RU2663732C1 - Method of automatic selection and packaging of microbiological objects - Google Patents

Abstract

FIELD: biology.SUBSTANCE: invention relates to a method for automatically selecting and packaging microbiological objects, which can be used to work with biological objects in the size from 0.1 mm to 0.5 mcm, located in an aqueous medium, such as chromosomes, spermatozoa, bacteria, fragments of plant and animal tissues, spores of fungi, pollen and other objects visible in an optical microscope. After the microbiological object has been diluted with the aqueous medium inherent in its composition, the resulting solution is filled with long capillaries by immersing the capillary packet into solution and freezing the filled capillaries. After that, the batch of capillaries is loaded into the receiving cassette, from which the next capillary is placed in the field of view of the microscope and its contents are viewed by moving the capillary, while using the computer to automatically search and select the desired objects. Upon detection, the computer remembers the image and the linear coordinate of the object. After viewing the whole capillary, the fragments containing the selected objects are automatically separated, fixed on a packing tape and supplied with an identification mark.EFFECT: method allows to formalize and automate the routine work associated with the analysis and analysis of a large number of samples, as well as streamline its accounting and storage.7 cl, 3 dwg

Description

The method can be used to work with a class of biological objects ranging in size from 0.1 mm to 0.5 μm, located in the aquatic environment, such as chromosomes, sperm, bacteria, fragments of plant and animal tissues, fungal spores, pollen and other objects visible under an optical microscope . The method allows for the automatic selection and packaging of microbiological objects for the purpose of their transportation, sale and storage.

According to the claimed method, microbiological objects are packed in glass or plastic capillaries, the inner diameter of which corresponds to the size of the object. The transparent capillary wall during the search and identification process allows you to examine the target object in a microscope.

Conserved biological objects are found in their characteristic composition of the aquatic environment. Drying, as well as any preservative media, distort and spoil many biological objects.

In order to stop the current biological processes, as well as fix their position, the objects in the process of selection, packaging and storage are in a frozen state. Deep freezing during transportation and storage allows the drugs to remain in a usable condition for a long time.

During the selection process, a solution with microbiological objects is placed in long capillaries, which after freezing are viewed under a microscope in order to search and select the desired objects. A package of tubes is lowered into the target solution, they are filled due to the hydrophilicity of the inner surface of the capillaries, if necessary, it is possible to use hydrostatic, hydrodynamic and electrokinetic filling methods. Thus, the entire volume of the absorbed solution will be further examined for the presence of suitable objects.

In the process of searching and selecting objects, the next capillary in the field of view of the microscope moves along its axis and, when a potential object is found, turns to identify and evaluate it. Together with the solution, garbage, foreign matter, damaged and defective objects get into the capillaries - they must be excluded during the search process, and not the necessary sections of the capillaries should be discarded.

Manual viewing of capillaries is time consuming; this routine work is supposed to be excluded. The identification and evaluation of the microbiological object is carried out automatically by the computer by comparing the visible image with a number of graphic images stored in the computer's memory.

As such graphic images, several typical microscope images selected by the operator when manually viewing objects of a given batch can be used.

In case of sufficient similarity of the image of the next object with the reference image, the computer remembers the image and the linear coordinate of the found object. In special cases, the final decision on the suitability of the drug is made by the operator after viewing the image file.

After viewing the entire capillary, sections with the selected objects are automatically cut out, placed on the packaging tape and provided with an identification mark. Unused sections of capillaries are thrown away.

For particularly valuable and rare objects, the identification mark is accompanied by a file with an image of a specific packed object.

Typically, biological micro-samples are examined under an optical microscope on a stage under a coverslip. This is convenient, but the selection of such samples is difficult, especially automatic selection. Meanwhile, there is experience in viewing small biological objects in thin glass capillaries. Distortions arising when viewing an object through a cylindrical wall of a capillary are of a natural nature and can be eliminated by software when transferring an image to a monitor.

In addition, the purpose of automatic viewing is not research, but only the identification and evaluation of biological objects, and this simplifies the task. If necessary, you can arrange the production of flat (flattened) capillaries or plastic capillaries. It is known that the freezing temperature of water in thin capillaries made of hydrophilic material is significantly lower than in an open vessel. This is due to the physics of the interaction of water molecules with the glass wall of the tube. The smaller the inner diameter of the capillary, the lower the freezing temperature of the water in it. So from practice it is known that in the pores and capillaries of a good concrete stone, water does not freeze during the most severe frosts, and in thin glass capillaries it was possible to keep water liquid at a temperature of minus 70 degrees Celsius. In addition, in order to preserve the mechanical strength of a capillary with a small inner diameter, it is necessary to keep the outer diameter large enough so that the ratio of the diameters increases. And this means that a relative increase in the internal diameter that occurs when water freezes does not destroy the thick-walled tube. So the sperm in the capillary with an inner diameter of 10 μm can be preserved even at the temperature of liquid nitrogen.

Areas of application of the technique.

Sperm. The most advantageous is the use of the described technology in the field of artificial insemination by the method of intracytoplasmic sperm injection (ICSI).

- the packaging method allows you to extract from the sperm bank only a few samples necessary for the procedure without defrosting the bulk of the biological material.

- the thawed sample is evaluated for viability and is directly transferred to the working tool (capillary and operating needle sizes are agreed).

- the customer is initially supplied with only healthy healthy cells evaluated according to morphological characteristics.

- in case of difficulty in obtaining sperm from the patient, all sperm can be stored and subsequently used for several attempts at fertilization.

- sperm from voluntary donors, packaged according to the proposed methodology, can be laid for long-term storage. Checking the genetic merits of biological material can be carried out according to the results of the birth and development of the child and successful material can be used in the future for a long time.

- in livestock farming while inseminating animals, the ICSI method is still little used, the usual method requires a large amount of sperm, and the high cost of elite sperm slows down the development of the industry. The progressive technique reduces the need for elite material by two to three orders of magnitude, and the possibility of long-term storage allows you to track the characteristics of the hereditary traits of manufacturers.

- Of commercial interest is the creation of a sperm bank of celebrities and people who have achieved outstanding results.

- preservation of sperm for rare and endangered species of animals.

Meristems and proto-feeds. The inventive method of packaging and storage of biological micro-objects can be used in the field of microclonal propagation of plants - vegetative growing in conditions.

- automatic cryopreservation of apical meristems into plastic capillaries of an appropriate size will reduce the cost of manual labor in preparing elite planting material.

- it becomes possible to create banks of primary undifferentiated plant cells of plants of many species for scientific and practical purposes.

DNA storage base. For reliable DNA storage, it is convenient to use small pieces of the tissue of an object as a carrier, from the cells of which, if necessary, hereditary material is extracted.

Scientific bases of biological samples. Ease of conservation and ease of storage allow you to create a single database of biological materials for various purposes.

- in the conservation of rare and endangered species of animals.

- tissue samples from fossil biological materials.

- sites of individual genes and DNA fragments intended for multiplication by PRC methods (without viewing through a microscope).

- studying proccess. A mobile fleet of microbiological samples can be created in a wide range of directions - cell preparations obtained by centrifugation of cell fractions, bacteria, insects, small aquatic fauna.

In FIG. 1 shows a capillary that is viewed under a microscope in order to identify the desired biological objects. In FIG. 3 is a logical diagram illustrating an example implementation of the proposed method.

1. Dilution of the solution, tinting (if necessary).

2. Filling the capillaries with a solution.

3. Download a batch of capillaries in the receiving cartridge of the device.

4. Installing another capillary on the table.

5. Advance of the capillary until the discovery of another object.

6. Is the object detected?

7. View records for the current capillary.

8. Are there objects found in the capillary?

9. An attempt to identify the found object.

10. Does the object satisfy the given conditions?

11. Image recording and fixation of linear coordinates.

12. The movement of the capillary in the cutting zone.

13. Setting the capillary to the position of the next object according to the previously saved linear coordinate.

14. The separation from the capillary of the fragment containing the object.

15. Fixing a fragment on a transport tape, printing an identification tag.

16. Are there more objects in the remaining part of the capillary?

17. Removing the capillary in a garbage container.

18. Are there more capillaries in the drawer?

19. Bringing the installation to its original state.

An example of a device for implementing the method. (see fig. 2)

1 - microscope stage

2 - microscope

3 - movable table

4 - test capillary

5 - step motor

6 - wave transmission

7 - lead screw

8 - nut

9 - transport tape

10 - laser cutter

11 - cutting zone.

A movable table 3 is mounted on the microscope stage 1. The studied capillary is mounted on this table 4. The table can move slowly or accelerated under the microscope lens 2. The table is driven from the stepper motor 5 through wave transmission 6. After analyzing the contents of the capillary, the movable table moves to the zone cutting 11. Here the capillary with the help of laser 10 is cut into individual microcontainers, which are fixed on the transport tape 9.

In FIG. 3 shows a diagram of the algorithm of the device according to the described method. On the flowchart, separate operations for working with objects are indicated and relationships are described that characterize the sequence of actions for implementing the described method.

Claims (7)

1. A method for the automatic selection and packaging of microbiological objects, including diluting the microbiological object with its aqueous composition, filling long capillaries with a solution by immersing a packet of capillaries in the resulting solution, freezing the filled capillaries and selecting objects, characterized in that after freezing a batch of capillaries is loaded in the receiving cartridge, install another capillary on a movable table located on the microscope stage, by moving the cap In the field of view of the microscope along the axis of the capillary, the illary is viewed and its contents are automatically searched and selected using a computer by comparing the visible image with a number of reference images stored in the computer’s memory; when the desired object is detected, the computer remembers the image and the linear coordinate of the found object, after viewing of the entire capillary, fragments containing the selected objects are automatically separated from the capillary, fixed on the packaging tape and provided with an identification tag after which the process is repeated with respect to the next capillary. 2. The method according to p. 1, characterized in that they use glass or plastic capillaries, the inner diameter of which corresponds to the size of the microbiological object. 3. The method according to p. 1, characterized in that the size of the microbiological object is from 0.1 mm to 0.5 microns. 4. The method according to p. 1, characterized in that to fill the capillaries with a solution using the hydrostatic, hydrodynamic or electrokinetic filling method. 5. The method according to p. 1, characterized in that as the reference samples using several standard images from a microscope, selected by the operator when manually viewing this batch. 6. The method according to p. 1, characterized in that for particularly valuable and rare objects, the identification mark is accompanied by a file with an image of a specific packed object. 7. The method according to p. 1, characterized in that the separation from the capillary of the desired fragment is carried out using a laser.

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