DE102006055987A1 - Process for automatic focussing of the objective of an optical image recording unit onto a receiving object useful in fluorescence microscopy gives more accurate results and avoids false results due to the presence of impurities - Google Patents

Abstract

Process for automatic focussing of the objective of an optical image recording unit onto a receiving object on a sample carrier, where the receiving object consists of a number of partial receiving objects spatially separated from each other involving:(a) determination of the number of partial receiving objects in a first receiving position, (b) determination of the number of partial receiving objects in n further receiving positions, (c) determination of the receiving positions containing the maximum number of receiving objects, and (d) adjustment of the objective into the receiving position.

Description

The The invention relates to a method for focusing optical image capture devices.

at devices and facilities with automatic image acquisition and image interpretation spatially extended Objects, autofocusing is a necessary requirement. In Microscopy, for example, has the task of reducing the number or the Density (number per unit area) marked spores on a specimen under a fluorescence microscope.

They are devices, preferably microscopes, for the electronic image acquisition of large object fields or many samples ( DD 229218 A5 ), in which the recording positions for recording partial images via motor drives in the recording plane (x-, y-) and the focus adjustment (z-) are set automatically (eg. DE 103 59 780 A1 . DE 695 08 248 T2 ). Furthermore, facilities are known whose objects to be examined are recorded by an electronic camera and evaluated in an image processing unit, which differ in shape, size and / or color. The objects in the samples are often not ideally arranged in a plane of sharpness (bulging of the specimens, unevenness of the slides, finite height of the preparations, and the like).

It is problematic with prior art means (e.g., maximizing contrast, as in FIG DE 103 30 714 A1 described, o. Ä.) To set the focal plane for reliable detection of the objects to be determined during automatic scanning of the samples constantly. In the presence of impurities or spurious objects that have spatial dimensions or arrangement in other focal planes, known methods are very inaccurate and distort the result.

task The invention is to the disadvantages of the prior art remove. It is intended in particular a method for improved Autofocusing of optical imaging devices specified be that particular the inclusion of spatial allowed extended objects.

These The object is solved by the features of claim 1. Advantageous embodiments of Inventions result from the features of claims 2 to 14th

• (a) das Bestimmen der Anzahl von Teilaufnahmeobjekten in dem Aufnahmeobjekt in einer ersten Aufnahmeposition mit einem ersten Abstand z₁ des Probenträgers von dem Objektiv;
• (b) das Bestimmen der Anzahl von Teilaufnahmeobjekten in dem Aufnahmeobjekt in n weiteren Aufnahmepositionen, bei denen sich der Abstand z_n des Probenträgers von dem Objektiv von dem Abstand z₁ unterscheidet, wobei n größer oder gleich 2 ist;
• (c) das Bestimmen der Aufnahmepositionen z_v, die die maximale Anzahl von Teilaufnahmeobjekten aufweist, aus den weiteren Aufnahmepositionen und der ersten Aufnahmeposition; und
• (d) das Bringen des Objektives in die Aufnahmeposition z_v.

According to the invention, a method is provided for automatically focusing the objective of an optical image capture device onto a photographic object on a sample carrier, wherein the photographic object consists of a plurality of partial imaging objects that are spatially separated from each other

• (a) determining the number of partial imaging objects in the photographic object in a first recording position with a first distance z _{1 of} the sample carrier from the objective;
• (b) determining the number of partial imaging objects in the photographic subject in n further imaging positions in which the distance z _{n of} the sample carrier from the objective differs from the distance z ₁ , where n is greater than or equal to 2;
• (c) determining the pickup positions z _v having the maximum number of split subjects from the further pickup positions and the first pickup position; and
• (d) bringing the lens into the pickup position z _v .

The steps (b) and (c) are expediently carried out in parallel with each other. This means that after each determination of the number. Partial recording objects in the recording object in the recording position z _i (i = 2 to n), the number of partial recording objects in this recording position z _{i is} compared with the number of partial recording objects in the previous recording position z _i-1 . If the number of partial recording objects in the recording position z _{i is} greater than or equal to the number of partial recording objects in the recording position z _i-1 , the next recording position n _{i + 1 is} approached. If the number of part receiving objects in the receiving position z _i is less than the number of part receiving objects in the receiving position z _i-1, a peak was found at part receiving objects in the receiving position z _{i -1.} The number of part-shot objects in the recording position z _i-1 is then regarded as the maximum number of part-recording objects, and the recording position z _i-1 is used as the recording positions z _v . Step (b) is thus carried out with successive recording positions along the z-axis until a maximum of partial recording objects has been determined.

Preferably is n larger or equal to 3, stronger preferably larger or 20, even stronger preferably larger or equal to 50 and strongest preferably larger or equal to 80.

Thus, according to the invention, the distance between the objective and the object to be photographed is changed stepwise along the z-axis, while the position of the object to be photographed in the x, y plane remains unchanged. It is expedient to start with a minimum distance between the objective and the photographing object in the z-direction (first recording position) and to increase this distance incrementally by displacing the objective in the z-direction. In this case, in the first recording position (minimum focal plane) and after each step (further focal planes) by means of an image capture unit pictures of the subject (additional shooting positions). In each picture, the number of partial recording objects is determined by means of a predetermined evaluation algorithm. By means of the evaluation algorithm, the recorded images can be evaluated on the basis of predefined features, for example with regard to shape, size and color, of the objects imaged in the images, in order to distinguish partial recording objects from interfering objects. The evaluation of the images can take place immediately after each image has been taken or only after all images have been taken.

The distance between the lens and the subject in which the image with the highest number of partial subjects has been obtained is selected as the photographing position z _v . This recording position z _v is the optimal focal plane. The optimal focus plane is then adjusted and an image of the subject is taken.

Out the evaluation of the images with regard to the given properties For example, a diagram can be obtained that the number the part-shot objects recognized in an image opposite the Distance between lens and subject in z direction shows. From the diagram it can be seen at which distance between lens and Recording object, the maximum number of partial recording objects detected has been.

• (e) das Bestimmen der Anzahl von Teilaufnahmeobjekten in dem Aufnahmeobjekt mit einer ersten Belichtungszeit b₁;
• (f) das Bestimmen der Anzahl von Teilaufnahmeobjekten in dem Aufnahmeobjekt mit m weiteren Belichtungszeiten b_m, die sich von der ersten Belichtungszeit b₁ unterscheiden;
• (g) das Bestimmen der Belichtungszeit b_v, die die maximale Anzahl von Teilaufnahmeobjekten aufweist, aus den weiteren Belichtungszeiten b_m und der ersten Belichtungszeit; und
• (h) das Einstellen der Belichtungszeit auf die Belichtungszeit b_v.

After the determination of the optimum focal plane z _v , an improvement of the image acquisition can be achieved by carrying out the following steps following step (d):

• (e) determining the number of partial imaging objects in the photographic subject with a first exposure time b ₁ ;
• (f) determining the number of partial imaging objects in the photographic subject with m further exposure times b _m , which differ from the first exposure time b ₁ ;
• (g) determining the exposure time b _v , which has the maximum number of partial imaging objects, from the further exposure times b _m and the first exposure time; and
• (h) setting the exposure time to the exposure time b _v .

The steps (e) and (f) are expediently carried out in parallel with each other. That is, after each determination of the number of sliced objects in the slidable object at an exposure time b _i (i = 2 to m), the number of sliced subjects at this shutter speed b _{i is} compared with the number of sliced subjects at the previous exposure time m _i-1 , If the number of part-shot objects at the exposure time b _{i is} greater than or equal to the number of part-shot objects at the exposure time b _i-1 , the next exposure time b _{i + 1 is} used. If the number of partial imaging objects at the exposure time b _{i is} smaller than the number of partial imaging objects at the exposure time b _i-1 , then a maximum of partial imaging objects was found at the exposure time b _i-1 . The number of part-shot objects at the exposure time b _i-1 is then considered to be the maximum number of part-shot objects, and the exposure time b _i-1 is used as the exposure time b _v . Step (e) is thus carried out with successive exposure times until a maximum number of partial acquisition objects has been determined.

Preferably is m greater or equal to 2, stronger preferably larger or equal to 20, even more preferred bigger or equal to 50 and most preferred bigger or equal to 80.

Therefore is next to a gradual change the focal plane in addition a gradual change the exposure time provided. It is expediently first Set a first exposure time and then gradually changed. At each exposure time, an image of the subject with the image capture unit recorded.

In Each image is assigned the number of partial objects by means of a determined evaluation algorithm. By means of the evaluation algorithm can the captured images based on predetermined characteristics, such as in terms of shape, size and color, the objects formed in the images are scored to be partial objects of disturbing objects to distinguish. The evaluation of the images can take place immediately after taking every picture or only after taking all pictures.

The exposure time at which the image with the highest number of sub-shot objects was obtained is selected as b _v for the shot object. This exposure time b _v is the optimum exposure time. The optimum exposure time is then set and an image of the subject is taken.

Out the evaluation of the images with regard to the given properties For example, a diagram can be obtained that the number the part-shot objects recognized in an image opposite the Distance between lens and subject in z direction shows. The diagram shows the distance between the lenses and the recording object, the maximum number of partial recording objects detected has been.

The inventive method is thus characterized in that an optimal focal plane z _v (distance between the lens and the object to be photographed in the z-direction) and in one embodiment of the invention additionally an optimum exposure time b _v at the inventively determined optimum focal plane z _{v be} determined. This is achieved by gradually changing the focal plane and, as soon as the optimal focus plane has been determined, by stepwise changing the exposure time. In the initial position z ₁ or b ₁ and after each step, an image of the object is taken. The obtained images are evaluated in terms of the number of partial recording objects according to predetermined characteristics.

If it is a recording object having an extension in the x, y plane, which can not be completely detected by means of an image recording, sub-images of the recording object can be made, which are then combined to form a complete image of the recording object. For this purpose, the image positions (x, y) for which partial images are to be recorded are approached successively according to a predetermined algorithm with the previously determined focus setting (z _v ), and an image acquisition is performed for each image position (x, y). Since the focus plane and luminance can change within the large subject, a re-adjustment of the optimum focal plane z _v and, if desired, the optimum exposure time b _{v is} performed after a predetermined number of frames. After passing through all the image positions (x, y), a complete electronic image of the object to be examined in the memory of the image acquisition unit (computer) is obtained, in which all parts to be searched are optimally recognizable.

The Partial recording objects of the recording object are spores, for example a mushroom.

The The invention will be described below with reference to the drawings explained in more detail. Show

1 a schematic representation of a device for automatic image acquisition;

2 an image of the image capture with objects to be counted and contaminants;

3 a diagram showing the number of detected spores as a function of focus adjustment; and

4 a diagram showing the number of detected spores as a function of the exposure time.

To 1 the optical image capture device consists of a microscope device 1 with a lens of a lighting unit 2 and an image capture unit 3 , For example, a recording camera, the microscope images to an evaluation 4 sends. From the evaluation unit 4 becomes an x, y, z positioning table depending on the evaluation result 5 controlled by a motor, on which the sample to be examined 6 as a recording object.

For carrying out the method according to the invention, the sample 6 by means of the positioning table 5 moved to a first picking position z ₁ . For this purpose, the positioning moves the sample step by step in the z-direction, wherein after completion of a step in each case a new recording position z _{i is} reached. In each of the n recording positions, an image is taken with the image acquisition unit 3 performed and determined with predetermined image recognition algorithms, the objects to be counted.

2 shows by way of example an image recording by means of the image acquisition unit 3 was obtained from a sample, in addition to round spores 7 also impurities 8th contains. The area where the diameters of the spores lie is known. The microscope should be focused so that the maximum number of spores are detected with a given diameter. Disturbing objects and / or impurities 8th should be disregarded when focusing. In each image acquisition, which is obtained in each recording position, thus the number of round spurs is determined.

At a great distance from the focal plane, no spores are found, as these are shown in the picture very dark, out of focus and large. The closer the positioning device 5 the sample 6 moved into the optimal focal plane z _v , the more pronounced the spores 6 imaged and meet the search criteria. The number of found spores is a maximum in the optimal focal plane ( 3 ). Will the sample 6 moved by the adjusting device further from the optimal focal plane, the number of spores found again decreases. The z position with the maximum of spores is saved. After a passage of the adjustment, the optimal focal plane (z _v ) is set.

After setting the optimum focus plane z _v according to the procedure described above, a further optimization of the image acquisition by varying the exposure time b of the image acquisition unit 3 be performed. Analogous to 3 one obtains a curve with a maximum with variation of the exposure time. In the case of underexposure (small exposure time only a few (very bright) spores are detected.) With larger exposure time, more image spurs are detected by the image processing unit radiate and neighboring elements (in accumulations) are no longer recognizable as individual elements or by over-radiations individual elements no longer fulfill the search criteria for spores. The optimal exposure is achieved by detection of the maximum ( 4 ) and set by controlling the evaluation device and / or the lighting unit.

According to a predetermined algorithm, for example by matrix scanning, the image positions (x, y) are approached with the previously determined focus setting (z _v ) and the image recordings are performed. As the focal plane and luminance within the. large sample, after a selectable number of image acquisitions, a readjustment is made according to the above-described procedure. After passing through all the image positions (x, y), a complete electronic image of the sample to be examined is obtained in the memory of the image acquisition unit (computer), in which all objects to be searched for are optimally recognizable.

1

microscope unit with lens

2

lighting unit

3

Image capture unit

4

evaluation

5

positioning

6

recording object

7

Part engulfing objects (Spores)

8th

obstructions

Claims (14)

A method of automatically focusing the objective of an optical image capture device on a photographic subject on a slide, the photographic object being comprised of a plurality of partial photographic objects spatially separated, comprising (a) determining the number of partial photographic objects in the photographic object in a first photographic position a first distance z _{1 of} the sample carrier from the objective; (b) determining the number of partial imaging objects in the photographic subject in n further imaging positions in which the distance z _{n of} the sample carrier from the objective differs from the distance z ₁ , where n is greater than or equal to 2; (c) determining the pickup positions z _v having the maximum number of split subjects from the further pickup positions and the first pickup position; and (d) bringing the lens into the pickup position z _v . A method according to claim 1, characterized in that steps (b) and (c) are carried out in parallel and the number of part-shot objects in the photographing object in a photographing position z _i-1 with the number of part-scan objects in the photographic subject in a photographing position z _i and that when the number of part-shot objects in the shot object in the shot position z _{i is} smaller than the number of split shot objects in the shot object in the shot position z _i-1 , the shot position z _{i-1 is used} as shot positions z _v . A method according to claim 1 or claim 2, characterized characterized in that Determination of the number of part-acquisition objects in step (a) and in step (b) in each case an image of the object with an image acquisition unit is recorded. Method according to claim 3, characterized that this Image evaluated by means of a predetermined recognition algorithm becomes. A method according to claim 3 or claim 4, characterized characterized in that Picture according to given characteristics, which have the partial objects should be evaluated. Method according to one of the preceding claims, characterized in that, following step (d), the following steps are provided: (e) determining the number of partial recording objects in the photographic object with a first exposure time b ₁ ; (f) determining the number of partial imaging objects in the photographic subject with m further exposure times b _m , which differ from the first exposure time b ₁ ; (g) determining the exposure time b _v , which has the maximum number of partial imaging objects, from the further exposure times b _m and the first exposure time; and (h) adjusting the exposure time to the exposure time b _v . A method according to claim 6, characterized in that steps (f) and (g) are carried out in parallel and the number of part-taking objects in the taking object at an exposure time b _{i-1 is} compared with the number of part-taking objects in the taking object at an exposure time b _i and that when the number of pieces of partitions in the subject at the exposure time b _{i is} less than the number of slices in the subject at the exposure time b _i-1 , the exposure time b _{i-1 is used} as the exposure time b _v . Method according to Claim 6 or Claim 7, characterized in that, in order to determine the number of part-recording objects in step (e) and in Step (f) is taken in each case an image of the object with an image capture unit. Method according to claim 8, characterized in that that this Image evaluated by means of a predetermined recognition algorithm becomes. A method according to claim 8 or claim 9, characterized characterized in that Picture according to given characteristics, which have the partial objects should be evaluated. Method according to one of the preceding claims, characterized characterized in that Partial objects of the object to be photographed are spores. Method according to one of the preceding claims, characterized in that, when partial images of the photographic object are recorded at different measuring points x _i , y _i , the method steps for each of these measuring points are repeated. Method according to one of the preceding claims 1 to 11, characterized in that, when Subpictures of the subject at various measuring points xi, yi are recorded, the process steps after recording of sub-images at a predetermined number of measuring points be repeated. A method according to claim 12 or claim 13, characterized characterized in that Partial images in the image acquisition unit of the optical image acquisition device be assembled into a full screen.