Method in the Preparation of Samples for Microscopic Examination, and Apparatus for Checking the Coverslipping Quality of Samples

Abstract

The invention relates to a method in the preparation of samples for microscopic examination onto which a coverslip is applied. The method is notable for the fact that the coverslipping quality is checked automatically and at least partly optically. The invention further relates to an apparatus for carrying out the method, and to an apparatus for checking the coverslipping quality of samples onto which a coverslip is applied.

Claims

1. An apparatus, comprising: an optical imaging apparatus configured to automatically check coverslipping quality of samples onto which a coverslip is applied, wherein the optical imaging apparatus is configured to automatically check the coverslipping quality at least partly with dark field illumination; and an automatic evaluation apparatus configured to evaluate at least one image generated by the imaging apparatus to determine whether one or more of: the coverslip is damaged after a coverslipping operation; the coverslip exhibits a fracture after a coverslipping operation; air inclusions are present in a coverslipping medium; air inclusions are present between the coverslip and the sample; and/or surface irregularities of the coverslip are present; and wherein the evaluation apparatus is further configured to automatically check the image generated by the imaging apparatus with regard to a staining quality of a hematoxylin-eosin stain and/or a color intensity.

2. The apparatus according to claim 1, wherein the image generated by the imaging apparatus includes a microscopic image of the coverslip and/or of the coverslipping medium.

3. The apparatus according to claim 1, wherein the image generated by the imaging apparatus is a grayscale image.

4. The apparatus according to claim 1, further comprising an illumination apparatus configured to: a. illuminate the coverslip and/or the coverslipping medium upon generation of the image and/or b. generate a bright field illumination and/or c. generate a dark field illumination.

5. The apparatus according to claim 1, wherein the imaging apparatus generates both an image with bright field illumination and an image with dark field illumination, and the evaluation apparatus compares the image with bright field illumination and the image with dark field illumination.

6. The apparatus according to claim 1, wherein the imaging apparatus generates both an image with bright field illumination and an image with dark field illumination, and the evaluation apparatus checks as to whether a correspondence is present between the image with bright field illumination and the image with dark field illumination for a detected detail in one of the image with bright field illumination or the image with dark field illumination.

7. The apparatus according to claim 1, wherein the optical imaging apparatus acquires images at an image acquisition angle and/or an illumination angle, the image acquisition angle and/or the illumination angle being adjustable and/or steplessly adjustable.

8. The apparatus according to claim 1, wherein the evaluation apparatus is configured to calculate, from a measured average brightness and/or brightness distribution of a reference image of a coverslipped specimen slide without a sample, an exposure time for generating the image.

9. The apparatus according to claim 1, wherein the evaluation apparatus is configured to calculate, from a measured average brightness and/or brightness distribution of a reference image of a coverslipped specimen slide without a sample, an exposure time for generating the image in such a way that a predetermined or predeterminable brightness for the background is achieved upon generation of the image.

10. The apparatus according to claim 1, wherein the evaluation apparatus carries out, on a basis of a measured brightness and/or brightness distribution of a reference image of a coverslipped specimen slide without a sample, a shading correction of the image.

11. The apparatus according to claim 1, wherein the apparatus is configured to generate an overview image.

12. The apparatus according to claim 1, wherein the apparatus is configured to read out a barcode attached to a specimen slide or to a coverslip.

13. The apparatus according to claim 1, further comprising multiple testing stations, wherein the imaging apparatus is configured to check the coverslipping quality in chronologically successive fashion under different conditions and/or at the different testing stations.

14. The apparatus according to claim 1, further comprising an automatic handling apparatus that conveys a coverslipped sample to be investigated into a scanning region of the imaging apparatus.

15. The apparatus according to claim 1, further comprising an automatic handling apparatus that sequentially removes coverslipped samples to be investigated from a receiving apparatus and transfers them into a scanning region of the imaging apparatus.

16. The apparatus according to claim 1, further comprising an automatic handling apparatus that sequentially removes coverslipped samples to be investigated from a receiving apparatus, transfers the coverslipped samples into a scanning region of the imaging apparatus, and deposits each of the coverslipped samples back into the receiving apparatus after each of the coverslipped samples has been checked.

17. The apparatus according to claim 1, wherein the imaging apparatus is part of a coverslipping machine and/or is integrated into a coverslipping machine.

18. The apparatus according to claim 1, wherein the apparatus is automatically configured to receive at least one sample equipped with a coverslip from a coverslipping machine, or to remove at least one sample equipped with a coverslip from a coverslipping machine.

19. The apparatus according to claim 1, wherein the apparatus is further configured to, based on results of the evaluation apparatus, re-coverslip a defective coverslipped slide or exclude the defective coverslipped slide from at least one microscopic examination and to provide a non-defective coverslipped slide to a subsequent laboratory process.

20. A sample processing system, comprising: an apparatus for checking coverslipping quality comprising an optical imaging apparatus configured to automatically check coverslipping quality of a sample onto which a coverslip is applied, wherein the optical imaging apparatus is configured to automatically check the coverslipping quality at least partly with dark field illumination, and wherein the apparatus for checking coverslipping quality is configured to automatically check an image generated by the optical imaging apparatus with regard to a staining quality of a hematoxylin-eosin stain and/or a color intensity; and a higher-level data processing system: configured to receive data regarding coverslipping quality, and/or a barcode attached to a specimen slide or to a coverslip, from the apparatus for checking coverslipping quality, and/or; with which the apparatus for checking coverslipping quality exchanges data, and/or; with which the apparatus for checking coverslipping quality exchanges data regarding coverslipping quality or whereabouts of the sample, or regarding patient data to be associated with the sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In the drawings:

[0050] FIG. 1 shows an exemplifying embodiment of an apparatus for checking the coverslipping quality of samples onto which a coverslip is applied,

[0051] FIG. 2 schematically shows the generation of an image of a coverslipped sample under bright field illumination when an air inclusion is present,

[0052] FIG. 3 schematically shows the generation of an image of a coverslipped sample under bright field illumination when a fracture of the coverslip is present, and

[0053] FIG. 4 schematically shows the generation of an image with dark field illumination.

DETAILED DESCRIPTION OF THE INVENTION

[0054] FIG. 1 is a schematic general depiction of an exemplifying embodiment of an apparatus for checking the coverslipping quality of samples 1 onto which a coverslip 16 (not depicted in FIG. 1) is applied. The apparatus comprises a handling apparatus (not depicted in further detail) that removes a respective coverslipped sample 1 to be investigated from a stacking shelf 2 and places it in a scanning region on a sample stage 3. After each sample 1 has been investigated, the handling apparatus places the investigated sample 1 back into stacking shelf 2. These operations are indicated by the horizontal double arrow.

[0055] Stacking shelf 2 can be displaced in a vertical direction, so that a further sample 1 to be investigated can be brought to the level of sample stage 3 and then removed. This operation is indicated by the vertical double arrow.

[0056] The apparatus for checking coverslipping quality comprises a first light source 4 for bright field illumination. First light source 4 emits a first illumination light bundle 5 that is directed by a beam splitter 6 and by a deflection mirror 7 to the coverslipped sample 1 to be investigated. Detected light 8 (drawn with dashed lines) reflected from the sample travels along the same light path in the opposite direction, i.e. via deflection mirror 7 back to beam splitter 6, passes through the latter, and lastly arrives at detector 9, which is embodied as a CCD camera. Detector 9 is located on an electronic circuit board 10 that carries an evaluation electronics system (not further depicted).

[0057] Also provided are optical components, in particular a telecentric optic, for guiding and shaping the illumination light and detected light, said components being indicated merely schematically and in exemplifying fashion as lens 11.

[0058] The apparatus furthermore comprises a further light source 12, drawn in merely schematically, for dark field illumination. Further light source 12 can be, in particular, a semi-annular light source.

[0059] Provision can be made, for example, that the apparatus for checking coverslipping quality outputs an optical or acoustic signal as soon as a coverslipping defect is discovered.

[0060] FIG. 2 schematically shows the generation of an image, in particular a grayscale image, under bright field illumination when an air inclusion 13 is present under coverslip 14 of a sample 1 located on a specimen slide 15. A light source 4 that emits light toward the coverslipped sample 1 serves for bright field illumination. The light reflected from the coverslipped sample 1 is detected with a detector 9 that is embodied as a camera.

[0061] Additional reflections occur in particular at the transition between air inclusion 13 and the material of sample 1 or its coverslipping medium (adhesive), and cause this region to appear particularly bright in the grayscale image as compared with the background.

[0062] FIG. 3 schematically shows the generation of an image, in particular a grayscale image, of a coverslipped sample 1 under bright field illumination when a fracture 17, filled with coverslipping medium (adhesive), of coverslip 14 is present. Especially because of the irregular surface structure of fracture 17 and of adhesive 18 that has emerged in this region, the light emitted from light source 4 is reflected in a different direction from the light that strikes the coverslipped sample 1 in a region in which a coverslipping defect is not present.

[0063] The distances, angles, and optical properties are selected in such a way that none of the light reflected in the region of fracture 17, or only a small portion of that light, arrives at detector 9. The consequence of this is that the region of fractured 17 appears particularly dark in the grayscale image relative to the background.

[0064] Be it noted that in the context of bright field illumination, the angle of incidence of the illumination light does not necessarily need to be equal to 0?. In particular, angles of incidence in the range from 0? to 30? relative to the incidence perpendicular, i.e. relative to the vertical in terms of the (unconfirmed) coverslip surface, are also used in practice in bright field illumination. The position of the detector in this context should preferably be selected so that no reflected light, but instead exclusively refracted and/or scattered light, arrives at the detector.

[0065] FIG. 4 schematically shows the generation of an image, in particular a grayscale image, with dark field illumination. The illumination light of light source 16 is incident relative to sample 1 at a flat angle, i.e. at a large angle of incidence relative to the surface normal of the coverslip. Detection occurs with a vertical view onto sample 1.

[0066] The image that is generated shows bright, sharply delimited objects with high contrast, enabling identification of coverslipping defects, in particular automatic identification of coverslipping defects. In particular, an image of this kind generated under dark field illumination makes it possible to check the results of the evaluation of a previously performed investigation with bright field illumination. In particular, it is possible to check whether, for a detected detail of the one image, a correspondence is present in the other image.

[0067] The invention is not to be limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the invention.

PARTS LIST

[0068] 1 Coverslipped sample [0069] 2 Stacking shelf [0070] 3 Sample stage [0071] 4 First light source for bright field illumination [0072] 5 Illumination light bundle [0073] 6 Beam splitter [0074] 7 Deflection mirror [0075] 8 Detected light [0076] 9 Detector [0077] 10 Electronic circuit board [0078] 11 Lens [0079] 12 Further light source [0080] 13 Air inclusion [0081] 14 Coverslip [0082] 15 Specimen slide [0083] 16 Light source for dark field illumination [0084] 17 Fracture of coverslip 14 [0085] 18 Emerged adhesive