position of the sample or lens. The linear displacement of r with z is then used to determine the constant m from the equator r - r₀ = m(z - z₀) . In the microscope embodiment shown in Figure 4, a laser beam (1) is directed up an objective lens 2 in an epi-illumination mode via a dichroic mirror 4. The path may be adjusted to be off axis using a translatable lens or mirror inserted in the laser beam 1 before the dichroic mirror 4. The surface is a glass slide 5 off which the beam is reflected and this light is collected by the same objective lens 2. Most of the light is reflected back out of the microscope by the dichroic mirror 4 but a small fraction of the light 8 is transmitted and detected on a sensitive CCD camera 3 where the displacement r of the beam can be measured.

Alternatively, the displacement r persists in the beam 7 deflected off the dichroic 4 and could be constantly monitored by an external camera, linear diode array or quadrant detector 6 and the focus drive constantly adjusted via a feed back loop. In this case r - r₀ is minimised physically using a feedback loop control circuit known in the art making z = z₀ without prior knowledge of m. For example in the case of the quadrant detector the reference beam might be set up such that when the surface is at the planer focus of the objective lens the reflected beam is evenly distributed across all four quadrants. If the surface drifts away from the focal plane then the reflected reference beam will be displaced resulting in an uneven distribution of light across the four quadrants of the detector. The change in distribution can be used to provide a signal to drive the lens or sample stage by an amount and in such a direction that the light distribution across the quadrant detector becomes even again. At this point the surface will be back at the focal plane of the lens. An embodiment of this latter kind will allow continuous sample scanning in the xy plane whilst maintaining the focal plane of the lens at the surface of the sample and without the need to acquire an image of the displaced reference beam prior to performing the focussing steps.

It is not necessary for the relationship between the relative movement of the lens and the displacement of the reflected beam to be linear. The precise nature of the relationship will depend on the optics and other features and proportions of the apparatus used.

Claims

CLAIMS :

1. Apparatus for detecting the distance of a surface from a lens having a central axis, the apparatus being arranged: to project an incident light beam through the lens such that the incident beam is incident on and reflects from said surface to form a reflected beam collected by said lens; such that the illumination beam enters the lens displaced from the central axis; and to detect a displacement of a point of incidence of said reflected beam in a detection plane.

2. The apparatus of claim 1, further arranged such that the incident beam entering the lens is substantially parallel to the central axis.

3. The apparatus of either of claims 1 or 2, further arranged such that the incident beam entering the lens is either slightly convergent or slightly divergent.

4. The apparatus of any preceding claim comprising a detection element located in the detection plane.

5. The apparatus of claim 4 wherein the detection element is one of a CCD array, a light sensitive diode array and a light sensitive quadrant detector.

6. The apparatus of claim 4 or 5 further comprising a detection lens arranged to focus the reflected beam to a point near to the detection plane.

7. The apparatus of any of claims 4 to 6 further comprising electronic circuitry coupled to the detection element and adapted to control the position of the lens in response to the detected displacement of the reflected beam.

8. The apparatus of any preceding claim further comprising a polarisation dependent optic arranged to separate the reflected beam from the incident beam.

9. The apparatus of any preceding claim further comprising a dichroic beam splitter adapted to direct the incident beam into said lens and to direct the reflected beam towards said detection plane

10. The apparatus of any preceding claim further comprising said lens, said central axis of said lens being arranged to be normal to the surface.

11. An autofocussing arrangement for a microscope comprising the apparatus of any preceding claim.

12. A microscope comprising the apparatus of any of claims 1 to 10 wherein the lens is an objective lens of the microscope.

13. The microscope of claim 12 wherein the point of incidence of the reflected light beam on the detection plane is used to automatically control movement of the objective lens along the lens axis.

14. The microscope of either of claims 12 or 13 wherein the microscope is a total internal reflection microscope .

15. A device for determining the distance of a plane surface from a first reference plane comprising: means for providing a light beam incident on the surface at an angle with respect to the surface normal, the light beam being at least partially reflected from the surface; and means for measuring the displacement of the point of incidence of the reflected beam on a second reference plane.

16. A device according to claim 15, wherein the surface, the first reference plane and the second reference plane are substantially parallel.

17. A device according to claim 15 or 16 which is an auto focussing device for a microscope wherein the surface is a sample plane and the first reference plane is a focal plane of an objective lens of the microscope.

18. A device according to any one of claims 15 to 17, wherein the means for measuring the displacement of the point of incidence of the reflected beam on the second reference plane comprises a CCD camera, linear diode array or a quadrant detector.

19. A method for determining the distance of a plane surface from a first reference plane comprising the steps of: providing a light beam incident on the surface at an angle with respect to the surface normal, the light beam being at least partially reflected from the surface; and measuring the displacement of the point of incidence of the reflected beam on a second reference plane.

20. A method according to claim 19, wherein the surface, the first reference plane and the second reference plane are substantially parallel.

21. A method according to claim 19 or 20 which is a method of automatically focussing a microscope wherein the surface is a sample plane and the first reference plane is a focal plane of an objective lens of the microscope.

22. A method according to claim 21, wherein a reference position is obtained in a set up step by focussing on an object at the surface and recording the position of the point of incidence of the reflected beam on a second reference plane.

23. A method according to claim 21 or 22 wherein the light beam is directed through an objective lens of the microscope towards the surface with the axis of propagation spaced from the optical axis of the

- objective lens, such that the beam is redirected towards the optical axis at an angle to the reflective surface.

24. A method according to claim 21, wherein the light beam is directed down an objective lens of the microscope towards the surface with the axis of propagation spaced from the optical axis of the objective lens, such that the beam is redirected towards the optical axis at an angle to the reflective surface, and therein a reference position is calculated from the focal length of the objective lens and the off-axis displacement of the reference beam.