DE10359780B4 - Method for optical image acquisition - Google Patents

Abstract

An optical imaging process comprising the steps
- Setting at least 3 measuring points (x _i , y _i ) on the object to be recorded;
- Determining the corresponding sharpness levels (z _k ) for each of these measuring points;
- generating a height profile from the determined planes of sharpness (z _k ) for at least a portion of the subject by interpolation and / or extrapolation;
- taking partial images in the interpolated and / or extrapolated regions of the height profile, wherein for each partial image whose focal plane (z _k ) is determined from the height profile;
- Assembling the image to be generated from the recorded partial images.

Description

The The invention relates to a method for generating an image of a Object by means of an optical system, the image of several Compose sub-images is.

One Such method may in particular for the investigation of biological Samples are applied. Such biological samples are z. B. by marking contaminations with fluorescent substances and then separating the liquid generated by the contaminations by filtering. The filters are the carrier the marked contaminations. Goal of such an investigation it is in many cases determine if there is contamination, such as a microbiological contamination with bacteria such as E. coli.

at Examination of extremely low or uncontaminated samples there are particular difficulties. A problem in particular the difficulty of having the optical system, such as a light or fluorescence light microscope to focus on the sample. This is particularly difficult because the microbiological contamination of the Sample extraordinary may be low, but this contamination is nevertheless of relevance for example the suitability of water from which the sample was taken as drinking water can be. Known methods for automatic focusing can therefore in these cases usually not be used successfully.

in the With regard to the possible low levels of contamination must also be sufficiently large samples be examined so that several Recordings of the sample must be made, which in connection to a full picture assembled the sample Need to become.

in the State of the art are microscopes for electronic image capture greater Object fields are known in which the shooting positions for recording from subpictures over motor drives in the recording plane (x, y plane) and focus adjustment (z-axis) can be adjusted.

Furthermore, microscopic devices are known whose objects to be examined are preferably illuminated with UV light and fluorescent objects are recorded by an electronic camera. Such a device is used for example in EP 0 339 582 A2 described.

One Another problem is that the samples are often not ideal are just (bulge of the preparations, Unevenness of the slides, u. ä.) so that when automatically scanning the sample once Accuracy level for one safe detection of contaminations is insufficient, which is why contaminations outside the sharpness level overlooked could become.

moreover is in such cases a determination of the focal plane a sub-image by known methods very time consuming and associated with long or multiple exposures. For the investigation Fluorescent substances is the time consuming, in particular multiple searches of the focal plane, however disadvantageous, because with increasing exposure of the sample the fluorescent substances lose their luminosity, making them invisible and that Falsify the result of the investigation.

DE 101 01 624 A1 describes a method for automatic focusing of an optical system. This is a further development of the so-called tip-stop method, in which the focusing of an objective onto a specimen by a stepwise movement of the specimen (or the objective lens of the objective) along the optical axis of the objective lens into a position of maximum contrast on it optical axis takes place. This in DE 101 01 624 A1 However, the method described fails when it is to be used in the study of extended transparent samples containing very little or no contamination or other formations.

DE 195 02 472 A1 discloses a method for producing a deep-focus image of an object, wherein a structure is projected onto the object and exposures of the entire object are made in multiple planes of sharpness. Areas of the object located in the respective focal plane are seen sharply, wherein in each focal plane two whole images of the object are taken, namely an image of the object on which the structure is imaged and an image of the object without the structure. The structure is a regular grid, so that the image that shows the object with the structure (raster image) in a plane of sharpness can be decomposed into image areas. The gray value differences of the image areas are determined in order to obtain a contrast image, wherein a contrast value is assigned to each image area. The contrast values of an image area in a particular position in a focus plane are compared with the image area in the same position in other planes of sharpness to determine the image area with maximum contrast. If an image area has the maximum contrast, then the corresponding image area of the image that was recorded without structure in the same focal plane, selected. These image areas are then combined, so that an overall sharp representation of the object is obtained. The elevation profile results from the level membership of the image areas used for the representation.

In US 6,449,087 B2 a laser scanning microscope is used which has a "sectioning" function. This feature detects a change in contrast when imaging an uneven sample. If a change in contrast is detected, the aperture stop is adjusted so that the depth of field corresponds to the height of the unevenness. The elevation profile is not used to determine the depth of field for capturing partial images that are outside previously determined measurement points. Moreover, the height profile for the object is not calculated, but determined by a laser beam along a line or a plurality of lines. A selection of measuring points thus takes place. This can result in sporadic contamination not being detected.

DE 196 53 413 C2 discloses a scanning microscope having a plurality of microlenses that are adjustable to different focal planes. DE 199 22 341 C2 describes a method for arranging the spatial coordinates of object points, wherein object coordinates in the terrain are determined by means of reference points.

task The invention is to the disadvantages of the prior art remove. It is intended in particular a method for generating a Image of extended objects by means of an optical system be given, which is an improved, especially faster and more precise focus on the object allows.

These The object is solved by the features of claim 1. Advantageous embodiments of Invention emerge from the features of claims 2 to 4th

• – (a) Festlegen von mindestens 3 Meßpunkten (x_i, y_i) auf dem Aufnahmeobjekt;
• – (b) Ermitteln der zugehörigen Schärfeebenen (z_k) für jeden dieser Meßpunkte;
• – (c) Erzeugung eines Höhenprofils aus den ermittelten Schärfeebenen (z_k) für zumindest einen Bereich des Aufnahmeobjektes durch Interpolation und/oder Extrapolation;
• – (d) Aufnehmen von Teilbildern in den interpolierten und/oder extrapolierten Bereichen des Höhenprofils, wobei für jedes Teilbild dessen Schärfeebene (z_k) aus dem Höhenprofil bestimmt wird;
• – (e) Zusammensetzen des zu erzeugenden Bildes aus den aufgenommenen Teilbildern.

According to the invention, there is provided an optical image forming method comprising the steps

• - (a) defining at least 3 measuring points (x _i , y _i ) on the object to be photographed;
• - (b) determining the respective planes of sharpness (z _k ) for each of these measurement points;
• - (c) generating a height profile from the determined planes of sharpness (z _k ) for at least a portion of the subject by interpolation and / or extrapolation;
• - (d) taking sub-images in the interpolated and / or extrapolated regions of the height profile, wherein for each sub-image whose focal plane (z _k ) is determined from the height profile;
• - (e) composing the image to be generated from the captured partial images.

The Procedure allows the recording of a large number of sub-images in a comparatively short time, there from the height profile the for determines the recording of the respective field required sharpness level can be. So it is not necessary, the sharpness level by using known methods for automatic focusing for each single image to be determined separately.

Of the Term "object" and the term "sample" are used in the present Description used synonymously.

The Number of measuring points should be distributed on the object so that a height profile out of the for each measuring point determined sharpness levels by interpolation between the measuring points and by extrapolation outside limited by the measuring points area can be calculated.

The expression "setting a number n of measuring points" means that both the number n of the measuring point and its position x _n , y _{n are defined} in the x, y plane.

The Measuring points are preferably chosen on the object so that they are uniform over the object are distributed. Particular preference should be given to measuring points located at the edge of the object and in its center, being edge and center of the object determined after measuring the object become.

Preferably in step (b), the sharpness level becomes for each measuring point by measuring the relative contrast in a stepwise movement of the Object or an objective lens along the optical system an optical axis (z-axis) of the objective lens measured and the maximum contrast of the object for this measuring point certainly.

For extended objects, the measuring points are expediently controlled by moving the object in the receiving plane (x, y plane) relative to the objective lens of the optical system. An extended object in the sense of the present invention is understood to mean an object which consists of several sub-formers. If, for example, the sample has a diameter of 1.5 cm, at least 176 partial images must be recorded for a partial image size of 1 mm ² , for which purpose the sample is scanned. The contaminations have a size of a few square microns. The size of a partial image depends on the resolution of the camera used.

Under Accuracy level gets the sharpness a sub-picture understood. To determine the focal plane can the well-known methods are used. Preferably however, in step (b), the sharpness level is determined by determining the maximum contrast determined.

to Capturing partial images of extended objects expediently makes the object in the receiving plane (x, y plane) relative to the objective lens of the optical system moves. The object should be scanned in this way be that for each other adjacent subregions of the object sub-images are included. The sharpness level for each Subarea or subpicture obtained for each subarea is to be, is from the height profile calculated.

optical visible marks are marks that are distinct from distinguish the objects to be detected, of which especially Markers with other colors and shapes are preferred through image analysis programs very easy from the contaminants can be distinguished. Suitable markers for producing such markings are, for example Cyanine.

Should Only a small number of structures or no structures in the Sample can be optically detected, it may also be appropriate optically visible markers, d. H. Markers to introduce into the sample, the position of the optically visible marks in the receiving plane of the optical system as a measuring point can be used. The measuring points used in the method according to the invention can all based on the use of such mark. However, it is also possible, in addition to measuring points, which result from the use of such mark, also Measuring points to use, arising from already existing in the object structures can be determined.

The Markers can evenly over the object be distributed or selected Places are applied to the object, preferably on the edge and in the center of the object.

The Procedure can be with any optical system that performs a movement of the Object in the recording plane (x, y plane) relative to the lens of the optical system and further movement of the object relative to the lens of the optical system along the optical axis (z-axis) of Objective lens of the lens allows.

A preferred embodiment The invention will be described below with reference to the accompanying drawings in more detail described. Show

1 a schematic representation of an optical system, which is suitable for carrying out the method of the present invention;

2 a schematic representation of a sample are applied to the optically visible markers; and

3 a schematic representation of a height profile, for the in 2 shown sample has been determined.

According to 1 is used as an optical system, a fluorescent light microscope 1 with lens 1.1 with an objective lens 1.2 used the microscope 1 furthermore a lighting unit 2 includes a stage 13 for receiving the object to be imaged 8th , motorized adjustment for moving the stage 13 on which the object 8th can be applied, in the x, y and z directions 3 (M1), 4 (M2), 5 (M3), an image capture unit 6 and a computer unit 7 for controlling the relative movements between the stage 13 and objective lens 1.2 , for determining the measuring points, storing the sharpness planes determined for the measuring points, calculating the height profile and, therefrom, the sharpness plane for the partial images and for joining the partial images to form an image of the object.

In 2 a preparation to be examined is shown. It consists of a slide 9 , an object 8th from a filter plate, on which marked contaminations 11 and optically visible labels introduced into the sample 12 are located.

This preparation is on the stage 13 applied. Then, the sample by means of the adjustment 3 . 4 moved to the first pickup position (x ₁ , y ₁ ) in the x, y plane in which a mark 12 contained in the sample. Subsequently, the adjusting device moves 5 the sample gradually in the z-direction. In each recording position z _{1, d} is an image recording with the image acquisition unit 6 performed and with suitable algorithms z. For example, the contrast in the obtained image is determined. The relative contrast is determined for each of these steps d in the z-direction and after passing through all the steps the position (z _{1, dmax} ) with the maximum contrast for the position (x ₁ , y ₁ ) is stored. Thereafter, this operation is performed for the recording positions with other marks, and the respective values (x ₂ , y ₂ , z _{2, d max} , x _n , y _n , z _{n, d max} ) are determined.

In the simplest case, an elevation profile is calculated from the values obtained by calculating a planar area _{segment which is spanned} by three adjacent values (x _n , y _n , z _{n, d max} ). An example of such a height profile is in 3 shown. When considering more than three measuring points, curved surfaces can also be calculated, which increases the accuracy of the focal plane.

To record the partial images from which the image is then to be composed, the acquisition positions (x ' _m , y' _m ) are then approached according to a predetermined algorithm (eg matrix scanning, circular scanning or other methods known to the person skilled in the art) associated focus point (z ' _m ) calculated from the previously determined height profile, by the adjusting device 5 approached, the sub-picture recorded for this recording position and in the computer unit 7 stored off. After passing through all recording positions (x ' _m , y' _m , z ' _m ), the (for example about 1000) partial images in the memory of the computer unit 7 put together so that you get a complete electronic image of the object 8th obtained with a sufficient sharpness in a very short time, which allows to perform an image analysis.

n

Anzahl der Meßpunkte

m

Anzahl der Teilbilder (= Anzahl der Aufnahmepositionen)

x,y

Koordinaten in der Aufnahmeebene

z

Koordinate parallel zur optischen Achse des Objektives

d

Anzahl der Schritte auf der z-Koordinate

d_max

z_d-Wert mit maximalem Kontrast

x_n, y_n, z_n,dmax

Position der Meßpunkte

x'_m, y'_n, z'_n

Position zur Aufnahme der Teilbilder (Aufnahmeposition)

The symbols used have the following meaning:

n

Number of measuring points

m

Number of partial images (= number of recording positions)

x, y

Coordinates in the recording plane

z

Coordinate parallel to the optical axis of the lens

d

Number of steps on the z-coordinate

d _max

z _d value with maximum contrast

x _n, y _n, z _{n, dmax}

Position of the measuring points

x ' _m , y' _n , z ' _n

Position for recording the subpictures (recording position)

1

microscope

1.1

objective lens

1.2

lens

2

lighting unit

3

adjustment for the x-coordinate

4

adjustment for the y coordinate

5

adjustment for the z coordinate

6

Image capture unit

7

computer unit

8th

object (Filter platelets)

9

slides

11

contamination

12

optical visible marks

13

stage

Claims (4)

An optical image forming method comprising the steps of: - setting at least 3 measuring points (x _i , y _i ) on the photographic subject; - Determining the corresponding sharpness levels (z _k ) for each of these measuring points; - generating a height profile from the determined planes of sharpness (z _k ) for at least a portion of the subject by interpolation and / or extrapolation; - taking partial images in the interpolated and / or extrapolated regions of the height profile, wherein for each partial image whose focal plane (z _k ) is determined from the height profile; - Assembling the image to be generated from the recorded partial images. Method according to Claim 1, characterized in that, in order to determine the focal plane (z _k ) of each measuring point, the distance between object and objective is changed stepwise and the sequence of associated relative contrasts is measured, and the maximum contrast is selected from this sequence and thus the focal plane (z _k ) of the respective measuring point is determined. A method according to claim 1 or claim 2, characterized characterized in that markings introduced into the object and the measuring points are determined by this. Method according to one of the preceding claims, characterized characterized in that Approaching the measuring points and / or for receiving the partial images, the object in the recording plane is moved relative to the objective lens.