DE10362244B4 - Locating focal position during sample imaging involves acquiring reflected light intensity values with position sensitive receiver inclined to field aperture, determining center of contrast position - Google Patents

Abstract

The method involves forming an image of a field aperture (11) on the sample (4) and at least partly superimposing the image of an optical grid, acquiring intensity values for the light reflected from the sample with a position sensitive receiver (6) inclined relative to the field aperture and associating with positions on the receiver, deriving position-related contrast values and determining the position of the center of contrast on the receiver as the equivalent of the focal position. An independent claim is also included for the following: (a) an arrangement for determining focal position during imaging of a sample in accordance with the inventive method.

Description

The invention relates to a method for determining the focus position and the tilting of the focal plane relative to the receiving surface of a position-sensitive receiving device in the imaging of a sample.

In order to obtain precise images of a sample or a sample section by means of imaging optics, it is necessary to place the sample exactly in the focus position of the imaging optics. If the image is blurred, it is important to know by what amount and in which direction a change in the position of the sample relative to the imaging optics is to be caused and, if necessary, to derive corresponding control commands that can be used for refocusing.

In this context, triangulation methods, imaging methods with contrast evaluation and position determination by means of an inclined confocal slit are known.

In the triangulation method, a collimated laser beam is reflected in the pupil plane of an imaging objective and closed from the course of this laser beam relative to the imaging beam path to the Z position of the laser light reflected by the sample.

A major disadvantage of this procedure is that image errors occur due to the imaging of the laser light in different depths of the sample, and as a result the detected signal varies greatly over a given depth of field.

The accuracy in the determination of the focus position is disadvantageously dependent on whether the measurement result from the center or at the periphery of the capture range of a detector is determined. To compensate for this effect, the focus position must be determined in several iterative steps, with the result that this procedure requires a relatively large amount of time.

In imaging methods with contrast evaluation, the sample is illuminated with a grid-like intensity distribution by placing a grid in the field stop plane of the illumination beam path. The thus illuminated sample is imaged onto a receiving device. In this way, a series of images with different distances between the imaging optics and the sample is taken and from this series, the image with the highest contrast is determined. The distance between the imaging optics and the sample assigned to this image is considered to be the optimum focus distance.

The disadvantage is that many different Z-positions must be approached with high accuracy to record the image series, which relatively much time is required for this procedure.

When determining the position by means of a slanted confocal slit diaphragm, a slit diaphragm is placed in the field diaphragm plane of the illumination beam path and imaged onto the specimen. The light reflected from the sample is directed to a CCD line inclined relative to the slit, and the position on the CCD line where the reflected light has a maximum is determined. Since, in this procedure, the focus position can be calculated with a single image on the line, the result of the focus determination is available in a relatively short time.

However, there are disadvantages in that contamination on the sample or disruptive structures on the sample surface cause intensity fluctuations which can lead to erroneous measurement results. The main problem with this procedure is the very high adjustment effort in the mapping of the gap on the CCD line, because the column (or line) must be very narrow in order to achieve high accuracy can. In addition, a very well corrected imaging optics is required.

In particular, in the chip production increasingly finer structures and thinner layers are sought, which means that the requirements for the inspection procedures, with which the manufacturing accuracy is checked, are getting higher. Accordingly, an increasingly faster and more accurate focusing is required as possible without interrupting the production process.

A significant disadvantage of the known arrangements and procedures is that a tilt of the focal plane with respect to the receiving surface of a detector in the receiving device is not detected and corrected.

In the DE 101 27 284 A1 an autofocusing device is described for an optical device, in particular a microscope, which is extended with a diaphragm device whose diaphragm aperture is in the same direction as the direction of movement of a measurement location to be autofocused.

In the DE 37 28 257 A1 a method for photoelectric distance adjustment is described in particular for surgical microscopes, in which upon displacement of the object plane, the line of a matrix-shaped receiver arrangement is determined, which has the maximum average contrast. From the change in position of this line and from the direction and amount of emigration, a control signal for a motor control of the focusing is determined.

In the DE 101 12 639 A1 a microscope with an autofocusing device will be described. In this case, two or more diaphragms axially spaced apart in the direction of the beam path are present in one imaging device, which diaphragms are arranged in such a way that a plane lying between them is focused on a receiving device at the same time as the observation object in the image field. An evaluation device generates a control signal for actuating an actuating device as a function of the intensities.

In the US Pat. No. 6,677,565 B1 A method for high-speed autofocusing and tilting of an inspection surface in a microscope is described. An array of geometric figures is projected onto the surface to be examined. The superposition of the surface with the array is imaged onto a CCD camera and stored for subsequent analysis. In these analyzes, the angle and the distance are calculated, which must be adjusted to bring the surface to be measured in the focal plane of the optical system.

In the US 5 539 494 A There is described a device for determining focus conditions that can be used in photographic cameras, etc., and that is described in U.S. Pat US 5 539 494 A described method characterized in that, in particular, little memory is needed in the evaluation.

In the US 4,748,321 and in the DE 31 22 027 A1 Further autofocusing devices for lenses will be described. In the JP H07-87 378 A A device for determining the focus is described, which is based on a contrast measurement. Finally, in the DE 101 53 113 A1 a method and a device for determining the distance, in which a sensor arrangement is arranged obliquely in the region of the focus plane of an image recording device.

On this basis, the invention has the object to further develop the known methods so that both a largely accurate determination of the focus position and a tilt of the focal plane can be registered.

This object is achieved by a method according to claim 1 or according to claim 2. Advantageous embodiments of the method are listed in subclaims.

In contrast to the relevant prior art methods, the focus position is not obtained as the equivalent of the intensity maximum on the receiving device, but the overlay of the aperture image on the sample with the image of an optical grating makes it possible to determine position-related contrast values and those of the determination based on the current focus position.

This has the significant advantage over the previously known method that the adjustment effort is significantly reduced. In addition, the results in the determination of the focus position are no longer so distorted by background light, impurities on the sample or interfering sample structures.

In a preferred embodiment variant of the method, it is provided that position-related contrast values I (y _i ) according to the function ∀i I (y _i ): = | I (x _i ) - I (x _{i + n} ) | be determined. Herein, I (x _i ) means intensity values assigned to a position x _i on the receiving means, while I (x _{i + n} ) are intensity values associated with an adjacent position x _{i + n} . In this case, n is preferably selected in the range from 1 to 20.

Subsequent to the determination of the position-related contrast values, a location is determined according to the function as an equivalent for the current focus position P _f

Here, all the contrast values I (y _i ) are included, which are greater than a predetermined minimum value I _min .

This is already achieved in many applications, a high accuracy compared to the prior art with reduced adjustment effort.

für alle Kontrastwerte I(y_i), die größer sind als ein vorbestimmter Mindestwert I_min, und hieran anschließend in einem zweiten Schritt als das Äquivalent für die aktuelle Fokusposition P_f ein Ort ermittelt nach der Funktion

für alle Kontrastwerte I(y_i), die größer sind als ein Mindestwert I´_min und die nicht weiter von dem Ort P_f´ entfernt sind als um einen vorgegebenen Abstand a. For even higher demands on the accuracy, first in a first step, a location P _f 'is determined according to the function

for all contrast values I (y _i ) that are greater than a predetermined minimum value I _min , and then, in a second step, as the equivalent for the current focus position P _f, a location determined by the function

for all contrast values I (y _i) which are greater than a minimum value I _'min and not further' remote from the location P _f as a predetermined distance a.

The respective specified for the first step and the second step minima I _min and I 'may be equal _min or different sizes.

As optical grating absorption grating in the form of stripe-shaped masks and as a receiving device using a CCD matrix. The positions x _i are characterized in this case by the continuous numbering of the sensor elements on a CCD line of the CCD matrix, hereinafter referred to as pixels. For example, if the CCD line is 2400 pixels, its positions are defined as x ₁ through x ₂₄₀₀ . The distance a between two pixels on the CCD line is then indicated with a number of pixels, with a being preferably set in the range of 10 to 1000.

In a further embodiment, which can be used in particular in automatic production control, is measured from the distance b measured on the receiving device between a location on the receiving device, which corresponds to the ideal focus position P _f "on the receiving device, and the location of the current focus position P _f , generates a control signal for automatic focusing.

Here, the control signal is preferably used to change the distance Az between the sample and the imaging optics, to the places shown on the CCD line P _f with the predetermined reference to a device calibration location P _f coincide''.

Such adjusting devices are known from the prior art and therefore need not be explained in detail here. In particular, stepper motors which are coupled to a sample table have proved to be suitable as drives for such adjusting devices.

With regard to the determination of a tilt of the focal plane with respect to the receiving surface of a detector in the receiving device is preferably provided that a rectangular aperture and as a receiving device using a CCD matrix and the optical grating consists of two in the lattice plane mutually inclined lattice masks, each of which has a plurality of mutually parallel strip-shaped masks. It is provided that an image is generated on the sample of each of the strip-shaped masks and the position of the contrast centroid is determined by the method of the method described above, by means of the CCD matrix for each mapped mask.

In this case, the positions of the contrast centroid on each mapped mask are the equivalent of a current focus position. According to the invention, the course and the inclination of the two connecting straight lines are determined by the contrast centers and from this the tilt angle of the focal plane with respect to the receiving surface of the CCD matrix, as will be explained in more detail below.

Here too, it is conceivable that an actuating signal is obtained from the tilt angle of the focal plane with respect to the plane of the receiving surface of the CCD matrix, and this is compensated via an adjusting device which influences the inclination of the specimen relative to the optical axis of the imaging beam path Tilt angle is used.

In each of the aforementioned embodiments of the invention, the gap width of the optical gratings is predetermined as a function of the reflectivity of a selected sample section and / or of the intensity of scattered or reflected and therefore interfering light. To increase the accuracy in the determination of the contrast center of gravity, the gap width should be set the smaller, the lower the reflectivity of the sample section or the more intense interfering scattered or reflected light.

Furthermore, it may be advantageous to firstly average the intensity values of adjacent pixels over a period of the imaged grid for further processing of the intensity values for position-related contrast values, then to determine the deviations of the intensity signal I _ist from a given intensity signal I _soll and finally to Depending on the resulting difference to make a correction of the contrast values.

As a result, advantageously only one intensity value, namely the averaged intensity signal I _ist , must first be determined. Based on the deviation of this intensity signal I _is from an expected intensity signal I _{is to} be then the contrast values corrected so that interference that are caused by dirt particles in the optical beam path or by a non-uniformly reflecting sample are compensated.

The averaging of the intensity values of adjacent pixels can be carried out according to the function

for all x _i with n = 2 ... 100, where n preferably corresponds to the number of pixels per grating period.

• – in den Beleuchtungsstrahlengang eine durch eine Gittermaske überlagerte Spaltöffnung gestellt ist,
• – ein aus dem Abbildungsstrahlengang abgezweigter Detektionsstrahlengang auf eine CCD-Zeile gerichtet ist,
• – die CCD-Zeile mit der optischen Achse des Detektionsstrahlengangs einen Winkel α ≠ 90° einschließt,
• – die CCD-Zeile mit einer Auswerteeinrichtung zur Ermittlung von positionsbezogenen Intensitätswerten I(x_i) und von positionsbezogenen Kontrastwerten I(y_i) in Verbindung steht, und
• – die Auswerteeinrichtung zur Ermittlung der Position des Kontrastschwerpunktes auf der CCD-Zeile als ein Äquivalent für die aktuelle Fokusposition ausgebildet ist.

To carry out the method steps described so far, for example, an optical arrangement is provided, which may be formed in particular as a microscope assembly, comprising: a light source for generating an illumination beam path, an imaging optics through which the illumination beam path is directed to the sample and a camera arranged in the imaging beam path, wherein

• In the illumination beam path a gap opening superposed by a grating mask is provided,
• A detection beam path branched off from the imaging beam path is directed onto a CCD line,
• The CCD line encloses an angle α ≠ 90 ° with the optical axis of the detection beam path,
• The CCD line is connected to an evaluation device for determining position-related intensity values I (x _i ) and position-related contrast values I (y _i ), and
• - The evaluation is designed to determine the position of the contrast center on the CCD line as an equivalent to the current focus position.

The gap opening is advantageously positioned together with an aperture opening which defines the circumference and the size of an image section on the sample in the field stop plane of the arrangement, wherein the aperture is aligned centrally with the optical axis of the illumination beam path and the gap opening is located at the periphery of the illumination beam path ,

In one embodiment variant of this arrangement, which is particularly suitable for the observation and examination of samples with strongly scattering surfaces, the aperture should be aligned perpendicular to the optical axis of the illumination beam path, while the gap opening with the optical axis encloses an angle corresponding to the angle the CCD line is inclined to the optical axis of the detection beam path, wherein the optical path length between the sample and the gap opening is as large as the optical path length between the sample and the CCD line.

für alle Kontrastwerte I(y_i), die größer sind als ein vorbestimmter Mindestwert I_min. In this case, the evaluation device may comprise, for example, a difference former, the position-related contrast values I (y _i ) according to the function ∀iI (y _i ): = | I (x _i ) - I (x _{i + n} ) | determined, each with the intensity values I (x _i ) and I (x _{i + n} ), which are the positions x _i and x _{i + n} associated, and wherein the evaluation device is further equipped with an arithmetic circuit, the contrast center of gravity as the equivalent of the current focus position is determined by the function

for all contrast values I (y _i ) greater than a predetermined minimum value I _min .

In this case, the value n determines the distance between adjacent positions on the CCD line to each other. In this respect, n is variable and is given according to the application with n = 1 ... 20.

In particular, for the application in connection with devices for automatic biochip production, in which the control of the manufacturing accuracy runs independently, an arithmetic circuit is provided in the evaluation, which is designed to determine the distance b between the location, which the current focus position P _f on the CCD line, and a location corresponding to the ideal focus position P _f "on the CCD line. This gives a measure of the deviation from the ideal focus position.

Accordingly, it is furthermore advantageous if the evaluation device is configured such that it ascertains an actuating signal from the deviation from the ideal focus position and is connected to a setting device for correcting the focus position, for example a change in the distance Δz between the sample and the imaging optics causes. Thus, the distance between the current focus position and the ideal focus position can be influenced, the adjustment is made so long and so often until the distance b goes to 0 and thus the ideal focus position is set.

To achieve optimal results in determining the current focus position as well as in determining the shelf to the ideal focus position can, it is advantageous if the frequency of each used for imaging on the sample grid of the optical resolution limit of the imaging optics is adjusted. Preferably, in each case five pixels of the CCD line should correspond to a grating period.

When imaging planar sample surfaces, the method or the arrangement described so far can also be used for leveling purposes by determining the inclination of the sample surface relative to the optical axis of the objective beam path and corrected to a predetermined value, preferably to an angle of 90 ° to the optical axis becomes.

For this purpose, the arrangement is to be equipped with a sample table which is displaceable in the coordinate directions X and Y perpendicular to the optical axis. This makes it possible to focus on three different points of the sample surface that are not on a straight line. For each of these three points, the current focus position is determined on the CCD line and determines the storage of the ideal focus position and / or the focus distance.

The inclination of the sample surface relative to the optical axis of the objective beam path is calculated from the geometrical combination of the shelves or the focal distances and / or the inclination of the sample surface is changed by tilting the sample by means of an adjusting device so that the shelves between current and ideal focus position or Focusing distances are the same for all three points and thus the sample surface is aligned perpendicular to the optical axis of the lens path.

The adjusting device, which can be embodied, for example, as a three-point linear adjustment or two-axis rotary adjustment, is controlled as a function of the current inclination and a positioning command to be generated therefrom until the undesired inclination has been corrected.

The invention will be explained below with reference to embodiments which disclose further features essential to the invention. In the accompanying drawings show:

1 an optical arrangement in which the slit opening of a diaphragm overlaid with the structure of a bar grating is imaged onto the surface of a sample,

2 a detail 1 concerning the diaphragm arrangement in the field diaphragm plane,

3 the symbolic representation of a CCD line used as receiving device,

4 an example of an intensity distribution over the CCD line,

5 depending on the intensity profile 4 resulting course of contrast values,

6 the equipment of the optical arrangement with an evaluation device and a delivery device for automatic focusing,

7 the typical intensity curve when imaging a glass / water interface onto the CCD line,

8th depending on the intensity profile 7 resulting course of contrast values,

9 the design of the optical arrangement with inclined receiving device and inclined gap opening,

10 the optical arrangement according to the invention, in which, in addition to the precise determination of the focus position, a statement about the tilting of the sample plane relative to the receiving surface in the receiving device is made,

11 an enlarged view of a diaphragm assembly 10 .

12 the indication of the contrast centers in the pixel rows of a CCD array of the arrangement 10 .

13 an example of the connecting line through the contrast centers

In 1 an imaging optical system is shown, for example, a microscope assembly, which is to be used for recording biochips. In biochip detection with the aid of microscopes, in particular in fluorescence microscopy, the finding of the exact focus position is particularly problematic.

The not belonging to the invention according to the microscope arrangement 1 essentially comprises a source of illumination 1 , which has an illumination beam path with the optical axis 2 generated, and a lens 3 through which the illumination source 1 on the surface of a sample 4 , in this case the surface of a biochip, is imaged. The light reflected or also scattered by the sample surface passes as an imaging beam path that has an optical axis 5 has, to a receiving device 6 , For example, a CCD camera.

In this case, the sample surface by means of imaging optics, which from the lens 3 and a tube lens 7 exists on the receiving surface of the receiving device 6 and can be observed or evaluated.

Illumination beam path and imaging beam path are at the splitter surface 8th a beam splitter 9 distracted or split.

In the aperture plane 10 the illumination beam path is a diaphragm arrangement 11 put in the 2 is shown enlarged.

Out 2 it can be seen that the diaphragm arrangement 11 an example square aperture 12 that defines the image field. Outside the aperture 12 but inside of the circular boundary 13 marked illumination beam path, is the gap opening 14 a slit diaphragm positioned with a grating 15 is superimposed.

When lighting the sample 4 becomes the one with the structure of the grating 15 superimposed slit opening 14 on the surface of the sample 4 displayed. This gets from the sample 4 Reflected or scattered light in the imaging beam path back to the beam splitter 9 and will be there at the splitter surface 8th again deflected in the direction of the incoming illumination beam path, thereby passing a tube lens 16 , is at the splitter surface 17 another beam splitter 18 decoupled from the illumination beam path and onto a CCD line 19 directed.

lens 3 and tube lens 16 act as imaging optics and form the superimposed with the grid structure gap opening 14 on the CCD line 19 from. The CCD line 19 is relative to the aperture arrangement 11 inclined. While the aperture arrangement 11 including the gap opening 14 perpendicular to the optical axis 2 the illumination beam path is aligned, closes the CCD line 19 with the optical axis 33 of the detection beam path at an angle not equal to 90 °, preferably from 45 °.

In 3 is the CCD line 19 symbolically represented. It can be seen that the CCD line 19 has a plurality of receiving elements arranged in line, hereinafter referred to as pixels 20 designated. Every pixel 20 is on the CCD line 19 assigned a fixed position x _i . Indicates the CCD line 19 for example, a number of 2400 pixels 20 on, it is assumed that at one end of the CCD line 19 the position x _i = 1, at the opposite end of the CCD line 19 on the other hand, the position x _i = 2400 of one pixel 20 is busy.

At the output of each pixel 20 An intensity signal I (x _i ) is available, ie each pixel 20 provides information about the intensity of the imaging radiation at its assigned position x _i . Because the gap opening 14 including the grating 15 on the slanted CCD line 19 is shown, there is exactly one position on the CCD line where the grating 15 sharply depicted. This position, which corresponds to the current focus position P _f , can be based on one or more associated pixels 20 determine.

First, for each position, x _{i of} a pixel 20 an intensity value I (x _i ) of the recorded radiation is determined. This results from the CCD line 19 an intensity course as in 4 shown. Out 4 It can be seen that the intensity of the imaging radiation in the range of pixels x ₇₀₀ to x _{1200 is} much stronger than in the other areas.

However, the position with the maximum intensity is not searched for and defined as the current focus position, but the intensity values I (x _i ) are first processed further to position-related contrast values. The position-related contrast values I (y _i ) are determined, for example, according to the function ∀iI (y _i ): = | I (x _i ) - I (x _{i + n} ) |.

That is, the difference of the intensity value I (x _i ) of a position x _i and an intensity value I (x _i + n) of a position x _{i + n} adjacent thereto is formed in each case. N is variable and should be chosen between 1 and 20.

Each difference thus obtained is assigned as a contrast value I (y _i ) to a position x _i . This results in a course of the contrast values I (y _i ) as in 5 shown.

Out 5 It can be seen that the contrast values I (y _i ) in the range y ₆₀₀ to y _{1100 are} substantially greater than in the remaining areas of the CCD line 19 ,

für alle Kontrastwerte I(y_i), die größer sind als ein vorbestimmter Mindestwert I_min. Im vorliegenden Fall wurde I_min beispielsweise mit einem Betrag vorgegeben wie in 5 eingezeichnet. In a next step, the contrast center is now determined from the contrast values I (y _i ) according to the function

for all contrast values I (y _i ) greater than a predetermined minimum value I _min . In the present case, for example, I _{min was} given an amount as in 5 located.

This contrast center of gravity can be a location on the CCD line 19 assign, which in the selected example is approximately at the position y ₈₅₀ . Thus, the current focus position P _{f is} determined.

In an advantageous embodiment, which is likewise not included in the invention, in a next step the current focus position P _f on the CCD line 19 compared to an ideal focus position P _f '' determined from a calibration of the microscope assembly, for example by means of a smooth sample surface, and assumed to be at position y ₁₂₀₀ .

In the example shown 5 a distance of 350 pixels results between the positions P _f and P _f ". The CCD line 19 has a certain distance d between every two pixels 20 on; From this, it can be deduced that in this case the current focus position P _f is approximately 350 times the distance d from the ideal focus position P _f '' and a sharp image of the sample surface on the receiving device 6 to obtain, the focus position must be corrected.

This correction is usually done by changing the distance Δz between the sample 4 and the lens 3 ,

In practice, for reasons of accuracy, the distances d between the individual pixels on the CCD line used are determined by measurements, and the arrangement is calibrated as a function of the measurement result and used in the calibrated state for the sample evaluation.

For the purpose of processing the intensity values I (x _i ) in the manner described above and also for automatic focusing, the microscope arrangement not according to the invention is as in FIG 6 still shown with an evaluation 21 and a delivery device 22 equipped, with the CCD line 19 with the evaluation device 21 and the evaluation device 21 with the delivery device 22 connected via signal paths.

The evaluation device 21 comprises a difference former for determining the position-related contrast values I (y _i ) and an arithmetic circuit for determining the contrast center of gravity as an equivalent for the current focus position P _f . Furthermore, the evaluation device has 21 via means for specifying a value n = 1 ... 20 and a minimum value I _min .

In addition, in the evaluation device 21 , According to the embodiment 6 Accordingly, an arithmetic circuit provided for determining the distance b between the current focus position P _f and the location on the CCD line 19 which corresponds to the ideal focus position P _f ''. At the same time, this arithmetic circuit is also able to generate a positioning command via the setting device 22 a change in the distance Δz between the sample 4 and the lens 3 by an amount equivalent to the distance b in a predetermined direction R causes.

Should the current focus position P _f on the CCD line 19 in the direction of R, a control command must be generated, which ensures that the distance between lens 3 and sample 4 is increased. Conversely, setting commands must reduce this distance if the current focus position P _f is to be shifted in the opposite direction to the direction R. If the current focus position P _f with the ideal focus position P _f '' overlaps after execution of the setting commands, the automatic focusing is completed.

In a special method variant, which is likewise not covered by the invention and which meets a higher requirement for the accuracy of automatic focusing, it is provided that the evaluation device 21 first taking into account all the contrast values I (y _i ) that are greater than a predetermined minimum value I _min , a location P _f 'on the CCD line 19 determined, and only in a subsequent step, the current focus position P _{f is} determined taking into account all contrast values I (y _i ) that are greater than the predetermined minimum value I _min , and also no more than by a predetermined distance a from the location P _f 'are removed.

With this two-step method, the influence of disturbances in the focusing is avoided by determining a corrected contrast centroid and based on the determination of the current focus position P _f .

If the focus position is to be determined with respect to an interface on or in the sample belonging to a multi-interface layer system, measures may be taken to reduce the contrast for the in-plane 4 increase maxima and minima. This is achieved, for example, by adjusting the grid gap width.

Thus, the width of the maximum can be changed by the width of the grid gap. If a very narrow grid gap is selected, the intensity distribution also becomes narrower. This results in "confocal" conditions and the light reflected from other interfaces does not get onto the CCD line 19 , In this way, the contrast of the CCD line 19 imaged lattice stripes are increased so much that much less reflective glass / water interfaces in the vicinity of glass / air interfaces can be precisely focused and thus accurately measured with respect to their distance from each other.

In this regard shows 7 the intensity curve when imaging a glass / water interface on the CCD line 19 , In this case, the disturbances to be detected in the range x ₅₀₀ to x ₁₁₀₀ are caused by adjacent scattering boundary surfaces which have a distance of approximately 150 μm from each other.

Further processing of in 7 shown intensity values I (x _i ) in the manner described leads to position-related contrast values I (y _i ), as in 8th shown.

In the previously explained, non-inventive embodiments of the arrangement, both the aperture 12 as well as the gap opening 14 as components of the aperture arrangement 11 perpendicular to the optical axis 2 aligned the illumination beam path. This can be used in the evaluation of samples 4 , which have a slightly scattering surface, achieve very good results.

In 9 In contrast, the arrangement is shown in a likewise not inventive embodiment, in particular for the investigation of samples 4 is suitable with relatively strong scattering surfaces.

As in the previously explained exemplary embodiments, the embodiment also shows 9 in the field diaphragm level 10 positioned aperture arrangement 11 a gap opening 14 and a shutter 12 on, here too the gap opening 14 the determination of the focus position is used and the aperture 12 the size and size of an image on the sample 4 Are defined.

Here is the aperture 12 approximately centric to the optical axis 2 aligned the illumination beam path, and the gap opening 14 is located at the periphery of the illumination beam path.

As a special feature here closes the gap opening 14 with the optical axis 2 an angle that has the same amount as the CCD line 19 against the optical axis 33 of the detection beam is inclined while the aperture 12 still perpendicular to the optical axis 2 the illumination beam path is aligned.

In addition, the optical path between the sample 4 and the gap opening 14 as long as the optical path between the sample 4 and the CCD line 19 ,

Such a diaphragm arrangement 11 For example, can be technically realized by a panel 11.1 with the gap opening 14 and a panel part 11.2 with the aperture 12 be made separately and then joined together.

The requirement of the same optical path lengths between the gap opening 14 and the sample 4 on the one hand and the sample 4 and the CCD line 19 on the other hand, in the illustrated case, it is satisfied if the point of intersection in which the optical axis is located 2 , optical axis 33 and splitter surface 17 of the beam splitter 18 meet, from the intersection of the optical axis 2 with the gap opening 14 as far away as the intersection of the optical axis 33 with the CCD line 19 ,

In the 10 illustrated inventive arrangement is suitable for applications in which in addition to the precise determination of the focus position and a statement for tilting the sample plane relative to the receiving surface of the receiving device 6 to meet.

For the sake of clarity, in 10 as far as possible again the same reference numerals for the same assemblies used as in 1 ,

The difference in arrangement 10 to the arrangement 1 is that a second illumination source 23 is provided, from which a second illumination beam path 24 goes out and to the test 4 is directed. The illumination beam path 24 becomes at the splitter layer 25 an additional beam splitter 26 towards the sample 4 deflected and in the beam splitter 9 with the optical axis 2 of the illumination beam path merged by the illumination source 1 emanates.

In the field diaphragm level 27 there is an aperture arrangement 28 , in the 11 is shown enlarged. The aperture arrangement 28 is from two partial apertures 28.1 and 28.2 composed, both in the illumination beam path 24 juxtaposed are the image field that is used to image on the sample 4 is provided, complete completely.

The lighting sources 1 and 23 are operated alternatively, ie the illumination source 1 is used to sample surface for observation and evaluation on the CCD camera of the receiving device 6 image while the illumination source 23 merely to determine the current focus position and to determine the tilt angle of the sample 4 against the receiving surface of the receiving device 6 is being used.

Each of the two partial apertures 28.1 and 28.2 consists of a number of parallel stomata 29 and 30 each of which as well as the gap opening 14 in the arrangement 1 is superimposed with a grid structure. Here are the stomata 29 the partial aperture 28.1 perpendicular to the stomata 30 the partial aperture 28.2 aligned.

The grid structures are each perpendicular to the stomata 29 respectively. 30 aligned, ie the grating vectors are parallel to the respective gap opening 29 respectively. 30 ,

An inclined CCD line 19 , as in the arrangement 1 , is not required here. The aperture arrangement 28 is applied to a CCD matrix in the receiving device 6 pictured after that by the sample 4 upcoming picture light the lens 3 , the beam splitter 9 , the tube lens 7 and the beam splitter 26 happened.

In 12 Symbolically, the CCD matrix is represented by the image of the stomata 29 respectively. 30 claimed pixel lines 31 respectively. 32 ,

It will be any of the stomata 29 including the grid structure on a pixel line 31 imaged on the CCD matrix and causes intensity signals I (x _i ), which can be tapped at the signal output of each pixel. This is true in the same way for the stomata 30 , including the grid structure on each pixel line 32 be imaged on the CCD matrix.

As above for a single CCD line 19 based 1 In this variant of the method, the determined intensity values I (x _i ) are also linked to position-related contrast values I (y _i ), in exactly the same way as already described on each of the pixel rows 31 respectively. 32 can determine a contrast center of gravity, each indicating the current focus position P _f .

The contrast centers can be used for each partial screen 28.1 respectively. 28.2 connect through straight lines, as in 13 shown separately. The two straight lines are defined by the corresponding straight line equations y ₁ = m ₁ * x ₁ + n ₁ or y ₂ = m ₂ * x ₂ + n ₂ .

The quantities n ₁ and n ₂ in the calibrated state give a measure of the current focus position. If n ₁ or n ₂ correspond to a predetermined distance value, the imaging optics are focused.

Furthermore, on the basis of the mathematical relationships, the quantity m ₁ , which is the slope of the straight line y ₁ = m ₁ × x ₁ + n ₁ and thus the deviation from the parallel alignment to the direction of the pixel rows 31 respectively. 32 describes the first tilt angle and from m ₂ the second tilt angle of the focal plane with respect to the plane of the sample 4 certainly.

It is assumed that in the calibrated state of the arrangement, the quantities m ₁ and m _{2 are} proportional to a tilt angle of the focal plane with respect to the plane of the sample 4 ,

Will the level of the sample 4 deliberately tilted until the two variables m ₁ and m ₂ correspond to a desired value, preferably the value "zero", the tilt is corrected.

LIST OF REFERENCE NUMBERS

1

lighting source

2

optical axis of the illumination beam path

3

lens

4

sample

5

optical axis of the imaging beam path

6

receiver

7

tube lens

8th

splitter surface

9

beamsplitter

10

Field stop plane

11

diaphragm arrangement

12

aperture

13

limit

14

gap opening

15

gratings

16

tube lens

17

splitter surface

18

beamsplitter

19

CCD line

20

pixel

21

evaluation

22

advancing

23

lighting source

24

Illumination beam path

25

splitter surface

26

beamsplitter

27

stop plane

28

diaphragm arrangement

28.1, 28.2

partial stops

29, 30

stomata

31, 32

pixel lines

33

optical axis of a detection beam path

Claims (4)

Method for determining the focus position and the tilting of the focal plane relative to the receiving surface of a receiving device when imaging a sample ( 4 ), wherein as a receiving device, a CCD matrix is used as a two-dimensional, spatially resolving surface detector, which is arranged perpendicular to an optical axis, in which - two in a field stop plane next to each other in the same plane arranged partial apertures ( 28.1 . 28.2 ), each of which has a plurality of parallel stomata ( 29 . 30 ), to the sample ( 4 ), wherein the stomata ( 29 ) of a partial panel ( 28.1 ) perpendicular to the stomata ( 30 ) of the other partial panel ( 28.2 ) and - each of the stomata ( 29 . 30 ) of the two partial diaphragms ( 28.1 and 28.2 ) is superimposed with the structure of a line grating whose grating vectors are parallel to the respective stomata ( 29 . 30 ), - for each of the stomata ( 29 . 30 ) with the superimposed grating by means of pixel lines ( 31 . 32 ) of the CCD matrix along the through the formation of the respective gap opening ( 29 . 30 ) directions separately intensity values I (x _i ) for that of the sample ( 4 ) Reflected light are obtained - from the intensity values I (xi) position-related contrast values I (y i), wherein the position y _{i i} corresponds to the position x, according to the function ∀iI (y _i ): = | I (x _i ) - I (x _{i + n} ) | each of the difference of an intensity value I (x _i ), which is assigned to a position i on the receiving device, and an intensity value I (x _{i + n} ), which is assigned to an adjacent position i + n, with n = 1 ... 20, are subsequently determined from these position-related contrast values I (y _i ) for each image of a slit opening (FIG. 29 . 30 ) on each of the pixel lines ( 31 . 32 ) of the CCD matrix separately whose respective contrast center of gravity is determined, which indicates the current focus position P _f , according to the function

for all contrast values I (y _i ) which are greater than a predetermined minimum value I _min , and furthermore - the contrast centers of the images of the respective stomata ( 29 . 30 ) on the respective pixel lines ( 31 . 32 ) for each of the partial screens shown ( 28.1 . 28.2 ) are separately connected to each subpanel ( 28.1 . 28.2 ) to obtain a connecting straight line separately so that two straight-line equations y ₁ = m ₁ .x ₁ + n ₁ and y ₂ = m ₂ .x ₂ + n ₂ are obtained, and from whose respective slopes m ₁ and m ₂ the tilt angle Focus plane is determined, and the sizes n ₁ and n _{2 are} a measure of the current focus position. Method for determining the focus position and the tilting of the focal plane relative to the receiving surface of a receiving device when imaging a sample ( 4 ), wherein a CCD matrix as a two-dimensional, spatially resolving surface detector is used as the receiving device, which is arranged perpendicular to an optical axis, in which - two in a field stop plane next to each other in the same plane arranged partial apertures ( 28.1 . 28.2 ), each of which has a plurality of parallel stomata ( 29 . 30 ), to the sample ( 4 ), wherein the stomata ( 29 ) of a partial panel ( 28.1 ) perpendicular to the stomata ( 30 ) of the other partial panel ( 28.2 ) and - each of the stomata ( 29 . 30 ) of the two partial diaphragms ( 28.1 and 28.2 ) is superimposed with the structure of a line grating whose grating vectors are parallel to the respective stomata ( 29 . 30 ), - for each of the stomata ( 29 . 30 ) with the superimposed grating by means of pixel lines ( 31 . 32 ) of the CCD matrix along the through the formation of the respective gap opening ( 29 . 30 ) directions separately intensity values I (x _i ) for that of the sample ( 4 ) Reflected light are obtained - from the intensity values I (x _i) position-related contrast values I (y _i), wherein the position corresponds to the position y _i x _i, according to the function ∀iI (y _i ): = | I (x _i ) - I (x _{i + n} ) | each of the difference of an intensity value I (x _i ), which is assigned to a position i on the receiving device, and an intensity value I (x _{i + n} ), which is assigned to an adjacent position i + n, with n = 1 ... 20, are subsequently determined from these position-related contrast values I (y _i ) for each image of a slit opening (FIG. 29 . 30 ) on each of the pixel lines ( 31 . 32 ) of the CCD matrix separately their respective contrast center of gravity is determined, which indicates the current focus position P _f , wherein in a first step, a location P _f 'is determined according to the function

for all contrast values I (y _i ) which are greater than a predetermined minimum value I _min and, in a second step, as the equivalent of the current focus position P _f, a location is determined according to the function

for all contrast values I (y _i ) that are greater than the minimum value I _min and that are no wider than one predetermined distance a from the location P _f 'are removed, and further - the contrast centers of the images of the respective stomata ( 29 . 30 ) on the respective pixel lines ( 31 . 32 ) for each of the partial screens shown ( 28.1 . 28.2 ) are separately connected to each subpanel ( 28.1 . 28.2 ) to obtain a connecting straight line separately so that two straight-line equations y ₁ = m ₁ .x ₁ + n ₁ and y ₂ = m ₂ .x ₂ + n ₂ are obtained, and from whose respective slopes m ₁ and m ₂ the tilt angle Focus plane is determined, and the sizes n ₁ and n _{2 are} a measure of the current focus position. A method according to claim 1 or claim 2, characterized in that derived from the tilt angle of the focal plane with respect to the plane of the receiving surface of the CCD matrix, a control signal for compensating the tilt angle and the plane of the sample ( 4 ) is tilted until the quantities m ₁ and m ₂ correspond to a desired value. A method according to claim 1 or claim 2, characterized in that the gap width of the optical grating is determined as a function of the reflectivity of a selected sample section and / or the intensity of scattered or reflected light, wherein in order to increase the accuracy in the determination of the contrast centroid and thus the current focal position, the smaller the reflectivity of the sample section or the more intensively scattered or reflected light is the gap width is set.

Images