FIELD OF THE INVENTION

The invention relates to an autofocussing system for an optical microscope and also a method for autofocussing a sample in a microscope. The invention finds particular application in the high-throughput screening of biological molecules.

BACKGROUND OF THE INVENTION

A variety of autofocussing systems are known in the art. Autofocussing systems are widely used in photography for miniature cameras, reflex cameras and video cameras. Autofocussing systems have been developed to determine surface topographies of, e.g., semiconductor wafers (JP 60102736). There have been various attempts to make autofocussing systems applicable for optical microscopy.

The international patent application WO-A-2003/060589 describes an apparatus for detecting a distance of a surface from a lens for use in a microscope autofocussing arrangement. A light beam is directed into the lens, displaced sideways from a lens axis. The displacement of the beam emerging from the lens and reflected from the surface is used to determine the distance between an object and the lens or to operate a focussing actuator. The incident light beam will typically be generated using a 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 lateral displacement depends on the distance of the surface from the lens. The relationship between the relative movement of the lens and the displacement of the reflected beam is not necessarily linear. The precise nature of the relationship will depend on the optics and other features and proportions of the apparatus used. The method and apparatus described in the '589 application is dependent on the various lens systems in the microscope. A change in magnification needs a new auto focus measurement. The described method is not suitable for high throughput screening processes.

The international patent application WO-A-2004/029691 teaches an enhanced autofocussing process. The taught method comprises the injection of a collimated light beam into a lens system which is made of an objective lens and a tube lens, as is the case in a microscope. The collimated light beam is emitted in a transversal manner onto the tube lens. The position of the light beam emitted by the tube lens and reflected by a surface of an object is detected. Thereby a signal allowing the quantitative determination of the distance between a reference plane and the surface is determined.

The knowledge of absolute value of the distance between the reference plane and the direction allows a fast movement of a sample into the desired focus position. However, this method is not fast enough for high throughput screening processes.

The German Patent Application DE-A-102 17 404 teaches a different way to determine the focus position. The focus plane is determined by a relative motion in a Z direction between the microscope stage and the objective. Images are read in by a camera during the relative motion, and a microscope control device and a computer are provided for evaluation and determination of the focus position. The computer determines, on the basis of the image data, the contrast value of each individual image. From the series of contrast values the focus position is derived. The ^λ 404 application tries to reduce the time for an autofocussing step by reading images by a camera during a zooming process with only very brief stops for image grabbing and parallelly calculating contrast values. It makes use of the optical systems of the microscope. The method described in the '404 application needs at least five images for focus determination. Acceptable times for single autofocussing processes can be achieved by the method of the ^λ 404 application, However, the throughput is not sufficient for high throughput screening applications.

The described autofocussing systems typically make use of parts of a microscope or are integrated parts of the used microscopy systems and therefore it is difficult to use them on different microscopes.

For biotechnological applications it is often desired to screen a large number of samples on one or more sample holders. It is difficult to obtain the necessary high throughput for fast screening of such biological samples with the current autofocussing techniques. Typical times for a single autofocussing step of systems such as described in the ^Λ 404 patent are in the time range of 0.5s to 10s. The

prior art addresses the problem of a high throughput by improving each single autofocussing step in the high throughput screening process. Furthermore the current autofocussing techniques use the optical path of the microscope to determine the focussing position. Therefore interference of light used for autofocussing on filters, lenses and apertures can occur.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide an apparatus and method for fast autofocussing of samples and in particular biological samples.

It is a further object of the invention to provide an apparatus and method for autofocussing that can be used for different types of microscopes and lenses.

It is yet a further object of the invention to provide an autofocussing apparatus and method that bypasses the regular optical path of the microscope.

It is a further object of the invention to provide an apparatus and method for fast screening of samples, in particular biological samples.

It is a further object of the invention to provide an apparatus and method which prevents the crashing of the lens system or objective of a microscope into the sample.

These and other objects are solved by providing a microscope system comprising at least one lens and a stage movable at least in a first direction and in a second direction. The microscope system has an attachment for at least one sample holder and a distance measurer for measuring a plurality of distances between a reference point and a plurality of points on a first surface of the at least one sample holder. The first surface could be a lower surface or an upper surface or an other reference surface of the sample holder. The microscope system comprises a memory for storing the plurality of distances and a controller for positioning the at least one lens at a plurality of focussing positions. The plurality of focussing positions are calculable from the plurality of distances. The focussing position is usually not the at the measured distance but the focussing position is chosen on or within the sample.

The disclosed construction of the microscope system allows to separate a step of determination of a focus position, essentially a step of determination of the distance from the lens to an object, from a step of focussing on the object. The separation of the steps allows the optimization of a distance measurer in respect of fast screening without being limited by the microscope apparatus. Distance measurers independent of the microscope and also completely independent from the microscopy techniques can be used. The distance measurement can be improved in terms of speed and precision and there is no necessity to adapt the distance measurement process to the microscope system. The separation of the steps in combination with the storage of the distances in the memory also allows the use of the distance data several times for several scans of the samples on the one or more sample holders, e.g. with different magnifications of the microscope without the necessity to determine the focus position again for every scan. The crashing of the lens into the sample can be prevented by first determining the distance and then moving the sample into focus. The controller can use stored offset values to stop any movement of the lens towards the sample before the lens comes in touch with the sample.

A method for focussing an image of a sample held on a sample holder comprising the following steps is disclosed: a first step of measuring and storing a plurality of distances between a reference point and a plurality of points on a surface of the sample holder and a second step of adjusting the distance between a lens and the sample using the plurality of distances such that the image is substantially in focus. The two steps allow the separation of distance measurement from focussing. Thereby measurement techniques for distance determination independent from the microscope can be used.

For the purpose of screening samples on the sample holder the steps of measuring the distance between the reference point and a point on the surface of the sample holder; storing a distance value in a memory; and moving a distance measurer relative to the surface of the sample holder can be repeated until a plurality of distance values between the reference point and points on the surface of the sample holder are stored in the memory. Then the stored distance value for the point on the surface is read out from the memory; the lens is moved relative to the surface of the sample holder using the stored information of the distance value; and the image acquisitions are performed or images are taken using the microscope system. The steps of read out, movement and image

acquisition can be repeated until a plurality of image acquisitions of the samples on the one or more sample holders are obtained by the microscope system.

The stored distance values can be used for several measurements of the samples on the sample holder with different magnifications of the microscope. Therefore with a single scan of the distances several microscope screenings can be conducted.

DESCRIPTION OF THE DRAWINGS

Fig. 1 shows an inverted optical microscope incorporating the distance measurer of the invention.

Fig. 2 shows a distance measurer used in an embodiment of the invention.

Fig. 3 shows a flow diagram illustrating the steps of autofocussing the microscope system in accordance with an embodiment of the invention.

Fig. 4 shows a map of the surface of the sample holder.

Fig. 5 shows a flow diagram with the steps of the method in a screening process.

DETAILED DESCRIPTION OF THE INVENTION

Fig. 1 shows an inverted optical microscope 10, e.g. an Olympus 1X81 wide field microscope. The invention is not limited to this type of inverted optical microscope. The inverted optical microscope 10 has a light source, preferably of visible light, 20 which shines onto a sample holder 30. The sample holder 30 is held on a stage 40. A cover slip may be used as the sample holder 30. The sample holder 30 can be moved in both directions substantially in the plane of the stage 40. Movement of the sample holder 30 is carried out by the use of computer-controlled stepper motors 45 or by manual adjustment as is known to the skilled person.

A lens 50 is positioned on the opposite side of the sample holder 30. In the depicted embodiment of the invention, the lens 50 is incorporated into a lens turret 60 with further lenses 70. The lens turret 60 can be turned about an axis 80. The lens turret 60 also includes a distance measurer 90. The distance measurer comprises a light source 100, preferably a laser beam, and a detector 110, preferably a charge coupled device. In one example the charge coupled device is supplied by the Keyence Corporation, e.g. the sensor Keyence LK-OIl in combination with a sensor controller Keyence RD 50RW. The detector 110 is connected to a controller 120 which has a memory 130. The controller 120 controls the stepper motors 45 and the memory 130 records the position of the stage 40 as well as values of light detected by the detector 110 as will be explained in detail later.

The sample holder 30 is made of glass and has typically a thickness of 170±l μm. As is shown in Fig. 2, a plurality of samples 140 is mounted on an upper surface

32 of the sample holder 30. The sample holder 30 also has a lower surface 34.

The plurality of samples 140 comprise gelatine with additives in which biological material, including cells, are present. The additives in the gelatine provide an experimental environment to observe the reactions of biological material. Although described in this disclosure in terms of the observation of biological materials, it is to be noted that the invention is more generally applicable and can be attached to any suitable microscope.

As example an embodiment of the method for autofocussing the sample will now be described with respect to Figs. 2 and 3. In this example, the sample 140 is viewed using the lens 50. However, the sample 140 could be focussed by any one of the further lenses 70. In a first step 300 the sample holder 30 is placed and attached to the stage 40. Although the surface of the sample holder 30 is produced with very tight tolerances, the effect of attaching the sample holder 30 to the stage 40 rs to introduce stress into the sample holder 30 so that perturbations of the surface of the sample holder 30 are formed. The sample holder 30 may also not be in exactly the same plane as the stage 40 as will be shown with respect to Fig. 4.

The distance measurer 90 is then brought into position substantially below the sample holder 30 in step 310. The distance measurer 90 emits a laser beam 115 from the light source 100 (step 320). The laser beam 115 is reflected from the lower surface 34 of the sample holder 30 back to the detector 110 (step 330) as

is shown in Fig. 2. The detector 110 records the position of the reflected laser beam to determine by means of a triangulation method the distance travelled by the laser beam between the light source 100 and the detector 100 (step 340). This measured value is stored in the memory 130 together with the position of the sample holder 30 at which the measured value is determined (step 350). In step 360 the sample holder 30 is moved to a new position by means of the stepper motors 45 (or possibly by manual adjustment) and steps 320 to 350 are repeated. The steps 320 to 360 from measuring the distance to movement of the sample holder 30 to a new position take about 0.6 s to 2 s. Most of the time is needed for the repositioning of the sample holder 30.

Steps 320 to 360 are repeated at regular intervals over the whole of the lower surface 34 of the sample holder 30 to obtain a "map" of the lower surface 34 of the sample holder 30 as is illustrated in Fig. 4.

The number of separate measurements carried out is dependent on the accuracy desired, the size of the sample holder 30 and also on the capability of the stage 40. Typically measurements are repeated approximately every 0.5 μm. The results are stored in the memory 130 to produce the "map" of Fig. 4 in the memory of the sample holder 30.

The distance measurer 90 is then replaced by the lens 50. In the embodiment shown in Fig. 1, both the distance measurer 90 and the lens 50 are mounted on the same lens turret 60. Thus in this example, the lens turret 60 need only be rotated about its axis 80. However, the invention is not limited to the mounting of the lens 50 and the distance measurer 90 on the lens turret 60. It would also be possible for the distance measurer 90 to be mounted on a holder which is moved into and out of position. It is, however, preferable to mount both the distance measurer 90 and the lens 50 on the same lens turret 60 to minimise problems with thermal fluctuations in the relative positions of the distance measurer 90 and the lens 50.

The controller 120 also controls the movement of the lens 50 and the further lenses 70 in the direction substantially perpendicular to the stage 40. Using the map in the memory 130 and knowing the thickness of the sample holder 30 the controller 120 can calculate the position of the lens 50 (and/or further lenses 70) such that the cells in the plurality of samples 140 are in focus. Any plane within or outside of the samples 140 can be chosen as a focal plane by an offset value.

An offset value is defined as the distance between the required focal plane and the lower surface 34 of the sample holder. This offset value is stored in the memory 130. As is shown in Fig. 2, the dimensions of the plurality of samples 140 is substantially but not exactly consistent between each ones of the plurality of samples 140. In the majority of cases, the depth of field of the lens 50 is such that the cells of interest within the plurality of samples 140 are in focus. In the event that the cells of interest are not in focus, then fine focussing methods can be used to adjust the focus of the lens 50.

The method of the invention finds particular application in high-throughput screening applications. Each one of the plurality of samples 140 could contain, for example, part of the human genome - or some other biological material. Each sample holder has roughly 400 samples 140 positioned on the upper surface 32. Each one of the samples 140 has different conditions applied to it and each of samples 140 is viewed to see the effect of the different conditions. In this example each one of the samples 140 is approximately 400 μm in diameter and has a height of between 20-25 μm. There may be more than one biological material in each one of the samples 140.

Having screened all of the samples 140, the user of the microscope 10 may wish to view one or more of the samples 140 in greater detail. Since the controller 120 stores the map of the sample holder 30 it can automatically calculate the correct focussing distance for any one of the samples 140 using the stored offset value and calculate the correct focussing distance quickly and accurately. This is also applicable when the user chooses to view the sample 140 at a greater magnification using another one of the further lenses 70.

Using the prior art system in which each lens 50 was focussed individually on the biological material within the sample, the time required for focussing was around 4s for each one of the samples 140. Using the method of this invention, the time required for focussing is around 0.6s.

The invention has the further advantage that the controller 120 can be so programmed that the lens 50 can never be brought into contact with the sample holder 30 since the distance between the surface of the lens 50 and the sample holder 30 is known.

In a further embodiment of the invention, the stage 40 can have several sample holders 30. A map of the lower surface 34 of each of the sample holders 30 can be initially created and stored in the memory 130 before the samples 140 are viewed using the lens 50.

Fig. 4 shows two maps of the lower surfaces 34 of two sample holders 30. The sample holders 30 used for these measurements were microscope slides. The microscope slides are 2 x 5 cm in size. The microscope slides used for the measurements have, in comparison to other sample holders 30, a substantially flat lower surface 34. The variation of the lower surface 34, as depicted in Fig. 4, by curving and bending is in the range of 20 μm to 30 μm. The units on the height axis in Fig. 4 have to be read as .1 μm. Other sample holders, not shown in Fig. 4, showed deformations of the lower surface in a range of up to 200 μm.

Fig. 5 shows in a generalised manner the steps of a screening process for a plurality of the biological samples 140 according to the invention. In a first loop 400 a distance of a reference point of a microscope 10 to a point on a surface of the sample holder 30 is measured in step 410 by the distance measurer 90 to obtain a distance value. The surface referred to can be the upper surface 32 of the sample holder 30 or the lower surface 34 of the sample holder 30. In step 420 the distance value is stored in the memory 130. In step 430 the distance measurer 90 is positioned relative to a different point on the surface of the sample holder 30. The loop 400 is repeated until distance values for the plurality of points on the surface of the sample holder 30 are obtained and stored in the memory 130. Typically, the plurality of points on the surface of the sample holder 30 is distributed on the surface in such a manner as to allow a scanning of the surface of the sample holder 30.

In a second loop 500 in step 510 the distance value for a point on the surface is read from the memory 130 and the lens 50 is positioned relative to the point on the surface in step 520. The positioning of the lens 50 is in such a manner that an image of the sample 140 on the sample holder 30 can be acquired by the microscope 10, preferably the samples 140 are positioned substantially in the focal length of the lens 10. The image acquisition takes place in step 530. The loop 500 can be repeated until a plurality of images have been acquired from a plurality of samples 130 on the sample holder 30.

The loop 500 with steps 510, 520 and 530 can be repeated again with a different magnification after a change of the lens 50, e.g. by turning the turret 60, with the same distance values as obtained in loop 400.

Reference Numbers

10 Inverted Optical Microscope

20 Light Source

30 Sample Holder

32 Upper Surface

34 Lower Surface

40 Stage

45 Stepper Motor

50 Lens

60 Lens Turret

70 Further Lenses

80 Axis

90 Distance Measurer

100 Light Source

110 Detector

115 Laser Beam

120 Controller

130 Memory

140 Plurality of Samples

300 Step of sample holder placement and adjustment to stage

310 Step of positioning sample holder

320 Step of laser beam emission

330 Step of laser beam reflection

340 Step of distance determination

350 Step of storage in memory

360 Step of movement of sample holder to new position

400 First loop

410 Step of distance measurement

420 Step of distance value storage

430 Step of positioning of distance measurer

500 Second loop

510 Step of reading from memory

520 Step of positioning of lens

530 Step of image acquisition