WO2015110250A2

WO2015110250A2 - Method for creating an image of an object, and optical apparatus

Info

Publication number: WO2015110250A2
Application number: PCT/EP2015/000034
Authority: WO
Inventors: Markus Morrin; Hendrik Spahr; Gereon Hüttmann
Original assignee: Euroimmun Medizinische Labordiagnostika Ag
Priority date: 2014-01-23
Filing date: 2015-01-12
Publication date: 2015-07-30
Also published as: WO2015110250A3; DE102014002584A1

Abstract

Described is a method and an optical apparatus for creating an image of an object (103) located within a three-dimensional sample region (101), said method comprising the following steps: a) determining a depth profile (1375) of the sample region (101) at least at one lateral point (119) by means of optical low-coherence interferometry; b) identifying a peak (1353) in the depth profile resulting from the reflection off a point (120) on the object; c) determining an optical distance position (1354) of the peak (1353) representing the optical distance of the point (120) on the object from a reference point (136); d) calculating a geometrical distance position of the point on the object on the basis of the optical distance position (1354) and at least one previously known optical property of the sample region (101), said geometrical distance position representing a geometrical distance (gWI) of the point (120) on the object from the reference point (136); e) focusing a microscope (111) onto the point (120) on the object on the basis of the geometrical distance position (gWI) in order to create an image of the object (103).

Description

Method for imaging an object and optical device

The present invention relates to a method for imaging an object located within a three-dimensional sample area, in particular a biochip, and an optical device designed to carry out the method. In particular, the method relates to imaging fluorophores bound on a biochip.

Laboratory diagnostic studies are an indispensable foundation for modern medicine. There are now a variety of routinely feasible tests available, in the absence of the patient from samples of human or animal origin crucial information on the present disease, the prognosis or success of a treatment received can be. Responsibility to the patient dictates that the reliability and validity of the results of such examinations should be of the utmost importance. For this reason, it is necessary to resort to labor-intensive and technically complex analytical procedures, providing more reliable results compared to simpler procedures.

Such superior methods include immunofluorescence. Typically, a liquid sample obtained from the human or animal body with antibodies to be detected is applied to a biochip which is coated with an antigen-containing biological reagent and fluorescently labeled antibody is added. If the sample contains antibodies, a fluorescent complex of antigen, antibody from the sample and labeled antibody is formed on the biological reagent. It is then not possible to make a reliable diagnosis on the biochip merely by way of quantification of the fluorescence signal as with other methods, but with the aid of a characteristic pattern recognizable under the microscope.

BESTÄTJGÜWGSKOPIE To the detriment of patients, the considerable expense of microscopically examining individual samples limits the use of immunofluorescence and other microscopy-based diagnostic techniques. Not only must the sample be incubated with reagents in several steps and prepared manually for microscopic examination, but also the microscopic examination itself, which must focus on the biological reagent in a first step, is labor intensive and must be performed by trained personnel ,

Various approaches have been described in the prior art to reduce the high resource requirements for immunofluorescence.

On the one hand, there is the possibility of minimizing the amount of expensive reagents required by sophisticated incubation devices. For example, US20100124750 describes a device for carrying out analyzes with grooves, wherein a slide and a reagent carrier are moved in the connected state by means of a movement such that liquid located in the grooves moves alternately in the two longitudinal directions of the grooves. DE 10 2013 017802 describes a device for contacting an immobilized reactant with at least one liquid, wherein the liquid is supplied to a downwardly facing immobilized reactant via a close-fitting survey, so that the volume required for contacting the liquid with the reactant is minimized ,

On the other hand, approaches have been described in the art to automate the focusing step. WO 2012/025220 describes a device suitable for this purpose with a cross table for the automated examination of a biological sample, which is characterized in that the biochip, on the surface of which the sample is located, a visually recognizable marking (for example, a target, the high-contrast Having rings). For automatic focusing, the marking and its position are first determined. As a result, while the XY stage is being traversed within a predetermined position, a series of images having different focusses are taken. This series of images is transmitted to an evaluation unit, which determines a focal plane based on a comparison of the quality of the images. This approach has a number of disadvantages. First, the sample must be exposed several times before the actual analysis. It can not be just one Bleaching of the fluorophore in the fluorescent label come, but also to a deterioration of the sample, which may suffer from the image quality, with the result that the diagnosis is made difficult or even impossible. Furthermore, the biochip must be provided with an identification only for focusing. This increases the manufacturing effort and takes up space on the chip, which can not be used for sample material. In addition, taking and evaluating the series of images requires a relatively long time and a large memory footprint, which can complicate a high-throughput process. DE 10 2010 033 249 A1 discloses a microscopic device for imaging a sample substance, wherein short-coherence interferometry is used for determining the Z position of the sample substance in order to move the sample substance into the focal plane. In this case, after assignment of the interference caused by the sample substance to the depth position, a relevant information on a drive unit to actuators passed to either cause movement of the lens or the slide a shift of the focal plane in the Z direction so far that this in the Depth position is in which extends the male sample substance. However, it has been observed that such a method does not give satisfactory results in terms of correct focusing under all conditions.

Document DE 10 2006 027 836 A1 discloses an autofocus device for microscopy, in which a light modulator generates a two-dimensional intensity-modulated modulation object which lies in the autofocusing beam path in a plane conjugate to the focal plane of the objective and is imaged into the focal plane of the objective. The light modulator increases the complexity of the device.

US 2009/0279052 A1 describes an operation microscopy system which has an OCT measuring device in order to enable automatic focusing on cell structures. Based on a detected OCT signal, a main lens is automatically adjusted. This document does not disclose details of how an OCT signal is used for focusing, especially if there is still a highly reflective layer over the sample. Thus, there is a need for a method and apparatus for imaging an object within a sample area for focusing is improved over the prior art in terms of accuracy, speed and required resources.

The need is met by the subject matters of the independent claims. The dependent claims specify preferred embodiments of the invention.

In a particularly preferred embodiment of the present invention, a method is provided for optically imaging an object located within a three-dimensional sample area, comprising the following steps: a) determining, at least at a lateral point (or two-dimensional area), a depth profile of the sample area by means of optical short coherence interferometry; b) detecting a peak in the depth profile resulting from a reflection at an object point; c) determining an optical path length position of the peak representing the optical path length of the object point from a reference point; d) calculating, based on the optical path length position and at least one known optical property of the sample area, a geometric path length position of the object point representing a geometric path length of the object point from the reference point; and e) focusing a microscope on the object point based on the geometric path length position for imaging the object.

The method can be carried out in particular by means of an optical device according to an embodiment of the present invention.

The three-dimensional sample area may include a plurality of partial areas each having (group and phase) refractive indices different from a corresponding refractive index for air in a wavelength range (specifically, fluorescent light) used for microscopic imaging. In particular, the three-dimensional sample area can be delimited by at least one partial area, which has a refractive index which deviates from that of the air, from a spatial area which has only air. The three-dimensional sample area can comprise solid and / or liquid partial areas, in particular at room temperature. One of these subareas may include the object to be imaged. The lateral point may have an extension in two dimensions which corresponds to a cross-sectional dimension of a measuring beam which is suitable for the short-coherence Interferometry is used. The extent of the lateral point may be, for example, 5 μm to 10 m in each of the two dimensions.

The depth profile is based on an interference signal which results from interference of reference light with light which is reflected from the sample area at the lateral point at different depths. For this purpose, the sample area is illuminated with light which has a short coherence length. A constructive interference of the reference light with light originating from the sample area can occur only if the optical path length which has traveled the reference light coincides exactly with the optical path length up to the coherence length, which has traveled the light which originates from the sample area , Due to different partial regions within the sample area, which have different (phase) refractive indices, reflections occur at intermediate layers between different partial regions, which can be detected in the interference signal.

Short-coherence optical interferometry in this embodiment of the present invention is understood to be an optical interferometric method for determining structural information within a volume range of an object. Sometimes, short-coherence interferometry within this application is also referred to as optical coherence tomography (OCT) or white-light interferometry, although the short-coherence interferometry employed within the method of the invention need not necessarily perform tomography. Short-coherence interferometry requires, according to the method according to the invention, a recording of a depth profile at least at a lateral point. For the sake of brevity, however, short-coherence interferometry is also referred to as OCT.

For this purpose, an object arranged in the object or sample area is illuminated with a first part of the OCT measuring light generated by the light source. The first part of the measurement light interacts with matter within a volume region of the object, with part of the first part of the OCT measurement light returning from the object. The part of the OCT measuring light which has returned from the object is superimposed interferometrically with a second part of the OCT measuring light and registered by a detector. Various implementations of OCT, all of which can be applied by the method, differ in the way the object is scanned along a depth direction of the object and also in the way the overlaid light is registered. Depending on the material included in the examined object, a wavelength of the OCT measurement light and other physical properties, an intensity of OCT measurement light entering the object decreases exponentially, which can be characterized by a certain penetration depth. In particular, a degree of reflectivity within the sample region may depend on a refractive index and / or a gradient of the refractive index of the material within that sample region. In particular, the OCT measuring light can have a small coherence length which is approximately in the range of a few micrometers. The coherence length of the OCT measuring light represents an average length of a wave train in which different sections of the wave train are in a defined phase relationship. The first part of the OCT measuring light, which returns from the object, is only capable of interfering with the second part of the OCT measuring light if a difference in the optical path lengths passed by the two parts of the OCT measuring light is smaller than the coherence length of the OCT measuring light is. Due to this principle, the OCT system can obtain structural information about the sample area at a defined depth.

Embodiments of the invention include various variants of a short-coherence interferometry apparatus provided in the optical device adjacent to the microscopy system. These variations of an OCT system differ in how different depths within the object are scanned and how the overlaid light is detected.

In time-domain OCT (TD-OCT), the sampling of the sample area is performed at different depths by changing the optical path length which is passed through by the second part of the OCT measurement light (also called reference light).

For this purpose, for example, a reflecting surface, such as a mirror, be displaced, while at the same time an intensity of the superimposed light is detected.

A disadvantage of this method, however, is that a mechanical displacement of the reflective surface must be performed, resulting in inaccuracies of a

Amount of the displacement of the reflective surface as well as inaccuracies in the correct orientation of the reflective surface may be afflicted.

Also, the method of the reflector for taking a depth profile requires a relatively long period of time. Frequency Domain OCT (FD-OCT) is another variant of an OCT system that may be provided in the optical device according to an embodiment of the present invention. In this case as well, the second part of the OCT measuring light (reference light) is reflected on a reflecting surface, but this reflecting surface does not have to be displaced in order to scan different depths within the object for structure determination. Instead, structural information about the sample area at different depths is obtained by detecting intensities of the superimposed light as a function of a wavelength of the superimposed light. According to embodiments of the present invention, various embodiments of frequency domain OCT may be used in the method and the optical device. In particular, the embodiments of frequency domain OCT spectral domain OCT (SD-OCT), sometimes also called Fourier domain OCT, and swept source OCT (SS-OCT) can be used. In spectral domain OCT, the superimposed light from the reference light and the OCT measurement light returning from the object is spectrally decomposed using a spectrometer to spatially separate a plurality of spectral portions of the superimposed light. Intensities of these spatially separated multiple spectral portions of the superimposed light are detected, for example, by a spatially resolving detector, such as a CCD camera / line detector. Here, the spatially resolving detector may comprise a plurality of detector segments, each of these detector segments receiving a spectral portion of the superimposed light. The spatially resolving detector then provides electrical signals corresponding to the intensities of the multiple spectral portions representing a spectrum of the superimposed light. By analyzing the spectrum, such as by Fourier transform, a distribution of reflectivities within the object along the depth direction, ie, an axial direction, can be obtained, thus determining a depth profile.

For time-domain OCT as well as for spectral domain OCT, for example, a superluminescent diode can be used as the light source for generating the OCT measurement light. The light source for generating OCT measuring light can produce OCT measuring light with a peak wavelength between 800 nm and 1300 nm and with a spectral width between 5 nm and 100 nm, in particular between 15 nm and 30 nm. A coherence length of the OCT measuring light is inversely proportional to the spectral width of the OCT measuring light. In the case of spectral domain OCT affects the spectral width of the OCT measuring light with which the object is illuminated, an achievable axial resolution, ie a resolution along the depth direction.

In spectral domain OCT, for example, a diffraction grating or a plurality of diffraction gratings with further optical components can be used as the spectrometer for spatially separating the multiple spectral parts of the superimposed light.

Other embodiments of the present invention use another embodiment of Frequency Domain OCT, Swept Source OCT (SS-OCT). Here, the object is illuminated with OCT measuring light, which has a much smaller spectral width, and thus a much longer coherence length than in time domain OCT or spectral domain OCT. Here, during a measurement, the central wavelength of the illuminating OCT measuring light is changed over a range of wavelengths from about 10 nm up to 200 nm or more. During the sweeping of the center wavelength of the OCT measuring light, the OCT measuring light returning from the sample area, which is superimposed with reference light, is detected by a detector such as a photodiode. Thus, a spectrum of the superposed light can be obtained by temporally changing the center wavelength of the OCT measuring light and detecting the superimposed light. As in the case of spectral domain OCT, structural information about the object can be obtained by analyzing the received spectrum of the superimposed light, such as Fourier transform. In contrast to spectral domain OCT, however, no spectrometer is necessary and no spatially resolving detector.

Determining the depth profile l ann may include detecting interference light, in particular by means of a line detector, and evaluating the detected interference light, in particular performing a Fourier transformation. The depth profile may represent the intensity of reflectivity at various optical path length positions within the sample area. At a certain optical path length position, the OCT light originating from the sample area has traveled a certain optical path length, which however does not correspond to the geometric path length, since the conversion still has the refractive index or the refractive indices (in particular group refractive indices) of the materials or subregions within the sample area must be taken into account, which are traversed by the OCT light. A peak in the depth profile can be characterized by a (locally) high reflectivity at a certain depth compared to other positions in the depth profile. The peak can be detected by evaluating a characteristic, in particular a profile of the depth profile, in particular by determining one or more local maxima in the depth profile and associating them with previously known subregions or sublayers of subregions within the sample region. The reflection at the object point may in particular correspond to a reflection at a biochip to be imaged at a point.

Determining the optical path length position of the peak may include determining a position of a local maximum in the depth profile. In particular, the depth profile can be formed by a plurality of measured values which each have a reflectivity value and an optical path length position. Different measured values can be separated from each other by a specific sampling interval. Intermediate values can be interpolated. To determine the optical path length position of the peak, an adaptation of a function to the plurality of measured values can be carried out. The calculation of the geometrical path length position of the object point can be based on prior knowledge of the construction and / or the previously known optical property of the sample area. The at least one previously known optical property may in particular comprise one or more refractive indices (in particular phase refractive indices and / or group refractive indices) of one or more subregions within the sample area up to the object point. However, the depth profile may contain information about reflectivity, which is even beyond the object point. An area of the sample area lying on this side of the object point is understood to be a region which is traversed by the OCT light from an OCT light source within the sample area to the object point. An area of the sample area lying beyond the object point is understood to be an area which is traversed by the OCT light after reaching the object point within the sample area. The portion of the sample area beyond the object point may be considered in the depth profile to detect the peak, ie, to detect that increased reflectivity at a particular position in the depth profile results from reflection from the object point. This area lying beyond the object point becomes however, not needed for calculating the optical path length position of the object point. In some embodiments, the bottom of the slide may be used to calculate the refractive indices or as a reference. The focusing of the microscope may include a method of a main objective of the microscope along an optical axis (parallel to the depth direction) of the main objective, wherein the optical axis of the main objective is preferably parallel to a beam path of the OCT light, for short-coherence interferometry is used.

In a particularly preferred embodiment of the present invention, the object comprises a biological sample, preferably cell structure and / or tissue and / or tissue section with fluorescently labeled antibody, on a, in particular planar, biochip, the biological sample preferably comprising at least one from the following group: Thin section of biological tissue, preferably paraffinized and / or formalin-fixed or frozen section, cell smear, bacterial smear, viruses, protozoa and parasites, applied in solution, dried or coupled antigen drops, other antigens coupled to a surface and any nucleotide sequences. In particular, the object may be sensitive to light, which is why excessive irradiation of the object with light, in particular fluorescence excitation light, should be avoided. The determination of the geometric-pathlength position of the peak by means of short-coherence optical interferometry may comprise irradiation with OCT light, which does not have to have fluorescence excitation light and in respect of which the object does not have to be photosensitive. Therefore, damage to the object during the determination of the geometrical path length position of the object point (in particular of a point on the biochip) can be reduced according to the method according to the invention. In a particularly preferred embodiment of the present invention, the detection of the peak comprises registering a pattern in the depth profile and associating, preferably based on relative optical path length positions, pattern details to previously known structures in the sample area. In the depth profile, a distance or distances between maxima can or can be determined. These distances can be used to detect from which previously known boundary surface or intermediate layers results in a reflection which leads to a (local) maximum in the depth profile. The arrangement of different maxima in the depth profile can be considered as a pattern. The more maxima (other than the maximum associated with the peak) can be observed in the depth profile, the more reliably the peak can be recognized as being the local maximum resulting from a reflection from the object point.

In a particularly preferred embodiment of the present invention, an image plane offset is calculated from a depth dimension and a refractive index of the sample area to the object point, and the focusing is based on, in particular, a difference between, the geometric path position and the image plane offset.

The area of the sample area lying on the side of the object point may have a refractive index (in particular phase refractive index) which is greater than the refractive index of air. Therefore, light emanating from the object point used to image the object may be broken away from the solder as it passes from the sample area into air before it enters the lens. Therefore, for focusing, an object located in a sample area having a refractive index greater than that of air on the other side of the object point must be moved farther away from the objective along the optical axis of the objective, than an object located on that side of the object point Area of the sample area, which has a refractive index which is greater than that of air.

For correct focusing on the object, therefore, the refractive index or the refractive indices (in particular phase refractive indices) of the materials or partial regions of the sample area must be known and taken into account on this side of the object together with their respective depth extents.

In a preferred embodiment of the present invention, the sample area comprises subregions up to the object point and the image plane offset is determined according to FIG

where the d _{i are} geometric depth extents of the respective partial regions / up to the object point and the n _{j are} the phase refractive indices, preferably at a Central wavelength of the light used for imaging by means of the microscope, the respective partial areas are up to the object point.

The geometric depth expansions may be known at least approximately beforehand. Likewise, the phase refractive indices and the aperture angle of the lens may be known. The image plane offset can be calculated without the use of short-coherence interferometry results by using at least approximately known depth dimensions. Alternatively, the geometric depth extents may be calculated based on differences in optical pathlength positions determined by short-coherence interferometry, the phase refractive indices being assumed to be known.

The above formula (*) for calculating the image plane offset can be applied to lenses with a small aperture angle of the lens (small numerical aperture (NA)). In the case of high-NA lenses, in addition to the focus offset, the focus is elongated in the axial direction (depth direction, ie, z-direction) (spherical aberration) since the image plane displacement is increased

is dependent on the angle of incidence ^{a of} the beams. This effect can lead to a reduction in imaging quality. A lens with high NA may therefore have, for example, a so-called cover glass correction ring (in particular a motorized correction ring). Thus, the strength of the cover glass correction (strength of the spherical aberration) can be adapted to different coverslip thicknesses and additionally through refractive media (such as glass and / or glycerin). After measuring the (optical and / or geometric) layer thicknesses by the short-coherence interferometry, the optimal setting for such a correction ring can be calculated. The correction ring can be adjusted in particular as a function of the optical path length from the top of the cover glass to the object point. The focus distance of the lens may vary depending on the setting of the correction ring. This can be taken into account when focusing.

In a preferred embodiment of the present invention, a short-coherence interferometry apparatus used for optical short-coherence interferometry has an axial resolution of 1 pm to 15 pm, preferably 3 pm to 10 pm preferably 8 μητι to 10 μιη, wherein the geometric path length position of the object point with higher accuracy, preferably 0.03 μιη to 1 μιη, more preferably 0.1 μητι to 0.5 microns, as the axial resolution of the short-coherence interferometry Apparatus is determined.

Thus, no excessive requirements for an axial resolution of the short-coherence interferometer e-apparatus are provided, so that conventionally available equipment can be used. Nevertheless, due to the use of prior knowledge of the structure and refractive indices of the sample region, an absolute geometrical position of the object point (along a depth direction parallel to the optical axis of the microscope) can be determined with much higher accuracy than the resolution of short-coherence interferometry -Apparatus. In particular, the sample area may have boundary layers or intermediate layers between partial regions with different refractive indices, which have a greater mutual distance to achieve this accuracy of an absolute position determination than the resolution capability of the short-coherence interferometry apparatus.

In a preferred embodiment of the present invention, the imaging comprises exciting fluorescence of a fluorophore included in the object and detecting the emitted fluorescent light from the object.

The exciting of fluorescence can be achieved by illuminating the object with fluorescence excitation light. Suitable fluorophores are fluorescent molecules or groups of molecules which have a sufficiently narrow extinction and emission spectrum, are intense and stable. Numerous suitable fluorophores with their respective extinction or emission spectra are known to the person skilled in the art, for example the fluorophores from the group comprising hydroxycoumarin (325 or 386 nm), Cascade Blue (410 or 423 nm), DAPI (345 or 455 nm). , Texas Red (589 and 615 nm), fluorescein (495 and 519 nm, respectively), X-rhodamine (570 and 576 nm, respectively), Alexa Fluor series, for example Alexa Fluor 532 (530 and 555 nm), Cy dyes , z. Cy2 (489 and 506 nm), Cy3 (550 and 570 nm) or Cy5 (650 and 670 nm, respectively), DyLight dyes, eg DyLight549 (562 and 576 nm), ethidium bromide (493 and 620 nm, respectively) ), Hoechst 33342 (343 and 483 nm, respectively), Fluo-3 (506 and 526 nm, respectively) and the fluorescent proteins GFP (396 and 508), Y66H (360 and 442 nm), Y66F (360 and 508 nm, respectively) ), RFP (563 and 582 nm), EBFP (380 and 440 nm, respectively), ECFP (434 and 477 nm, respectively), EGFP (488 and 477 nm, respectively), 507 nm), as well as fluorescent derivatives and homologs thereof, all of which can be used in various embodiments according to the present invention. In a particularly preferred embodiment, the fluorophore is derived from the classes cyanine dyes, fluoresceins, rhodamines, most preferably fluorescein isothiocyanate (FITC). Depending on the application, fluorophores can be used underivatized, eg. B. DNA-binding fluorophores such as ethidium bromide. However, it is particularly preferable for the fluorophore to be part of a reagent which specifically interacts with molecules, cell or tissue constituents or structures of the samples to be examined, preferably a reagent from the group comprising antibodies, fusion constructs or fragments thereof, for example primary or secondary antibodies, Nucleic acids, anticalins, aptamers and the like. Suitable fluorophores and reagents labeled therewith are commercially available, for example from EUROIMMUN AG, Lübeck.

Wavelength ranges of the light used for the excitation may comprise a range around a wavelength of the extinction maximum of the fluorophore used +/- 20 nm, preferably +/- 10 nm, more preferably +/- 5 nm.

In a preferred embodiment of the present invention, the sample area in a region through which the object is irradiated comprises a capping glass and a liquid, preferably a pH-buffered glycerol solution. Such a designed sample area allows, in particular, a mapping of a biochip after a precise focusing. In this case, the cover glass can provide two detectable interfaces and a third interface beyond the liquid can represent a surface of the biochip to be examined. In a preferred embodiment of the present invention, the depth profile in addition to the peak in a radiated to the object area another peak, which originates from an upper surface of the coverslip, and still another peak, which originates from a bottom of the coverslip, on, wherein the another peak and the still further peak can be used to detect the peak and / or the geometric path position of the object. The further peak and the still further peak can facilitate detection of the peak and thus can improve the reliability of the method. In other embodiments, the accuracy of determining the geometric position of the object point may be improved. Advantageously, a conventionally used sample area can be used to examine a biochip without having to change the conventionally used sample area, in particular without having to attach visually recognizable markings or targets to the sample area. This can save costs. In a preferred embodiment of the present invention, calculating the geometrical path length position of the object point is further based on an optical path length position of the further peak, an optical path length position of the still further peak, a refractive index of the capping glass, a difference between Optical path length position of the still further peak and the optical path length position of the peak and a refractive index of the liquid.

From the difference of the optical path length position of the further peak (in particular top side of the cover glass) and the optical path length position of the still further peak (in particular lower side of the cover glass), the thickness of the cover glass can be calculated. From the difference of the optical path length position of the still further peak and the optical path length position of the peak, the thickness of the glycerol solution can be calculated. If the group refractive indices of the coverslip and the glycerol solution are then used, then the geometric path length position of the peak and thus taking into account the image plane offset, the focus position of the object point can be determined. Thus, only the evaluation of a depth profile and knowledge of the refractive indices of the coverslip and the glycerol solution is required for focus position determination of the object point.

In a preferred embodiment of the present invention, the optical path length position of the peak and / or the further peak and / or the still further peak is preferably determined by adapting a suitable fit function to an oversampled depth profile generated by zeropadding of the interferogram and / or taking into account a maximum signal and two-way neighboring signals of the respective peak in the depth profile, preferably by means of a frequency estimation method. Other mathematical methods are also possible. Zeropadding "padding with zeros ..." is a method for virtual magnification of the spectral resolution in the discrete Fourier transform. The appending of zeroes causes the interpolation of additional frequency values in the Fourier transform. For example, if the spectrometer or detector provides 200 pixel values, 824 zeros are appended. Thereafter, an FFT is performed over 1024 interpolation points.

A frequency estimation method makes it possible to increase the accuracy of the determination of the peak position in the FFT spectrum and is comparable to the mathematical center of gravity determination. A "classical" focus determination leads to artifacts, due to the convolution between window function and signal. In the frequency estimation method, the peak position determination is less subject to the artifact by the convolution between window function and signal when performing center of gravity determination over several pixels in the vicinity of the maximum value. Thus, in particular, an optical path length position of the peak can be determined with higher accuracy than the axial resolution of the short-coherence interferometry apparatus.

According to one embodiment of the present invention, the geometric path length position Z (Obj) of the object point is calculated according to:

Z (Obj) = Z (wP) / n (air) + [Z (nwP) -Z (wP)] / n (glass) + [Z (P) -Z (nwP)] / n (glycerol), where

Z (wP) is the optical path length position of the further peak,

Z (nwP) is the optical path length position of the still further peak,

Z (P) is the optical path length position of the peak,

n (air) is the (group) refractive index of air at the central wavelength of the light used for short-coherence interferometry,

n (glass) is the (group) refractive index of the coverslip at the central wavelength of the light used for short-coherence interferometry, n (glycerol) is the (group) refractive index of the glycerol solution at the central wavelength of the light used for short-coherence interferometry.

Thus, the geometrical path length position of the object point can be easily calculated, and then correctly focused, if the above-defined image plane offset is taken into account as well. In a preferred embodiment of the present invention, the method further comprises the step of: f) lateral movement of the object along a first line and preferably further along a second line, during which respective geometric path length positions of a plurality, in particular between 10 and 30 object points respectively in the first and second lines, are determined by object points.

A lateral method can be performed with a conventional sample holder with displacement capability. The lateral process can be carried out step by step, wherein at each standstill after a step, a geometric-Weglänge- position of the peak can be determined. The (depth) position can be stored in each case (in particular together with associated lateral position information). The second line may be laterally offset from the first line. Thereby, positional information about the object can be obtained at different lateral points, which can improve a (later) focusing on the object for imaging the object.

In a preferred embodiment of the present invention, a first straight line equation is determined on the basis of positions of the object points of the first line, a second straight line equation is determined based on positions of the object points of the second line, wherein a normal of the object is calculated from the first and the second straight line equation and the normal and a geometric-pathlength position of the object at a lateral point are used to focus the microscope at another lateral point.

A determination of a respective straight-line equation to the first line or the second line may include an adaptation of a respective straight line to the plurality of measured points such that an error term becomes minimal. As a result, measurement inaccuracies of the specific positions of the measured points can be partially compensated. The straight line equations can be determined from the geometric path length positions and / or the optical path length positions. The normal can generally be calculated using at least three positions or three-dimensional positions, not necessarily thus with the aid of a first straight line equation and a second straight line equation and thus also not necessarily with the aid of a survey along a first line and a second line. If a plurality of biochips are to be imaged, then at first all biochips for determining at least three geometric path length positions of three points each can be measured by means of short-coherence interferometry. In a subsequent pass, the biochips can be imaged sequentially, whereby it may happen that a lateral position approached for a biochip does not correspond exactly to a lateral position, which was previously measured by means of short-coherence interferometry with respect to its depth position, ie focus position , In this case, to determine the depth position, ie focus position, the approached lateral position, the geometric-path length position of the previously measured point which is closest to the current lateral position and with further reference to the determined normals (or their Inclination to the depth direction) which linearly associates a depth deviation from the depth position of the previously measured point with the distance therefrom.

According to one embodiment of the present invention, the reference point has a known distance along a microscope beam path from a position, preferably an object-side nearest optical surface, of an objective of the microscope, wherein preferably the objective is a 20x to 40x magnifying objective and the microscope preferably has a depth of field between 0.5 pm and 1, 8 pm.

The reference point may be a fixed point with respect to the short-coherence interferometry apparatus. The short-coherence interferometry apparatus may be fixed or movable, in particular pivotable, relative to the microscope. Using the distance of the reference point from the position of the lens, it is possible to deduce the geometric path length position of the peak to a travel length of the objective in order to bring the object into the focal plane of the objective. According to one embodiment of the present invention, the short-coherence interferometry apparatus comprises an illumination source (also referred to as OCT light source) for illuminating the object with illumination light (also referred to as OCT light), preferably in the near-infrared region, and a detector for detecting interference light, which is obtained by interference of the object reflected illumination light with reference light, wherein the short-coherence interferometry apparatus preferably has a grating for spectrally decomposing the interference light, and preferred is designed to record depth profiles at a sampling rate of between 1 kHz and 2 kHz.

In particular, the detector can be a line detector which can detect the interference light in a spectrally resolved manner. In particular, the short-coherence interferometry apparatus may be or include a spectral domain short-coherence interferometry apparatus. The measurement rate may be sufficiently high to measure a plurality of biochips arranged on a travel table sequentially for focus position determination and subsequently to image them by means of the microscope, using retrieved focus positions of the respective biochips,

In a preferred embodiment of the present invention, a measuring method is provided for measuring a plurality of biochips arranged laterally next to one another on a carrier, comprising the steps: a) step-wise lateral, preferably meander-shaped, method of the carrier relative to a microscope and short-coherence interferometry apparatus; b) in one step or continuously, performing a method according to any one of the preceding claims; and c) taking an image of a respective focused biochip by means of the microscope.

The meandering method allows a measurement of a two-dimensional field of objects in a relatively short time. Further, this type of method allows for measurement along a first line and along a second line. During the meandering process of the carrier relative to the short-coherence interferometry apparatus, it can be pivoted into a microscope beam path (or the microscope beam path can be swung out) so that microscopic imaging does not take place. Certain positions may be stored for each object of the plurality of objects or biochips, respectively, in a memory of the optical device. In a subsequent passage of a method of the carrier relative to the microscope, the geometric position length positions or resulting focus positions corresponding to the lateral position can be retrieved and the microscope can be focused based on a focus position of the respective biochip in the field of view of the microscope become.

According to one embodiment of the present invention, during the lateral process of the carrier relative to the short-coherence interferometer, at least one geometric path length position is determined for each biochip and included After determining the geometric-path length positions of the plurality of biochips, the lateral method of the carrier relative to the microscope, the method retrieves: retrieving the stored geometric-path length position associated with the current lateral position of the microscope relative to the carrier, focusing on the biochip for imaging by the microscope based on the retrieved geometric path length position. Thus, in particular, a plurality of biochips can be measured quickly and reliably. It should be noted that features described or provided individually or in any combination in connection with a method of imaging an object located within the three-dimensional sample area are also individual or in any combination for an optical device according to one embodiment of the present invention apply or can be applied.

In a preferred embodiment of the present invention, an optical device is provided for imaging an object located within a three-dimensional sample area. In this case, the optical device has a short-coherence interferometer apparatus, which is designed to determine a depth profile of the sample area at least at a lateral point by means of optical short-coherence interferometry, and a processor. The processor is configured to detect a peak in the depth profile resulting from a reflection at an object point, to determine an optical path length position of the peak representing the optical path length of the object point from a reference point, and based on the Optical path length position and at least one known optical property of the sample area, a geometric-path length position of the object point, which represents a geometric path length of the object point of the reference point to calculate. The optical device further comprises an actuator configured to focus a microscope on the object point based on the geometric path length position for imaging the object.

The optical device is in particular designed to carry out a method for imaging an object according to an embodiment of the present invention. The optical device may in particular comprise the microscope. Furthermore, the optical device may comprise a device for detecting a beam path of the short-coherence beam. To direct interferometry device for illuminating the object with OCT light on a field of view of the microscope or in a beam path of the microscope or swivel and selectively swing out.

In a preferred embodiment of the present invention, the optical device further comprises a controller coupled to the processor and the actuator for biasing the actuator to focus in response to an output signal of the processor to preferentially scan a lens along a depth direction.

This can be achieved in an automatic way focusing a variety of objects.

In a preferred embodiment of the present invention, the optical device further comprises a laterally movable sample holder, which is preferably controlled by the controller to the lateral method.

Thus, a plurality of objects, which are arranged laterally side by side, can be imaged.

In a preferred embodiment of the present invention, the optical device further comprises a memory for storing geometric-path length positions of a plurality of object points.

First, by means of the short-coherence interferometric apparatus, focus positions of a plurality of objects, in particular biochips, can be determined and stored in the memory. In a further passage, the biochips can be successively imaged by means of the microscope, for which purpose the respective focus positions are retrieved from the memory and used to focus the objective of the microscope.

The optical device may further comprise an objective for imaging the object, wherein the objective has in particular (eg with a numerical aperture greater than 0.7 or greater than 0.8) a cover glass correction device which is designed to reduce aberrations, in particular spherical aberration, when mapping object points due to To correct knowledge of position, thickness and refractive index of the sub-areas, the image plane offset in particular by

is calculated, the geometrical depth expansions of the respective

Partial regions i are up to the object point (120) and the ⁿ 'are the phase refractive indices, preferably at a central wavelength of the light used for imaging by means of the microscope, of the respective partial regions up to the object point and where α is the angle of the optical path to the optical axis of the microscope Is objective. In particular, the spherical aberration of the objective can be adjusted within a certain range by changing the setting of the cover glass correction device.

The objective can e.g. have a so-called cover glass correction ring (in particular motorized correction ring). Thus, the strength of the cover glass correction can be adapted to different cover glass thicknesses and additionally through refractive media (such as glass and / or the glycerol). When using such a lens, after finding the focal plane, the optimum setting of the correction ring must also be determined by "trial and error". After measuring the layer thicknesses by the short-coherence interferometry, the optimal setting for such a correction ring can be calculated. The focus distance of the lens may vary depending on the setting of the correction ring. If the cover glass correction changes the focus distance of the lens, this can be taken into account when focusing.

Embodiments of the present invention, to which the invention is not limited, will now be described with reference to the attached drawings.

Fig. 1 schematically shows an optical device for imaging an object located within a three-dimensional sample area according to an embodiment of the present invention; Fig. 2 schematically shows a short-coherence interferometry apparatus which can be used for example in the optical device of Fig. 1;

3 illustrates a sample carrier for a plurality of biochips arranged in a plurality of chambers and examined in accordance with a method of imaging an object within a three-dimensional sample area;

Fig. 4 is a side view showing a sample area which is imaged with a microscope of the optical apparatus of Fig. 1;

Fig. 5 is a side view of another sample area having a greater thickness for illustrating an image plane offset, which is considered in accordance with embodiments for focusing and imaging; Fig. 6 schematically illustrates interfaces within a sample area which, according to embodiments of the invention, are recognized to determine a focus position of a biochip;

FIG. 7 illustrates a focal plane determinable according to embodiments of the present invention; FIG.

Fig. 8 illustrates an offset spectrum and Fig. 9 illustrates an apodization spectrum taken into account in an evaluation of an interference signal; Fig. 10 shows a spectrum taken by a detector of the short-coherence interferometry apparatus;

Fig. 11 illustrates a recorded spectrum of Fig. 10 corrected by means of the spectra discrete in Figs. 8 and 9;

Fig. 12 shows a windowed spectrum according to embodiments of the invention;

13 illustrates a depth profile or an A-scan, which is used to determine a geometric path length position of an object point, in particular a biochip; Fig. 14 illustrates a phase refractive index and a group refractive index of glass from which a capping glass of the sample region is made;

Fig. 15 illustrates a phase refractive index and a group refractive index of a glycerin solution which is located between a capping glass and the biochip.

The figures show schematic representations. In this case, in structure and / or function similar or identical elements are marked with reference numerals that do not differ in the last two places. A description of an element occurring in a figure but not explicitly described can be taken in such cases, the description of this element in another figure.

FIG. 1 schematically illustrates an optical device 100 for imaging an object 103 located within a three-dimensional sample area 101 according to an embodiment of the present invention. The optical device 100 comprises a short-coherence interferometry apparatus 105, a processor 107 and an actuator 109. Optionally, the optical device 100 comprises a microscope 11, which comprises an objective 11, an imaging optics 115 and an image detector 117. The short-coherence interferometry apparatus 105 is designed to determine a depth profile of the sample area 101 by means of optical short-coherence interferometry, at least at a lateral point 1 19. In Fig. 1, this lateral point is shown as a lateral region 1 19, which has a certain lateral extent, about a few micrometers depending on the lateral resolution of the short-coherence interferometry apparatus 105. The lateral extent of the region 119 is in an x-direction or a y-direction, which are shown at reference numeral 121, which are perpendicular to a z-direction, which is designated by reference numeral 123. The z-direction 123 corresponds to a depth direction along which a depth profile is determined by short-coherence interferometry.

For this purpose, the short-coherence interferometry apparatus 105 has a light source 125 for emitting OCT-short with a short coherence length, approximately a few micrometers, which is guided along an optical waveguide 127 to an optical coupler 129. The optical coupler 129 divides the OCT ücht into two parts, of which a first part is guided along the light guide 127 to a reflector 131, which may be adjustable in its position for setting a reference optical length. A second part of the OCT Light is formed by the light guide 127 and after exiting the light guide by an illumination optical system 133 to a suitable cross-sectional dimension to irradiate the sample area 101 along the depth direction and z-direction 123, respectively. For this purpose, the OCT light is directed, for example, by means of a reflector 135 onto the sample area 101 at the lateral point or area 119. The reflector 135 is optional, the OCT light can be directed without this on the object. A central area on the reflector 135 defines a reference point 136 to which a geometric path length position in a depth profile relates. The reference point 136 may be located at other locations.

The sample area 101 comprises a capping glass 137 which has a front side 139 (ie located on this side of the object 103) and a rear side 141 (located on the side of the object 103 but located beyond the front side 139 of the capping glass 137). On the other side of the cover glass 137, the sample area 101 comprises a glycerol solution 143, which covers the entire space between the cover glass 137 and the object 103, i. an upper surface of the biochip 145, fills. The biochip 145 is attached to a chamber bottom 149 with adhesive 147. Within the only incompletely illustrated chamber with chamber bottom 149, a plurality of (not shown) biochip 145 may be included.

In a depth profile, which is determined by means of the short-coherence interferometry apparatus 105 after an evaluation by means of the processor 107, optical path length positions of the boundary layers 139, 141 and 103 can be determined, which can be stored in a memory 151. In particular, the processor 107 is configured to detect a peak, such as peak 1353 in FIG. 13, and to determine a corresponding optical path length position of the peak resulting from a reflection of the OCT light from the surface 103 of the biochip 145. Based on the optical path length position and at least one known optical property of the sample region 101, such as refractive indexes of the capping glass 137 and the glycerin solution 143 and the respective thicknesses of these elements, the processor 107 calculates a geometrical path length position of the object point 120 on the Surface 103 of the biochip 145. The processor 107 may output control signals or results of its calculations to a driver 155, which may drive the actuator 109 to move the lens 1 13 along the depth direction 123 or z-direction, so that the object point 120 is arranged in the focal plane of the objective 1 13. The OCT light reflected at different depths from the lateral region 19 is reflected at the mirror 135 and re-enters the light guide 127, which guides the reflected OCT light to the optical coupler 129 where the reflected OCT light with reference light , which has been reflected by the reference mirror 131, is brought to interference. The interference light is detected by a detector 130, which in one embodiment comprises an optical grating to split the interference light into wavelengths before it is detected by a line detector, for example. Electrical or optical signals corresponding to intensities detected by the detector 130 are applied to the processor 107, which determines a depth profile from these signals.

The optical device 100 illustrated in FIG. 1 further comprises a laterally movable sample holder 157 which can be moved relative to a base 159 along one or more lateral directions 121 (for example x-direction and / or y-direction). In particular, the controller 107 may supply appropriate control signals to the base 159 to laterally move the sample holder 157, on which the sample region 101 is disposed within a chamber. In particular, geometric path length positions of a plurality of surfaces 103 of biochips 144 may be stored in the memory 151 and retrieved from the memory 151 with respect to the respective lateral position of the currently examined biochip for focusing the objective 1 13.

The short-coherence interferometry apparatus within the optical device can meet the following specifications:

• A lateral movement speed of the microscope slide under the microscope objective of up to 60 mm / s is supported. The measuring frequency is at least 1 kHz.

The lateral resolution can be at least 100 μm due to the typical dimensions of the sample and image field.

^• The axial resolution is less than 15 μιη (in particular they may be greater than the Rayleigh length of the lens used in the fluorescence image to be). The accuracy of determining the position of the interface on the to be focused may be more accurate than the Rayleigh length of the lens of the microscope.

• The measuring depth is up to or about 2 mm.

The sample area 101 has a depth extent TA up to the object point 120. The opening angle of the objective is α. The geometrical path length position of the object point 120 is denoted by gWI, the image plane offset by δζ and the difference between geometrical path length position of the object point 120 and image plane offset is denoted by Δζ and entered in FIG. The distance between the lens 1 13 and the reference point 136 is designated A.

FIG. 2 schematically illustrates a short-coherence interferometry apparatus 205 that may be used, for example, in the optical device 100 of FIG. The short-coherence interferometry apparatus 205 includes an OCT light source 225 which emits OCT light 226 and directs it to a beam splitter 229. The beam splitter 229 splits the OCT light 226 into two parts, with a first part 228 being directed to a reference mirror 231 and a second part 230 being directed to the object area 201. OCT light 230 reflected from the object region 101 of different depths z (reference numeral 223) interferes with first part 228 of the OCT light 226 reflected at the reference mirror 231, and is spectrally decomposed at the optical grating 232. The spectrally decomposed interference light is detected by the line camera 230. From this, a wavelength-dependent intensity spectrum 232 can be obtained.

The sample is scanned confocally with a broadband light source, while the

Running time of the detected light is determined within the sample to the scattering and

To gain reflection properties of the sample in depth resolution. The transit time measurement is done interferometrically by superposition of the returning from the sample

Light with a reference beam, which has traveled a known path length.

A fast spectrometer records the interference phenomenon with wavelength resolution.

From this spectral interferogram, the depth information is obtained in the Fourier domain OCT by means of Fourier transformation. For example, the following specifications may apply to equipment 105 and 205, respectively: • Measuring frequency 1, 2 kHz

• Lateral resolution 15 μητι · Axial resolution 7 m

• Measuring depth 1, 6 mm

3 shows a sample carrier 350 with a plurality of chambers 352, in each of which a plurality of biochips 345 are contained, the biochips being imaged by means of a microscope according to an embodiment of the present invention. For this purpose, the sample carrier 350 is initially moved meander-shaped for determining depth positions of the individual biochips 345 by means of short-coherence interferometry, as indicated in the track 354. In this case, two lines 356 and 358 per biochip 345 are preferably traversed and the depth positions or geometric path length positions at a plurality of points 360 on each biochip are determined and stored, for example in the memory 151 illustrated in FIG. The chambers 352 may include a sample area 101 as illustrated in FIG. 1, each having a plurality of biochips 145, or sample areas as illustrated, for example, in FIGS. 4 and 5.

The slide 350 may be injection molded from PMMA. Depending on their size, they can have either 10 or 50 chambers, in each of which up to nine biochips 345 can be located. The biochips may be glued into the chambers 352 of the slide 350 and covered with a Schott D263T coverslip. The gap may be filled with glycerol 143 (see FIG. 1). The optical properties and geometric dimensions of the sample components are known: glued cover glass (biochip) (item 145 in FIG. 1)

Refractive index n (biochip) = 1, 5230

- thickness d = 145 ± 15 μιτι

- Lateral dimensions 1 * 1 mm ² to 2 2 mm ² · Loose covering glass (Number 137 in FIG. 1)

Refractive index n (glass) = 1, 5230 - thickness d = 170 ± 30 μιη

^• intermediate space filled with glycerin (numeral 143 in Fig. 1)

- refractive index n (glycerol) = 1, 4722

- thickness d = 20 ± 20 μιη

The lenses 113 used for the fluorescence images produce foci with a Rayleigh length in the micrometer range, so that due to the production-related fluctuations of the sample dimensions is not readily ensured that the sample surface is in focus of the lens.

Figs. Figures 4 and 5 illustrate a focal plane offset in refractive media. The biochip surfaces 403 and 503, which are to be focused on the fluorescence images, are not in air, but under the coverslip 437 or 537 and a layer of glycerol 443 or 543. Upon entry into these refractive media reduces the opening angle αθ (also referred to as α) of the beam path to α '. As a result, the focus shifts from its original position to larger working distances, depending on the layer thickness and refractive index of the media being passed through:

If the biochip 445 or 545 or its surface 403 or 503 positioned exactly at the working distance under the microscope objective, then still no sharp imaging is possible because the cover glass 437 and 547 and the glycerol shift the focus within the sample. It is therefore not enough to know exactly the position of the biochip or its surface 403 or 503 exactly. In order to position the biochip in the shifted focus, the exact interfaces of the overlying refractive media must also be known.

In Figs. 4 and 5, the distance between biochip surface 403 and 503 and microscope objective 413 and 513 is identical in both cases, nevertheless the biochip in FIG. 5 appears too high. The sample would have to be lowered to move the biochip 503 into the focus of the objective 513. By contrast, optical coherence tomography does not measure the actual geometric structures, but rather light propagation times within the sample, ie optical path lengths. By increasing the glycerol layer thickness, the light transit time to the biochip surface 403 or 503 is longer, so that in FIG. 5 it seems to be further from the lens 513. If only the position of the biochip surface 503 in the OCT image were to be adjusted, then in this example, one would have to lift the sample and thus remove the biochip even further from the focus than it already is. The different influences of the refractive media on the focal plane offset and the transit time measurement by means of OCT are taken into account according to embodiments of the invention.

For this purpose, first the three relevant interfaces air-glass 639, glass-glycerol 641 and glycerol biochip 603 are precisely measured, as illustrated in FIG. 6. Subsequently, the focus plane 703 is calculated for its depth position and orientation by a normal N (which is perpendicular to the plane 703), as illustrated in Fig. 7, in which the biochip must be positioned to obtain a sharp image.

Figs. FIGS. 8-13 illustrate processing steps of processing interference signals received by a detector of a short-coherence interferometry apparatus, such as detector 130 illustrated in FIG. 1 or detector 230 illustrated in FIG.

The offset error spectrum 861 illustrated in FIG. 8 (shown in a coordinate system with an abscissa 863 associated with the wavelength and an ordinate 865 associated with an intensity) is typically acquired at power-up of the device before the OCT light source, such as an SLD, and is later used for black balancing the recorded spectra.

The apodization spectrum 867 shown in FIG. 9 is recorded to determine the spectral intensity distribution of the OCT light source (for example, SLD). During recording, no light from the sample arm may fall onto the spectrometer. For example, a mechanical shutter may block the sample arm, or the microscope stage may be moved such that no object is within the focus range of the OCT imaging optics during acquisition of the apodization spectrum. Since the spectrum can change over time, it can be re-recorded at regular intervals. It is advisable to do this, for example, directly before measuring each slide. The two reference spectra 861 and 867, which are shown in FIGS. 8 and 9, respectively, can be averaged from 25 consecutive spectra. Subsequently, the slides can be measured by recording a spectrum 1069 at different lateral positions (x, y) of the samples, as illustrated in FIG. 10.

The further processing of the spectra may include the following steps:

1. Apodization and offset correction: Each recorded spectrum is included

the two previously recorded reference spectra charged. First, the uncorrected apodization spectrum is subtracted, and then it is divided by the offset-corrected apodization spectrum:

Such a corrected spectrum is illustrated in FIG. 11 as curve 1171.

2. Wavelength interpolation: The spectral interferograms are scanned approximately equidistantly in λ by the spectrometer. From the corrected spectra equidistant spectra must now be interpolated in the wavenumber k = 2ττ / λ. If one interpolates the spectrum at these non-integer indices, one obtains the linear spectrum sampled in k. For best picture quality, spline interpolation can be used.

3. Windowing: To suppress minor bands, the spectra with a

Hann window function, the result being shown in FIG. 12 as curve 1273. The values of the Hann window can be stored as a vector.

4. Fourier Transform: The corrected, windowed spectra becomes a

Fast Fourier transform applied. One gets (e.g., 1024) complex numbers.

5. Discard Negative Frequencies: The negative frequencies contain no additional information and can be discarded.

6. Forming Amounts: The amounts are formed from the Fourier coefficients at the (e.g., 512) positive frequencies. Logarithmically, they form a classic

OCT-A scan, as shown in FIG. 13 as depth profile 1375. The depth profile 1375 illustrated in FIG. 13 shows four local maxima, a peak 1353 with optical path length position 1354, which originates from the surface 103 of the biochip 145, another peak 1377 with optical path length position 1378 which extends from the top side 139 of the cover glass 137, an even further peak 1379 with optical path length position 1380, which originates from the lower side 141 of the cover glass 37, and an additional further peak 1381, which originates from a lower side 104 of the biochip 145. The calculation of the focal plane from the processed OCT data can be done as follows:

1. Identify Surfaces: The surfaces recognizable in the OCT data

are assigned to the respective interfaces. According to one embodiment of the present invention, the further peak 1377 of the air-cover glass interface is first identified, which is easy to carry out, since this further peak represents the highest and usually also the first peak of the A-scan or depth profile 1375. If the further peak 1377 of the cover glass top 139 is found, then the position of the peak 1353 and the still further peak 1379 can already be estimated approximately on the basis of the sample specifications, so that they are relatively easy to identify.

2. Frequency Estimation: After identifying the relevant peaks will be

determines the exact position. For this a parabola fit can be used by the maximum and the two neighboring values of a peak. Thus, the accuracy in the position determination of 7 pm can be increased to about 120 nm. For a more precise determination of the three peak positions (1377, 1379, 1353), a frequency estimation method can be carried out. The maximum of the amounts Azmax and the two direct neighboring values are also considered here:

-jwrai- - * nuct ij -z - 7 ~ ~ Ä '

According to other embodiments of the invention, the peak positions may be determined by other methods. The method defined above by the equation does not require much numerical effort and can improve the accuracy of the reflection determination from 120 nm to about 25 nm. Alternatively, the processed spectra could be padded with zeroes before the Fourier transform to obtain an oversampled A-scan. Here, the peak positions can be determined by parabolic adjustments with an accuracy of a few nanometers.

3. Biquadratic fit: To the locally detected interface positions

Be adapted to straight lines. If, in the data acquisition, the sample was not moved, but instead the deflection mirror 135 (if it functions as a scan mirror, for example) (see Figure 1), then due to the curvature of the field no parallels need to be adjusted but parabolas. Because of the increased robustness against outliers, an iterative biquadratic fit procedure can be used. The numerical overhead might be reduced by first removing any outliers from the data points and then applying a simple linear least squares fit.

4. Determine levels: Mathematically, the problem is

after determining two straight lines already overdetermined, because the two

Straight lines must have the same slope in order to be able to lie in one plane.

This could already be taken into account when fitting the straight line. For the determination of the plane 703 (see for example FIG. 7) three points are required, for example the first value P1 = (x1, y1, z1) of the first fit straight line, the last value P2 = (x2, y2, z2) of the first fit straight line and the center P3 = (x3, y3, z3) of the second fit line.

One of the three points, z. B. P1, can serve directly as a point A point E level. The normal vector N is calculated most simply as a cross product of the connecting vectors between A and P2 and A and P3, as is known to the person skilled in the art. Each point P of the plane 703 now fulfills a plane equation resulting from the normal vector N and the distance from the zero point, as is known to the person skilled in the art.

5. Calculate focal plane: At each point of the biochip can choose from the three interface positions

Z (wP), the optical path length position of the further peak, Z (nwP), the optical path length position of the still further peak,

Z (P), the optical path length position of the peak, the focus position of the biochip surface 103 are calculated according to:

Z (Obj) = Z (wP) / n (air) + [Z (nwP) -Z (wP)] / n (glass) + [Z (P) -Z (nwP)] / n (glycerol), where n (air) is the (group) refractive index of air at the central wavelength of the light used for short-coherence interferometry,

n (glass) is the (group) refractive index of the coverslip at the central wavelength of the light used for short-coherence interferometry, n (glycerol) is the (group) refractive index of the glycerol solution at the central wavelength of the light used for short-coherence interferometry. b) Calculation of the image plane offset:

The image plane offset δζ, which causes a plane-parallel plate of thickness d with refractive index n, is dependent on the opening angle α of the beam path. As a result, in the case of uncorrected objectives, the focus for the central rays does not coincide with that of the marginal rays if a plane-parallel plate (for example a cover glass) is located in the ray path:

An objective 113 can be corrected, for example, for a cover glass thickness of 170 μm. If the focus of the marginal rays is corrected to the central rays, then the image plane offset can be determined according to

be calculated. c) Calculation of the necessary height correction: The geometric position of the focal plane results from the working distance of the objective plus the image plane offset caused by the cover glass and the glycerol layer, when the biochip is in the Focus is located. The geometric position of the biochip surface was calculated in point a). If the sample is raised by the difference between the two values, then the biochip surface is in the shifted focus of the objective. The group and phase refractive indices for the cover glass 137 (Schott D263T Eco) and glycerol solution 143 are shown in FIGS. 14 and 15, respectively, as a function of the wavelength. The values for the phase refractive indices 1483 and 1485 are respectively smaller than the values of the group refractive indices 487 and 1489.

Claims

claims

Method for imaging an object (103) located within a three-dimensional sample area (101), comprising the following steps: a) determining, at least at a lateral point (1 19), a depth profile (1375) of the sample area (101) by means of optical short-coherence Interferometry, b) detecting a peak (1353) in the depth profile resulting from a reflection at an object point (120), c) determining an optical path length position (1354) of the peak (1353) representing the optical path length of the object point (120) from a reference point (136), d) calculating, based on the optical path length position (1354) and at least one known optical property (s) of the sample area (101), a geometric path length position of the object point, which represents a geometric path length (gWI) of the object point (120) from the reference point (136), e) focusing a microscope (111) on the object point (120) based on the geometri shift path length position (gWI) for imaging the object (103).

Method according to claim 1, wherein the object (103) comprises a biological sample, preferably cell structure and / or tissue and / or the tissue section with fluorescently labeled antibody, on a, in particular planar, biochip, wherein the biological sample preferably at least one of the group comprising a thin section of a biological tissue, preferably paraffinized and / or formalin-fixed or frozen section, a cell smear, bacterial smear, viruses, protozoa and parasites, applied in solution, dried or coupled antigen drops, others to a Surface-coupled antigens and any nucleotide sequences.

The method of claim 1 or 2, wherein detecting the peak comprises registering a pattern (1377, 1379, 1353, 1381) in the depth profile (1375) and associating, preferably based on relative optical path length positions, pattern details to previously known structures (139 , 141, 103, 104) in the sample area (01).

Method according to one of the preceding claims, wherein an image plane offset (δζ) from a depth dimension (TA) of the sample area (101) to the object point (120) and a refractive index (n) is calculated, and wherein the focusing based on a difference (Δζ) between the geometric path length position (gWI) and image plane offset (δζ) takes place.

Method according to the preceding claim, wherein the sample area (101) up to the object point (120) comprises partial areas (137, 143) and wherein the image plane offset (δζ) according to

where the d _{i are} geometric depth extents of the respective subregions / to the object point (120) and the n _{ the phase refractive indices, preferably at a central wavelength of the light used for imaging by means of the microscope, of the respective subregions are up to the object point. Method according to one of the preceding claims, wherein a short-coherence interferometry apparatus (105) used for optical short-coherence interferometry has an axial resolution of 1 μm to 15 μm, preferably 3 μm to 10 μm, more preferably 8 μm to 10 μm, wherein the geometric path length position (gWI) of the object point (120) with higher accuracy, preferably 0.03 μιη to 1 μηη, more preferably 0.1 μπι to 0.5 μιη, as the axial resolution of the short-coherence interferometer apparatus (105) is determined.

A method according to any one of the preceding claims, wherein the imaging comprises exciting, by fluorescence of a fluorophore included in the object (103) and detecting, preferably by a camera (1 17), the emitted fluorescent light from the object.

Method according to one of the preceding claims, wherein the sample area (101) comprises in a region irradiated to the object (103) a Eindeckgias (137) and a liquid (143), preferably a pH-buffered glycerol solution.

A method according to the preceding claim, wherein the depth profile (1375) adjacent to the peak (1353) in an area irradiated to the object (103) has another peak (1377) originating from an upper surface (139) of the Eindeckgias (137), and yet another peak (1379) originating from a bottom surface (141) of the capping glass (137), the further peak (1377) and the still further peak (1379) for detecting the peak (1353) and / or the peak geometric path length position (gWI) of the object (03) are used.

A method according to the preceding claim, wherein calculating the geometric path length position (gWI) of the object point (120) further comprises an optical path length position (1378) of the further peak (1377), an optical path length position (13). 1380) of the still further peak (1379), a refractive index (n (glass)) of the cover glass (137), a difference between the optical path length position of the still further peak (1379) and the optical path length position of the peak (1353) and a refractive index (n (air)) of the liquid (143).

A method according to claim 9 or 10, wherein determining the optical path length position of the peak (1353) and / or the further peak (1377) and / or the still further peak (1379) by matching a suitable fit function to a by zeropadding of the interferogram generated, over-sampled depth profile (1375) and / or taking into account a maximum signal and bilateral neighboring signals of the respective peak in the depth profile, preferably by means of a frequency-estimation method.

12. The method according to one of the preceding claims 9 to 1 1, wherein the geometric-path length position (gWI) Z (Obj) of the object point (120) is calculated according to:

Z (wP) is the optical path length position (1378) of the further peak (1377), Z (nwP) is the optical path length position (1380) of the still further peak (1379), Z (P) is the optical path length position Path length position (1354) of the peak (1353),

n (air) is the group refractive index of air at the central wavelength of the light used for short-coherence interferometry,

n (glass) is the group refractive index of the coverslip (137) at the central wavelength of the light used for short-coherence interferometry, n (glycerol) is the group refractive index of the glycerol solution (143) at the central wavelength of the light used for short-coherence interferometry.

13. The method according to any one of the preceding claims, further comprising the step: f) lateral movement of the object (103) along a first line (356) and preferably further along a second line (358), during which respective geometric-path length positions of a plurality, in particular between 10 and 30 object points (360) respectively in the first and second line, from object points (360).

14. The method according to the preceding claim, wherein based on positions of the object points of the first line a first

A straight line equation is determined, wherein a second straight line equation is determined based on positions of the object points of the second line, and wherein from the first and the second straight line equation a normal (N) of the object (103,703) is calculated and the normal and a geometric-path length position of the object at a lateral point for focusing the microscope at another lateral point.

Method according to one of the preceding claims, wherein the reference point (136) has a known distance (A) along a microscope beam path from a position, preferably an object-side next optical surface, of an objective (113) of the microscope (111), wherein preferably the objective ( 13) is a 20x to 40x magnifying lens and the microscope (111) preferably has a depth of focus of between 0.5 pm and 1.8 pm.

Method according to one of the preceding claims, wherein the short-coherence interferometry apparatus (105, 205) comprises an illumination source (125, 225) for illuminating the object (103) with illuminating light (230), preferably in the near-infrared region and a detector (130, 230) for detecting interference light obtained by interference of illumination light reflected by the object with reference light, and wherein the short-coherence interferometry apparatus (205) preferably has a grating (232) for spectrally decomposing the interference light and is preferably designed to record depth profiles at a measuring rate of between 1 kHz and 2 kHz.

A measuring method for measuring a plurality of biochips (345) arranged laterally next to one another on a support (350), comprising the steps: a) stepwise lateral, preferably meandering (354), movement of the support (350) relative to a microscope and a Short-coherence interferometry apparatus; b) in one step or continuously, performing a method according to any one of the preceding claims; and

c) taking an image of a respective focused biochip (345) by means of the microscope (1 1).

The measuring method according to claim 17, wherein during the lateral process of the carrier (350) relative to the short-coherence interferometry apparatus (105), at least one geometric-pathlength position is respectively determined for a biochip (345) and stored associated with lateral position information, and wherein the method further comprises: determining the geometric path length positions (gWI) of the plurality of biochips (345), lateral method of the carrier (350) relative to the microscope (11), Retrieving the stored geometric pathlength position associated with the current lateral position of the microscope relative to the carrier, and focusing on the biochip (345) for imaging by the microscope (11) based on the retrieved geometric path length; Position (gWI).

An optical device (100) for imaging an object (103) located within a three-dimensional sample area (101), the optical device comprising: a short-coherence interferometry apparatus (105) which is designed to have a depth profile (at least at one lateral point (119)) 1375) of the sample area (101) by means of optical short-coherence interferometry; a processor (107) configured to detect a peak (1353) in the depth profile (1375) resulting from a reflection at an object point (120), an optical path length position (1354) of the peak (1353 ), which represents the optical path length of the object point from a reference point (136), based on the optical path length position (1354) and at least one known optical property (n (glass), n (glycerin)) of the sample area ( 101), a geometric path length position (gWI) of the object point (120) representing a geometric path length of the object point (120) from the reference point (136); and an actuator (109) configured to focus a microscope (11) on the object point (120) based on the geometric path length position (gWI) to image the object (103).

The optical device of claim 19, further comprising a controller (155) coupled to the processor (107) and the actuator (109) for driving the processor Actuator in response to an output signal of the processor for focusing to drive preferably a lens (113) along a depth direction (123, z) to proceed.

The optical device according to claim 19 or 20, further comprising a laterally movable sample holder (157), which is preferably controlled by the controller (155) or the processor (107) for lateral processing.

The optical device according to one of claims 19 to 21, further comprising a memory (151) for storing geometric-path length positions of a plurality of object points (360).

The optical device according to one of claims 19 to 22, further comprising an objective for imaging the object (103), wherein the objective comprises, in particular, a cover glass correction device which is adapted to image aberrations, in particular spherical aberration, in the imaging of Object points due to the knowledge of position, thickness and refractive index of the sub-areas to correct, the image plane offset in particular by

is calculated, wherein the ¹ geometric depth expansions of the respective

Are partial regions i to the object point (120) and the ^η 'are the phase refractive indices, preferably at a central wavelength of the light used for imaging by means of the microscope, of the respective partial regions up to the object point and where α is the angle of the optical path to the optical axis of the microscope Is objective.