WO2012016753A1

WO2012016753A1 - Autofocus system

Info

Publication number: WO2012016753A1
Application number: PCT/EP2011/060081
Authority: WO
Inventors: Markus Sticker
Original assignee: Carl Zeiss Microimaging Gmbh
Priority date: 2010-08-03
Filing date: 2011-06-17
Publication date: 2012-02-09
Also published as: DE102010033249A1; EP2601551A1

Abstract

Microscopic device for imaging a sample substance (1), comprising: - a microscopic imaging system, and - means for automatically displacing the focal plane along the optical axis (4) of the imaging system into a Z-position, in which the sample substance (1) to be imaged is situated, characterized by - a sample beam (10) which emerges from an interferometer and is directed in the Z-direction through the sample substance (1), wherein the interference signals arising as a result of interference of the light of the sample beam (10) that is reflected or backscattered from optically active interfaces and/or structures with the reference beam of the interferometer are provided for determining the Z-positions of the interfaces and/or structures of the sample, - an evaluation device for determining the Z-position of the sample substance (1), - an adjusting device, designed for displacing the focal plane and the sample substance (1) relative to one another until the sample substance (1) is situated in the focal plane, and - a drive unit connected to the evaluation device and the adjusting device and serving for generating actuating commands for automatically displacing the focal plane into the Z-position determined.

Description

title

Autofocus System

Field of the invention

The invention relates to a method and a device for determining the Z position in the direction of the optical axis of a microscopic imaging system, in which a sample substance to be imaged is located, as well as for automatically focusing the imaging system on this Z position.

State of the art

Microscopic imaging systems are often used to digitally record a sample substance, for example a tissue section, which is stored on a microscope slide protected by a cover glass. If the lateral extent of the sample substance to be recorded is greater than the field of view of the imaging system or greater than a range which can be directly absorbed by a camera, then it is necessary to first consecutively extend several times, for example 220 μm x 165, perpendicular to the optical axis of the imaging system μιη record large areas of a sample substance. In the prior art, this is done with the aid of a so-called digital "slide scanner." The images of the individual regions are then combined to form a so-called "tiled image" as an overall image.

The problem here is that the depth of field of the optical imaging system decreases with increasing lateral optical resolution. If the sample is focused at a location with respect to a first region to be recorded, it is highly probable that the focus position after shifting the sample to accommodate an adjacent region is different, ie the sample is defocused there, unless an adjustment of the depth position or a renewed focus is made. Causes for the change in the focus position may be, for example, a non-perpendicular orientation of the slide to the optical axis, thickness variations in the slide or in the cover glass, deflections of the slide or thickness variations of the embedding between cover glass and sample substance. It is also possible that the distance between the sample substance to be recorded and the slide varies with lateral displacement.

Hence, there is a need to adjust the z-position of the sample from area to area during the scan to bring the sample into focus.

For this purpose it is known to generate a sample-related, so-called "focus map" before scanning, and then to move the sample or the lens in the axial direction during scanning, taking into account the instructions of this "focus map" to maintain the focus.

To generate the "focus map", several prior art locations are approached on the sample, and the focus position is determined by means of a software autofocus, whereby several images are recorded at different depths of the sample and, for example iteratively, the best Focus position determined on the basis of the image contrast.

Another known procedure provides to determine the focus position at predetermined time intervals during the scanning with the aid of a respective software autofocus and thereby correct any deviations ascertained. Both methods have the disadvantage that they take undesirably much time.

Another known method for focusing the sample is the so-called hardware autofocus, in which the Z-positions of reflective interfaces are determined, such as those of the cover glass top or the slide bottom. However, there is the disadvantage here that depth positions, for example the position of the sample substance to be imaged, can not be measured directly, because the backscattered amount of light is not sufficient for this purpose. The information about the position of, for example, the cover glass top or the slide bottom is often insufficient as a prerequisite for focusing the sample substance, since the distance of the sample substance to these interfaces is not constant. In a method using confocal detection, it must be possible to focus through the sample, which requires mechanical movement, which in turn takes an undesirably large amount of time and, in addition, can lead to inaccuracies in the measurement result due to the required mechanical positioning movements.

Description of the invention

On this basis, the invention has the object, a method and a device of the type mentioned in such a way that with higher efficiency than in the prior art, a determination of the Z-position of the sample substance is ensured with subsequent automatic focusing on this Z-position.

According to the invention, the Z-position of a sample substance to be imaged is determined in a microscopic imaging system and the automatic focusing on the sample substance is carried out in the following method steps:

Performing a depth scan in the Z direction through the sample substance by means of an interferometer, wherein

the light of an interferometer sample beam reflected or backscattered by optically effective interfaces and / or structures is brought into interference with the reference beam of the interferometer,

a determination of the Z-positions of the interfaces and / or structures of the sample is made on the basis of the resulting interference signals,

determining therefrom the Z-position corresponding to the position of the sample substance in the direction of the optical axis,

- The focal plane and the sample substance to be imaged are moved in the Z-direction relative to each other until the sample substance of the focal plane is, and then the sample substance is imaged.

As a sample in the context of the invention, for example, the entirety of a slide, a coverslip and the enclosed between them sample substance to understand. Preferably, the sample substance is recorded digitally. The interferometer used is preferably a short-coherence interferometer.

Short-coherence interferometry is a relatively young, high-precision optical method for examining surfaces and thin layers and scattering media, such as the retina of the eye, skin, tissue, etc. It is currently still in the medical and industrial development phase applications. The underlying measuring principle is based for example on a fiber optic Michelson interferometer; the essential components are a short-coherent light source, a seroptic beam splitter and a detector. A sample beam emerging from the beam splitter passes to the measurement object, and the detector detects the interference of the light of the sample beam reflected or scattered by the measurement object with the light from the reference arm. By shifting a mirror inserted into the optical path, different optical path lengths result, and as a function of this, interferences in the form of different intensities are displayed on the detector.

The detected signal is analyzed for depth and reflectance or backscatter potential information of a reflective or backscattering interface or structure in the sample.

White light sources or superluminescent diodes are particularly suitable as light sources. The wavelength ranges are typically in the range from 400 nm to 1600 nm. The temporal coherence of the light source is related to the spectral power density via a Fourier transformation, which leads to a short coherence length for a large spectral bandwidth. The size of the possible path length differences of reference and object light, which still reveal an interference, depends on the coherence length. Thus, with a short coherence length, high axial resolutions can be achieved even with a large depth of field of optics.

Thus, for example, at a bandwidth of 30 nm to 50 nm axial resolutions of 3 μιη to 10 μιη. Newer light sources with more than 100 nm bandwidth enable axial resolutions close to 1 μιη.

The sample beam of the short-coherence interferometer is preferably coupled parallel to the beam path for image acquisition into the microscopic imaging system, so that it is directed through the microscope objective to the sample substance. It is advantageous if the sample beam uses only a small portion of the aperture of the objective, so that the lateral extent and the depth of focus of the focus in the sample substance are as large as possible. An increase in the depth of focus can additionally be achieved with an axial lens.

The interference signals are analyzed with respect to the various interfaces and backscattering structures as well as the depth position of the sample substance, and the Z-position of the sample substance is determined based on the analysis result.

If the known Fourier domain method is used for this, a repetition rate of 20 kHz of the depth scan can be achieved. Because the accuracy of the so determined Depth position is many times higher than the coherence length, based on the knowledge of the depth position of the sample substance, the focus position in the sample can be adjusted so that the sample substance or certain structures of the sample substance are in the focal plane.

With the method according to the invention, the focus position can also be determined very quickly during the scanning. A prior generation of a focus map is no longer necessary. The time for the determination of the focus position as well as the subsequent focusing can be restricted in time to the duration of the image acquisition, if the invention is advantageously used, as will be explained in more detail below.

The invention can be configured with further method steps to the effect that even after the alignment of the focus position on the male sample substance from this outgoing optical information is used to control the correct focus and maintain.

For focusing, either the specimen or the objective can be moved, or internal focusing can be used, changing the beam divergence in front of the objective. For image acquisition, you can use either 2D sensors (Tile Scan), TDI sensors (Line Scan), 1 D sensors (Line Scan) or even single sensors (Point Scan). As a contrast method, bright field, fluorescence, phase contrast, DIC and dark field can be used in connection with the method according to the invention.

The invention further relates to a device for automatic displacement of the focal plane of a microscopic imaging system in a Z-position, in which there is a sample substance to be imaged. According to the invention, in this device, a sample beam emanating from an interferometer is directed in the Z direction through the sample substance,

the interference signal generated by the interference of the light reflected by optically active interfaces and / or structures or backscattered light of the sample beam with the reference beam of the interferometer for determining the Z positions of the structures and / or interfaces,

an evaluation device is provided, which is designed to determine the Z position of the sample substance from the set of determined Z positions,

an adjusting device is provided which is designed to shift the focal plane in the Z direction up to the Z position of the sample substance to be imaged; a control unit connected to the evaluation device and the adjusting device, which is designed to generate setting commands for automatically shifting the focal plane to the specific Z position, and a camera connected to the drive unit, designed to hold the sample substance after the automatic focusing.

In a first preferred embodiment of the device according to the invention, the illumination and imaging beam path of the imaging system pass through the objective of the imaging system together with the beam paths of the interferometer. On the image side of the objective, a beam splitter for coupling and decoupling the interferometer beam paths from the illumination and imaging beam path is provided. If the sample beam lies outside the image field, the coupling in and out of the interferometer beam paths can also be carried out by means of mirrors. In this case, a smaller proportion of the aperture of the objective is provided for the interferometer beam paths than for the illumination and imaging beam path, which advantageously has the consequence that the lateral extent of the focal plane and the depth of focus of the focus range are relatively large. To increase the depth of field of the focus, an axicon lens can be arranged in the illumination and imaging beam path. In this case, the axicon lens is arranged outside the beam path provided for image recording.

In a particularly preferred embodiment, a short-coherence interferometer with a super-luminescence diode as the light source is provided as an interferometer. The wavelength of the light emitted by the light source is in the range of 750 nm to 1600 nm.

To determine the Z positions, the Fourier domain method is preferably used, the sampling or repetition rate of the depth scan being, for example, 20 kHz.

When the sample substance is enclosed by a slide and a coverslip, in particular the slide surface, the cover glass back surface, the sample substance to be imaged and the cover glass surface are of interest with regard to the determination of the Z positions in the depth of the sample. With sufficiently high axial resolution of the interferometer, it is also possible to determine Z positions of structures within the sample substance and to automatically focus on these structures.

In an advantageous embodiment of the device according to the invention, as already described above, an overall image of the sample substance is obtained by adding several recorded adjacent to the optical axis of the imaging system adjacent areas of the sample substance and then assembled into an overall image. The image of the sample substance or of the individual regions of the sample substance advantageously takes place on a spatially resolving digital image sensor.

Brief description of the drawings

The invention will be explained in more detail with reference to an exemplary embodiment, which may also disclose additional features essential to the invention. In the accompanying drawings show: the basic structure of the device according to the invention for imaging a sample substance or a portion of the sample substance, examples of interference signals, which are obtained with the device of Figure 1 in a depth scan through the sample.

Detailed description of the drawings

In FIG. 1, a sample substance 1, for example a tissue section, is deposited on a microscope slide 2 and covered with a cover glass 3. The sample substance 1 extends in a plane X, Y perpendicular to the optical axis 4 of the illumination and imaging beam path 5 of a microscopic imaging system not shown in detail.

In the illustration according to FIG. 1, the sample substance 1 is located in the focal plane of an objective 6, which is part of the imaging system.

The illumination for the microscopic image acquisition takes place in the example chosen here according to the method of fluorescence microscopy in reflected light. Deviating from this, however, the application of bright field microscopy is conceivable, in which case the illumination takes place in transmitted light in the visible wavelength range.

The illumination light reflected or scattered by the sample substance 1 is imaged on the two-dimensional, pixel-resolved sensor surface of the image sensor 8 of a digital camera through the objective 6 via the imaging system, which further includes a tube lens 7. For the sake of clarity, FIG. 1 omits the representation of the light source and of a beam splitter for coupling the illumination light into the illumination and imaging beam path 5. The procedure for coupling is known from the prior art and therefore need not be explained in detail here.

In contrast to the prior art, however, a short-coherence interferometer 9 is provided according to the invention, from which a sample beam 10 originates and is coupled into the illumination and imaging beam path 5 via a beam splitter 11 so that it passes through in the Z direction as well as the illumination light the objective 6 is directed onto the cover glass 3, the sample substance 1 and the slide 2.

According to the invention, the short-coherence interferometer 9 is provided to carry out a depth scan in time before the taking of the sample substance 1 or the taking of a partial image of the sample substance 1, which serves to exactly determine the Z-position of the sample substance 1 or a region of the sample substance 1 to be recorded in order then to be able to align the focal plane accordingly.

This is done in such a way that the light reflected by boundary layers and structures inside or outside the sample of the sample beam 10 again passes through the lens 6 on the steel divider 1 1 and is deflected there in the direction of the Kurzkohärenzinterferom- ter 9. This light is made to interfere with the reference beam of the short-coherence interferometer 9, and the resulting interference signal is analyzed with respect to the depth and Z positions of the boundary layers and structures of the sample, respectively.

In the example carried out here, in particular, there are interferences due to the upper side of the slide 2 facing the sample substance 1, through the sample substance 1 itself, through the underside of the cover glass 3 facing the sample substance 1 and through the upper side of the cover glass 3 facing away from the sample substance 1.

An example of the interference signals that result is shown in FIG. It can be seen from FIG. 2 that the interferences caused by boundary layers or structures differ with regard to their amplitudes and are assigned to different depths in the sample in the Z direction.

For example, an interference 12 caused by the slide top, it corresponds to the depth position Z1. Of the sample substance 1, an interference 13 is caused, which corresponds to the depth position Z2. Interference 14 and 15, which are glass rear surface and the cover glass front surface, define the depth positions Z3 and Z4.

The amplitude also gives information about the optical properties of the relevant reflecting or backscattering interface or structure. However, these should not be considered here.

After assignment of the caused by the sample substance 1 interference 13 to the depth position Z2 a related information via a drive unit is passed to actuators, either by movement of the lens 6 or the slide 2 to cause a shift of the focal plane in the Z direction to the extent that this is in the depth position Z2 and thus in the plane X, Y, in which the sample substance 1 to be recorded extends. As soon as this has taken place, a triggering command is transmitted to the camera by the drive unit and an image of the sample substance 1 is taken.

If, as already mentioned, a sample substance 1 is to be taken whose lateral extent is greater than the image field of the imaging system or larger than an area that can be directly absorbed by a camera image, then it is necessary to have several perpendicular to the optical axis 4 of FIG Record imaging system extending areas n, n + 1, n + 2, etc. of the sample substance 1 and then join the shots of these individual areas to form an overall picture.

In this regard, a very advantageous application of the method according to the invention and the device according to the invention is to carry out the depth scan in an area n + 1 by means of the sample beam 10 of the short coherence interferometer 9, the Z position of the sample substance 1 in this area n + 1 and store this Z position assigned to the area n + 1 while the camera is still focused on the previous area n and recording the area n.

In this case, the sample substance 1 is moved continuously under the objective 6 in the X and / or Y direction, with a speed corresponding to the camera image repetition rate, so that in the next image acquisition the region n + 1 is in the image field of the camera, and the The Z-position of the sample substance 1 assigned to this region n + 1 has already been approached while the depth scan in the region n + 2 to be subsequently recorded already determines the Z-position of the sample substance 1 in the region n + 2, the region n + 2 stored and provided for automatic focusing in the subsequent recording of the area n + 2. This process continues until all areas of the sample substance have been collected.

For this purpose, the microscopic device according to the invention is designed such that the sample beam 10 of the short-coherence interferometer 9 is directed outside of the image field of the camera onto the sample substance 1. This is achieved by coupling the sample beam 10 at an angle into the objective 6.

Thus, the object underlying the invention is achieved and an autofocus system is provided which, in connection with a microscopic device for imaging in particular of tissue sections, ensures fast and highly accurate focusing.

LIST OF REFERENCE NUMBERS

1 sample substance

2 slides

3 coverslip

4 optical axis

5 illumination and imaging beam gang

6 lens

7 tube lens

8 image sensor

9 short-coherence interferometer

10 sample beam

1 1 beam splitter

12, 13,

14, 15 interference

I intensities

Z1, Z2,

Z3, Z4 depth positions

Claims

claims

Method for automatically displacing the focal plane of a microscopic imaging system in the Z direction along the optical axis into a Z position in which a sample substance to be imaged is located,

with the following process steps:

Performing a depth scan in the Z direction through the sample substance (1) through an interferometer, wherein

the light of an interferometer sample beam (10) reflected or backscattered by optically effective interfaces and / or structures is brought into interference with the reference beam of the interferometer,

a determination of the Z positions of the interfaces and / or structures of the sample substance (1) is carried out on the basis of the resulting interference signals, from which the Z position of the sample substance (1) is determined,

the focus plane and the sample substance to be imaged (1) are displaced relative to one another in the Z direction until the sample substance (1) of the focal plane is located, and

then the sample substance (1) is imaged.

The method of claim 1, wherein the sample substance is enclosed by a slide and a coverslip, the depth scan is performed by means of a Kurzkohärenzinterfe- rometers (9) and / or the sample substance (1) is digitally recorded.

Microscopic device according to claim 1 or 2, in which, after the alignment of the focus position on the sample substance to be recorded, optical information originating from the sample substance is used to control and maintain the correct focusing.

Microscopic device for imaging a sample substance (1), comprising: a microscopic imaging system, and

Means for automatically shifting the focal plane along the optical axis (4) of the imaging system to a Z-position in which the sample substance (1) to be imaged is located,

characterized in that

a sample beam (10) emanating from an interferometer is directed through the sample substance (1) in the Z-direction, the interference signals of the sample beam (10) reflected by optically active interfaces and / or structures and interfering with the reference beam of the interferometer generate interference signals for determining the Z position of the sample substance (1),

an evaluation device is provided, designed to determine the Z position of the sample substance (1),

an adjusting device is provided, designed to displace the focal plane and the sample substance (1) relative to one another until the sample substance (1) of the focal plane is located, and

a drive unit connected to the evaluation device and the adjusting device is present, configured to generate setting commands for automatically shifting the focal plane into the specific Z position, and

a camera connected to the drive unit is provided for receiving the sample substance (1) following the automatic focusing.

Microscopic device according to claim 4, in which the beam paths of the interferometer are directed through the objective (6) of the imaging system and on the image side of the objective (6) a beam splitter (1 1) is provided for coupling and decoupling the interferometer beam paths.

Microscopic device according to one of Claims 4 or 5, in which a smaller proportion of the aperture of the objective (6) is provided for the interferometer beam path than for the beam paths of the imaging system, so that the lateral extent of the focal plane and the depth of field of the focal region are relatively are big.

Microscopic device according to one of Claims 4 to 6, in which an axicon lens is arranged in the imaging system in order to increase the depth of focus.

Microscopic device according to claim 4 to 7, wherein

the sample substance (1) is recorded by fluorescence microscopy, and

for the interferometer beam paths light with wavelengths in the range of 750 nm to 1600 nm is provided.

Microscopic device according to one of Claims 4 to 8, in which a short-coherence interferometer (9) is provided as the interferometer. Microscopic device according to Claim 9, in which the Fourier domain method is provided for determining the Z positions, wherein the repetition rate of the depth scan is preferably 20 kHz.

Microscopic device according to one of claims 4 to 10, wherein the sample substance of a slide (2) and a cover glass (3) is included and in particular the determination of the Z positions of the slide surface, the cover glass rear surface, the sample substance (1) and the cover glass surface provided is.

Microscopic device according to one of claims 4 to 11, in which the recording of the sample substance (1) on a spatially resolving digital image sensor (8) of the camera is provided.

Microscopic device according to one of claims 4 to 12, wherein the inclusion of a plurality of aligned in the X or Y direction regions n, n + 1, n + 2, ... of the sample substance (1) and the subsequent assembly of these images into an overall image is provided.

A microscopic device according to claim 13, wherein

the automatic determination of the Z-position of the sample substance (1) is provided in an area n + 1 while the area n is being recorded,

the displacement of the sample substance (1) relative to the objective (6) is provided until the area n + 1 is in the image field for the recording,

for the range n + 1, the focus position is automatically brought into the Z position of the sample substance (1),

the automatic determination of the Z-position of the sample substance (1) is provided in an area n + 2 while the area n + 1 is being recorded, etc.