WO2014161585A1

WO2014161585A1 - Particle counting system adaptable to an optical instrument

Info

Publication number: WO2014161585A1
Application number: PCT/EP2013/057164
Authority: WO
Inventors: Óscar BASTIDAS GARCÍA
Original assignee: Bastidas García Óscar
Priority date: 2013-04-05
Filing date: 2013-04-05
Publication date: 2014-10-09

Abstract

The invention describes a microscopic counting system, designed for counting particles, in particular microorganisms, with a microscope or a magnifying glass. The system is adaptable to any microscope or magnifying glass. It allows analyzing biological samples and/or particles that have previously been prepared for observation and introduced into a counting chamber or other container (Neubauer Chamber, Thoma Chamber, etc.) but also can be used with microorganisms on its culture medium without exposing the culture (Flasks, Petri dishes, bioreactors, etc.). Using our own calibration method, the system allows calculating automatically or in a semi-assisted manner the cell concentration (or of particles) in the sample quickly and efficiently.

Description

PARTICLE COUNTING SYSTEM ADAPTABLE TO AN OPTICAL

INSTRUMENT

Technical field of the invention

The present invention is related to the field of cell and particle counting. In particular, it is related to the cell and particle counting systems with magnifying glass or microscope.

More specifically, is related to the counting systems with microscope or magnifying glass based on image analysis that carry out an automatic, semi-automatic or semi-assisted counting.

State of the Art

Various old techniques of cell count such as the one disclosed by the patent US- 5135302 (Flow cytometer) or by the patent US-1918351 (hemacytometer/Neubauer Chamber) are known. These techniques have various disadvantages and problems. Among which are highlighted the following:

Counting in Neubauer Chamber has the following disadvantages and problems:

(a) human counting errors;

(b) statistical errors;

(c) errors in the calculation of concentration, when applying the corresponding mathematical formula;

(d) low reproducibility / high variance in the measurements;

(e) very tedious and monotonous process for the laboratory technicians;

(f) difficult process for people with visual impairments;

(g) tiring process for the view of the laboratory technicians.

Counting in flow cytometer has the following disadvantages and problems:

(a) destruction of the sample when performing the counting;

(b) system with a high maintenance cost;

(c) system requires periodic calibrations;

(d) if the system is not used often, it is damaged;

(e) the system does not allow the visual observation of the samples by the technicians.

The counting chambers of the type described by US 1918351 (Neubauer Chamber) are chambers adapted with a bright field or a phase contrast microscope. They consist, generally, of a slide with a depression in the centre, at the bottom of which a grid with a given size has been marked with the help of a diamond, with a known separation between two consecutive lines. For counting the cells the reticle is observed with a microscope with the suitable magnification and the cells are counted.

Based on the number of cells counted, knowing the liquid volume that the reticle field holds, the concentration of cells per volume unit of the initial liquid sample is calculated.

One of the problems of this technique lies in the inaccuracy that occurs when the count is carried out, since a statistical formula that introduces certain error is used, and also the human error is present. This inaccuracy results in a lower reliability and reproducibility (reliability= absence of error on the measurement, reproducibility= precision= coherence in the measurements of the same concentration).

US 5135302 describes a Flow cytometer. Flow cytometry is a technique of cellular analysis which involves measuring the characteristics of light scattering and fluorescence that the cells have as they are passed through a beam of light. For their analysis by flow cytometry, the cells should individually be suspended in a fluid. When they pass through the beam of light, the cells interact with this causing light scattering. Based on the diffraction of the light frontally, the size of the cells that pass can be assessed and by measuring the reflection of the light laterally the granularity or complexity of these is assessed. In addition to light scattering, if the cells are placed in the presence of monoclonal antibodies marked with fluorescent molecules prior to their analysis, it can be evaluated which cells have the antigens complementary to the monoclonal antibodies used.

A major problem of this technique lies in the destruction of the sample that will be used to carry out the count, since by exposing the cells to the beam of light and to fluorescence the extracted sample is destroyed.

Various techniques based on other cell counting principles are also known.

Examples of this are patents and patent applications such as US 2007/0143033 that discloses systems and methods for counting particles by Beckam Coulter, US 2007/0012784 discusses the authentication of product by Thomas J. Mercolino, US 2008/0050619 "Fuel cell life counter and method of managing remaining life" by Life Technologies, US 3973194 from 1976 by Daniel W. McMorris and William J. Skidmore, US 5159642 from 1992 by Tokihiro Kosaka or US 5741648 from 1998 by George P. Hemstreet which describes a method of cell analysis using fluorescent quantitative image analysis.

Brief description of the invention

The present invention proposes a system adaptable to any microscope or magnifying glass. Currently, the cell automatic counting systems are closed machines that only allow performing cell counts. The claimed system leverages the existing microscopes and converts them in machines for counting cells allowing measurements and counts on a wider range. The mounting on the microscope, also allows saving space on the workbench.

Advantageously, the invention allows in addition the automatic counting in any container of known depth that can be observed in the microscope.

To achieve these objectives, several difficulties have had to be solved:

Problems with the illumination of some microscopes. According to the microscope, it may be necessary to include a light detector, and to include a brightness parameter associated with each cell profile. If the brightness with which the system was configured is changed when trying to perform the count the profile must be redefined.

Blur problems. There are microscopes which are defocused in a matter of seconds or by moving the plate. This problem has been mitigated by instantly showing the analysis on screen (if the analysis is wrong, it is usually a problem of defocusing and the user realizes it immediately and corrects it).

Problems of artifacts, dirt of the microscope and aberrations in the lenses in low quality, used, etc., microscopes. Exclusion areas can be defined to eliminate especially problematic areas of the screen and prevent their use in the count.

Thus the particle counting system is adaptable to an optical instrument and includes:

- Means of image acquisition for acquiring images from a container with a sample of particles through the optical instrument.

- Means of visualization for viewing images acquired by the capturing means associated with the sample.

- Means for processing the acquired images. Said processing means identify edges of possible particles, identify a plurality of regions of the image, at least partially defined by edges, to associate them with the background of the image or to associate them with a region with at least one potential possible particle. They also check if, indeed, said region contains at least one particle. This is done on the basis of the fulfilment of a condition based on at least one of the following geometric parameters: concentration of edges, maximum length, minimum length, perimeter, area or coincidence with a preset contour pattern. The processing means assign a number of particles greater or equal to 1 to said region and can count the particles contained in a plurality of regions. Optionally, the processing means are configured for assigning the number of particles to the region on the basis of a previous classification of said region. For example an extrapolation can be carried out if the number of particles in an area is known to assign the number of particles of the region.

Optionally, the processing means can convert the acquired image to a scale of shades according to its luminous intensity and wavelength.

Optionally, the means of visualization can also distinctly mark the particles counted.

Optionally, the means of visualization comprise a user interface for validating a recorded region or for allowing discarding it as recorded.

Optionally, the processing means can also assign a value in the scale of shades to the background of the image.

Optionally, the processing means can associate a particle size according to the number of pixels in the corresponding image.

Optionally, the processing means can calculate the concentration of particles per volume unit or per area unit when the sample is placed in a container of known dimensions.

Optionally, the processing means can exclude from an acquired image. The region of exclusion can be defined by a user through the interface of the means of visualization.

Optionally, the captured image can be converted to an image in greyscale.

The system is particularly applicable when the particles are biological microorganisms. The biological microorganisms can be, among others, cells, fungi, algae or platelets. Also protozoa, virus, bacteria, mites or spores.

The processing means can optionally carry out a selective count in the captured image when filtered when it is illuminated with light of a wavelength associated with a particular feature of the biological microorganisms, if said microorganisms were previously marked with a marker sensitive to such wavelength.

Optionally, a selective count in the acquired image can be carried out when it is illuminated with light of a wavelength associated with a particular feature of the biological microorganisms if said microorganisms were marked with a marker sensitive to said wavelength.

Optionally, the means of visualization can detect the illumination of the sample and for modifying the luminous intensity applied to the cell sample.

Optionally, the image capturing means comprise a digital camera. Optionally, the means of visualization of images comprise a touch screen.

Optionally, the system may include a mechanism for automatically moving the container of the sample.

Optionally, the image capturing means are calibratable, such that a pixel is associated with a real dimension value. Thus a correspondence is possible between size on the image and actual size.

Optionally, the processing means can calculate a correspondence between the total area of the screen covered by particles (confluence) and the concentration of particles per area or volume unit.

Optionally, the counting system can also include the optical instrument.

Optionally, this optical instrument may be a magnifying glass or a microscope.

When the instrument is a microscope it may optionally include an auto focus mechanism that focuses automatically.

Figures

Aspects relating to an embodiment of the invention are schematically represented in the following drawings.

Figure 1. Scheme of counting system adapted for a microscope (4).

Figure 2. Example of definition of an area of exclusion (14) for avoiding false detections.

Figure 3. Example of several operations carried out by the counting system with the schematic image produced by each operation: image capture (21), edges detection (22), delimitation of areas of possible particles (23), filtering based on geometric criteria (24) and resulting count (25).

Detailed description of the invention

In the next pages, the invention is illustrated in addition and without limitation by means of its integration in an optical microscope (4) (phase contrast, fluorescence, etc.) with coupling means to the digital camera (2).

However, for counting particles and in particular microorganisms the invention is applicable both to a magnifying glass and a microscope.

For example, said coupling means can be carried out in:

1) the trinocular of the microscope if it exists (usually a coupling from the digital camera to the C mount adaptor with a diameter of approximately 25 mm will be used, although also couplings for camera thread, bayonet mount, etc. are available).

2) if there is no trinocular, a coupling to one of the binocular lenses would be carried out, which have a diameter usually of 25 mm. This option is more uncomfortable because one of the lenses that allow the visualization with the microscope is disabled. In monocular systems there is no other possibility of visualization rather than the screen of the invention.

Another possibility is the adaptation to the image capturing camera in the microscope (if it exists).

The system may include also the following elements:

Image capturing camera (2).

Processing device (1) (PC or equivalent) with storage capacity.

- Visual interface / screen (3).

Communications cable between the camera and the processing device

Calibration device (it can be a Neubauer Chamber or a chamber wherein a reference measurement can be taken, a microscope gauge, etc.).

Sample holding chamber (6) (it can be a Neubauer Chamber, a Thoma Chamber, etc. The sole requirement is that the depth of the chamber must be known). The holding chamber can be washable or disposable.

Also it is necessary to have a microscope (4) for adapting the system. The microscope must be clean, to the possible extent, have light for illumination of the samples and have at least one optics, preferably of at least lOx.

The system can also be optionally coupled to the local data network through

Ethernet RJ45, WiFi or similar connection, with the object of:

1) performing backups on the server,

2) sending images to the data receiving centre for maintenance, quality control, out of calibration / malfunction of the system detection, updates, etc.

The option of use of identification credentials in the same device, to facilitate traceability is envisaged.

Advantages: complete traceability with user identification, entry barriers to external staff, increase of the responsibility in sampling by staff, improves the quality of the measurements as the employees will make greater efforts in the preparation of the sample, dilutions etc. since their measuring operations will be registered.

The system supports connection with other peripherals such as keyboard, mouse and/or plastic pen for Tablet PC.

The system even allows through a touch screen (3), to be used with the finger. The options are selected by pressing the touch screen and through the use of a virtual keyboard that appears on the screen when it is required. Neubauer Chamber Counting

The advantages of performing the automatic counting with respect to traditional solutions are:

1) Reproducibility. The error introduced by the system is restricted, and is systematic. It does not depend on the laboratory staff performing the count.

2) Reliability. Human errors are eliminated; taking more images reduces the statistical error.

3) Elimination of the tedious manual counting process. The measurement by manual counting with a microscope can take between 1 and 10 minutes of a laboratory technician, depending on the concentration. 4) Less maintenance, less setup and cleaning time than a flow cytometer.

5) Traceability (images and counts associated with a user and determined in time).

6) Recovery of images.

7) Visual detection of contamination.

The advantages of performing the semi-assisted count with respect to traditional solutions are:

8) Reliability. Certain human errors in the calculations of the concentration are eliminated.

9) Usability. Reduction of visual fatigue and it allows staff with certain visual impairments to use the microscope.

10) Usability. Reduction of intellectual fatigue. The system automatically saves the counted cells, and by using the marks on the screen of those already counted it allows the staff to "get distracted" without losing count.

11) Responsibility. The counts undertaken are recorded with the name of the laboratory employee who carried out the count. The poorly made counts can be related with a certain person, subsequently improving their habits through training, etc.

12) Less maintenance, less setup and cleaning time than a flow cytometer.

13) Traceability (images and counts associated with a user and determined in time). 14) Subsequent recovery of images, and of the counts together with the information of what has been considered as a cell / particle to be counted.

15) Contamination visual detection.

16) Count of elements with high visual complexity (adherent cells, very dirty samples, etc.) where the system cannot be configured to count automatically. 17) Used in conjunction with automatic counting, semi-assisted counting allows validating the calibration carried out for the automatic operation mode, and verifying that the system is operating within the acceptable operating ranges. The use of the system for the first time requires the following steps, in this order 1) Size calibration.

2) Definition of cell profile

3) Launch of cell count

In a second use, the cell count can be launched in a direct way, without performing the size calibration and definition of profile, provided that the same microscope is used, and the type of cell to be counted is the same (or that it has been previously defined). Each of the steps is described below.

CONFIGURATION AND CALIBRATION OF THE SYSTEM PRIOR TO CARRYING OUT THE COUNT.

1) A calibration of the system is carried out, where the following actions are performed

(A) Size calibration. A known distance in the microscope (4) is taken as reference to obtain the real distance to number of pixels on screen ratio (size calibration).

(B) Definition of the biological profile. For this are defined:

a. Depth of the container of count (6). (distance / depth calibration in the Z axis of the microscope (4))

b. Maximum and minimum size of cells (eventually)

c. Maximum and minimum illumination (it is not selected, it is detected automatically)

d. Sensitivity and contrast of the sample

e. Other morphological characteristics of the type of cell to be measured (eventually: roundness, form factor, etc.).

f. Features dependent on the wavelength of the element to be analyzed (for example, in the visible spectrum)

Next these settings are defined in more detail.

(A) SIZE CALIBRATION.

The calibration can be done in different ways, although it must always be done with an object of which we know the exact distance between 2 points with a microscope. Among others, the following elements of calibration can be used.

1) Standard microscope calibration plate. It is standard in some commercial microscopes. This is a plate where a pattern with lines is printed, where the distance between the lines is known.

2) A Neubauer Chamber, Thoma Chamber, Improved Neubauer Chamber, disposable chamber or any other type of chamber with known depth. In this type of chambers, there is a grille / grid on the microscope in the central part. This grid has been used historically as a reference for hand counts in the microscope. The distances and dimensions of the grid are generally written at the top part of the chamber.

3) Other systems. The system calibration can be done with any system, provided that the exact distance between 2 points visible with a microscope is known.

(B) DEFINITION OF THE BIOLOGICAL PROFILE.

The calibration of the biological profile determines the morphological characteristics of size, shape, texture, colour and/or absorbance in the visible spectrum (or invisible depending on the image sensor), and contrast in the sample.

The calibration of the biological profile is carried out always subsequently to the size calibration, since in order to perform this calibration, we must know the distances of the elements that we are visualizing on the screen, to be able to select the maximum and minimum range of geometric parameters of the biological elements that the system will count.

In the biological profile, the user selects the features of the elements of the image that they want to count.

- Sensitivity (value between 1 and 100). It determines the minimum contrast that the body or edge of the biological element (cell or equivalent) must have to be considered valid for count.

If a very high sensitivity is selected, the system will capture strange elements of the image, such as dirt from the camera or the microscope, artifacts, etc. producing false positives (detection of elements where there should not be any).

If a very low sensitivity is selected, the system will ignore elements of the image that should be taken into account in the count, producing false negatives (no detection of elements that should be detected).

- Geometric parameters (24): They determine the size (maximum length, minimum length, perimeter, area or coincidence with a preset contour pattern) that the biological element must have to be taken into account in the count (25).

- Light colour / absorbance calibration: This filter determines the range of acceptable colours counting the elements. For example, if the colour red is selected for the cells, because one wants to count only the cells that have absorbed a red dye, the system will ignore the cells/elements of colours very different to red, and will count the elements of the selected shade of red as well as similar shades (close in the colour and frequency spectrum).

The profile filters can be activated or deactivated. If the profile filter is deactivated, there will not be discrimination of the elements according to the characteristic of the profile. For example, if the colour filter is deactivated, the system will ignore colour when considering the elements for the count, counting all the elements of the image that meet the rest of the filters, and ignoring the colour. - Viability: The system allows a specific calibration for the measurement of cell viability (percentage of dead cells on total cells, live and dead). The measurement of viability can be deactivated. The system performs a simple counting and provides only the cell concentration in cells / ml, or activated, in which case the cell concentration will be provided in cells / total ml and the percentage of living cells in the sample, in percentage.

In the case of activation of the viability measurement, the specific colour filters for living (usually white) and dead cells (usually blue when using Trypan blue dye) must be defined.

- Used optics: The user selects the used optic. This is necessary to take into account the distances in the image, and make the relevant adjustments once the initial calibration has been carried out. The most common optics in optical microscopes are 4x, lOx, 40x and lOOx.

- Depth of the chamber / Container: As a rule, the commercial chambers (Neubauer, Thoma, etc) have a standard and known depth, and also said depth is written on the surface of the chamber.

To perform this calibration, the exact depth of the measurement container in mm must be introduced. The most common depths are 0.1 mm and 0.2 mm.

Therefore the chamber (image sensor) and the biological samples that you wish to measure are independent from the microscope used.

TYPE OF COUNTING [automatic] vs. [semi-assisted]

In cases in which the nature of the images or the type of cell to be counted do not allow to carry out an automatic count with sufficient reliability, the system allows configuring a profile for semi-assisted count.

In the case of selecting semi-assisted count in a cell profile, the system will ignore all the filters previously described and will not perform the automatic analysis of the images, but the user will be the one that will indicate manually or in a semi-assisted manner what they consider as a cell in each one of the images captured on the screen (by pressing with the finger or with the mouse).

THE CONFIGURATION OF THE BIOLOGICAL PROFILE STEP BY STEP.

While the characteristics of size calibration are common and do not vary, provided that the camera (image sensor) and the microscope are not changed, the characteristics of the biological profile change with each type of particle or cell to be measured. Therefore the user must define a different biological profile for each type of biological element that they want to measure. The system allows the storage of the features defined for each profile in the memory of the system, for later retrieval.

Example of profile name: hepatocytes -type-a-lOx-viability-Maria.

The operations for carrying out the calibration of the biological profile are the following:

1) Preparation of biological sample of the type of cell / microorganism to be measured.

2) Introduction of the sample in a Neubauer Chamber or similar. If necessary, a dilution has been previously carried out by introducing a sample into a test tube with an inoculating loop. This step is performed to achieve a proper concentration that allows the visual analysis on the screen. It is estimated that the system can perform a calibration if we can visualize on the screen or with a microscope between 1 and 2 cells as minimum and 50-100 cells as maximum, which corresponds to a concentration of between 200,000 cell / ml and 10,000,000 cell / ml (approximately).

3) Selection of the Configuration section in the device.

4) A Counting Profile (a set of parameters that will define what should and what should not be counted in each image) is selected.

5) The parameters corresponding to the type of cell / element that we want to count are selected.

6) If the microscope has dirt, or a part of the image appears blurred due to imperfections in the lens, the part of the screen having a problem will be eliminated by an Exclusion Area (14) (equivalent to a mask for ignoring problem areas).

7) After the adjustment of the parameters (and eventual configuration of the exclusion area) it can be visually checked that the system detects the cells of the image by drawing a coloured circle (10) on top of each cell. The verification that the system is well calibrated consists of manually counting the cells in the image, and checking that the system has drawn a superimposed circle on all of them.

8) In the case that all the cells / elements are not detected correctly, steps 5, 6, and 7 are repeated until at least 90 -95 of the cells of the image are detected correctly.

9) After the visual check, the microscope is moved and it is checked that the detection of cells is carried out correctly with 2 or 3 additional images. (Correct detection of at least 95% of the cells / elements)

10) The profile data are saved, and the system is ready to be able to carry out counts with this particular microscope, and with the type of element for which it has been configured.

11) This step is only necessary with adherent, overlapping cells, or cells with high level of agglomeration. In the case of adherent cells that tend to agglomerate against each other, the system must be configured to perform a calculation of extrapolation of the cell concentration from the confluence (or percentage of visual field occupied by cells). For this purpose a CONFLUENCE - CONCENTRATION internal ratio must be configured, through the following steps:

a. Measurement of the CONFLUENCE of the sample.

b. Measurement of the Cell concentration of the sample using an alternative method (e.g. manual counting, flow cytometer, etc), and introduction into the system of the value of said concentration.

From this ratio, the system will be able to calculate the cell concentration by means of the analysis of the confluence of the sample.

It is only required to perform this configuration the first time you work with a cell line.

BIOLOGICAL ELEMENTS COUNTING - AUTOMATIC MODE (WITH PREVIOUSLY CALIBRATED / CONFIGURED SYSTEM).

After completion of the calibration of the system (see previous paragraph) and definition of the biological profile of the element we want to count, the following steps are followed for carrying out a count.

1) The biological sample is prepared and introduced in the counting container

(Neubauer, Thoma, Howard ch., Slide + coverslip, or a proprietary container and made to measure for the system, a chamber with special calibration marks, a 24 or 96 wells plate, a Petri dish, a culture flask, etc.).

2) The PROFILE that has been previously configured in step 1) for this microscope and specific cell type is selected.

The images are taken with the digital camera (the device for moving the microscope tray is used, and the touch screen or the keyboard is used to indicate the system that the image can be captured). A number of images that can vary are captured. Several images are taken to reduce the statistical error (in the same way as in a manual counting with Neubauer Chamber, the custom is to measure 5 quadrants and perform an average of the same). In our case, taking more images entails a minimum effort for the user that translates into a significant reduction of the error.

As a general rule between 5 and 20 images will be taken, although we intend to take only 1 image with high resolution in a next version of the product.

If you want to reduce further the statistical error it is possible to take as many images as you wish, the statistical error being inversely proportional to the number of images taken.

3) The images are sent to the data processing system (1).

4) In the case that the images do not have the sufficient illumination or excessive illumination, the system will make the appropriate adjustments.

a. By means of the adjustment of the level of illumination in the captured image.

b. By means of an adjustment loop, where the processing unit sends a signal to the control unit of the intensity of the light source ordering to increase or decrease the intensity of the light source.

c. Returning to item 4 (and it is iterated until the illumination falls within the range).

d. If it were not possible to perform an automatic adjustment, it will be indicated to the user that the illumination levels are out of range and that the system is out of range for performing the count.

5) In the case that the focus level of the images is not appropriate, the system will make the appropriate adjustments.

a. By means of an adjustment loop, where the processing unit (1) sends a signal to the focus control unit of the microscope (4) ordering the microscope to get closer or away from the sample.

b. Returning to item 4 (and it is iterated until the focus falls within the range).

c. If it were not possible to perform an automatic adjustment of the focus (because the microscope does not include focus control), the system systematically indicates on each image analyzed the elements being recognized, so that if the system is not correctly focused, the user can see on the screen that the cells are not being detected correctly.

6) The analysis system processes the images, by applying:

a. size filters

b. filters on the morphology of the object c. filters on the wavelength that passes through the element / or is reflected by the element.

d. filters on the eccentricity of the element (similar to the eccentricity of an ellipse)

e. filters on the length of the contour of the object

f. filters on the area of the object

g. filters on the area to be analysed (areas of exclusion (14) )

h. elimination of the "background image (12)" (noise and dirt of the image that remain constant in each image)

7)

a. It calculates the number of cells in each image.

b. It performs an averaging of the same.

c. It subsequently multiplies by the average volume of the image (this average volume is calculated from the calibration in size and the depth of the chamber used - detailed in the previously defined biological profile).

The system displays on screen the results of the element count:

The system provides:

Cell or particle concentration per volume unit.

Cell or particle concentration per area unit

It can also provide:

- Total number of cells counted on screen.

- Degree of cell confluence (the cell confluence is the percentage of area occupied by the cells or particles with respect to the total percentage of the screen).

- Percentage of cells of a cell type or profile with respect to the number of total cells. - Percentage of cells of a cell type or profile with respect to the number of cells of another cell profile.

- Percentage of living cells with respect to total cells.

- Percentage of dead cells with respect to total cells.

- Total area analysed.

- Total volume analysed.

- Statistical error

8) The system stores the samples and images for subsequent consultation, generation of growth charts, etc. The display of results, images and graphics is done through the graphical interface (screen). BIOLOGICAL ELEMENTS COUNTING - SEMI-ASSISTED MODE (WITH PREVIOUSLY CONFIGURED / CALIBRATED SYSTEM).

The system allows the semi-assisted counting of elements with a microscope. In this mode of operation the system does not apply any filter defined in the biological profile nor performs any automatic analysis of the image (is the own user the one that does the counting manually, and their own intelligence is used to select the cells on the screen).

The steps to be followed in this case are:

1) size calibration. (Which is performed only once for each microscope)

2) definition of the biological profile (in this case only the depth of the chamber used and the optics have to be defined).

3) item 2) of the automatic method is carried out.

4) item 3) of the automatic method is carried out.

5) the images are taken with the digital camera (the device for moving the microscope tray is used, and the touch screen or the keyboard is used to indicate to the system that the image can be captured). A number of images that can vary are captured. Several images are taken to reduce the statistical error.

In semi-assisted mode, after the capture of each image the cells have to be marked manually on the screen using the mouse, the finger or the plastic pen. Whenever a cell has been marked, a semi-transparent circle is drawn on the cell to indicate to the user that the cell has already been counted.

6) The system displays on the screen the cell concentration (case of simple count) or the cell concentration together with the viability percentage (in the case of count with viability).

The system provides:

- Cell or particle concentration per volume unit.

It can also provide:

- Total number of cells counted on screen.

7) The system stores the samples and images for subsequent consultation, generation of growth charts, etc. The display of results, images and graphics is carries out through the graphical interface (screen).

In this case the data processing has been limited to the calculation of the cell concentration or the calculation of the total sum of cells marked by the user on the screen.

The calculation of the cell concentration can be done in this case thanks to the innovative calibration system of the system.

One of the most advantageous innovative components of the system is the coupling to any microscope on the market. This is achieved thanks to the following set of factors: a. the mechanical adaptation, which is done through common mechanical adapters, which usually exist on the market.

b. the size calibration.

c. the detection of changes in luminosity and the adjustments in brightness d. the detection of blur and focus adjustment.

e. the edge detector that prevents problems of illumination.

f. the exclusion areas.

The system can also be considered as a whole, attached to a specific and pre- calibrated microscope for the set of lenses of the microscope.

Numeric references

1. Processing unit.

2. Camera.

3. Touch Screen.

4. Microscope.

5. Eyepiece.

6. Sample container.

10. Edge of particle/cell.

11. Particle.

12. Photography background.

13. Inclusion area.

14. Exclusion Area.

21. Image capture.

22. Edge detection.

23. Delimitation of areas of possible particles/cells.

24. Filtering according to geometric criteria.

25. Count.

Claims

1. - Particle counting system adaptable to an optical instrument (4) comprising:

- means of image acquisition (2) configured for acquiring images from a container (6) with a sample of particles through the optical instrument (4),

- means of visualization (3) configured for viewing images acquired by the capturing means (2) associated with the sample,

- means for processing (1) the acquired images,

characterized in that

the processing means are configured for:

identifying edges (10) of possible particles,

identifying a plurality of regions of the image, at least partially defined by edges, to associate them with the background of the image (12) or to associate them with a region with at least one possible particle,

checking if said region contains at least one particle (11) depending on the fulfilment of a condition based on at least one of the following geometric parameters: concentration of edges, maximum length, minimum length, perimeter, area or coincidence with a preset contour pattern;

assigning a number of particles greater or equal to 1 to said region and counting the particles contained in a plurality of regions.

2. - Counting system according to claim 1, characterized in that the processing means are configured assigning the number of particles to the region on the basis of a previous classification of said region.

3. - Counting system according to claim 1 or 2, characterized in that the processing means (1) are configured for converting the acquired image to a scale of shades according to its luminous intensity and wavelength.

4.- Counting system according to any one of the previous claims, characterized in that the means of visualization (3) are also configured for distinctly marking the particles (11) counted.

5.- Counting system according to claim 4, characterized in that the means of visualization (3) comprise a user interface configured for validating a counted region or for allowing discarding it as counted.

6. - Counting system according to any one of the previous claims, characterized in that the means for processing (1) are also configured for assigning a value in the scale of shades to the background of the image (12).

7. - Counting system according to any one of the previous claims 4 to 6, characterized in that the means for processing (1) are configured for associating a particle size (11) according to the number of pixels in the corresponding image.

8. - Counting system according to any one of the previous claims, characterized in that the processing means for processing (1) are configured for calculating the concentration of particles (11) per volume unit or per area unit when the sample is placed in a container of known dimensions.

9. - Counting system according to any one of the previous claims, characterized in that the means for processing (1) are configured for excluding from an acquired image an exclusion region (14) according to that defined by a user through the interface of the means of visualization (3).

10. - Counting system according to any one of the previous claims, characterized in that the captured image is converted to an image in greyscale.

11. - Counting system according to any one of the previous claims, characterized in that the particles counted are biological microorganisms.

12. - Counting system according to claim 11, characterized in that the biological microorganisms are selected at least from the following:

- cells,

- fungi,

- algae,

- platelets,

- protozoa

- virus

- bacteria - mites

- spores.

13.- Counting system according to claims 11 or 12, characterized in that the means for processing (1) are configured for performing a selective counting in the image acquired when it is illuminated with light of a wavelength associated with a particular feature of the biological microorganisms if said microorganisms were marked with a marker sensitive to said wavelength.

14.- Counting system according to any one of the previous claims, characterized in that the means of visualization (3) are configured for detecting the illumination of the sample and for modifying the luminous intensity applied to the cell sample.

15. - Counting system according to any one of the previous claims, characterized in that the means of image acquisition (2) comprise a digital camera.

16. - Counting system according to any one of the previous claims, characterized in that the means of visualization (3) of images comprise a touch screen.

17.- Counting system according to any one of claims 8 to 16, characterized in that it comprises a mechanism to automatically move the container of the sample.

18. - Counting system according to any one of the previous claims, characterized in that the means of image acquisition (2) are calibratable, such that a pixel is associated with a real dimension value.

19. - Counting system according to any one of the previous claims, characterized in that the means for processing (1) are configured for calculating a correspondence between the total area of the screen covered by particles and the concentration of particles per area or volume unit.

20. - Counting system according to any one of claims 1 to 19, characterized in that it comprises the optical instrument (4).

21.- Counting system according to any one of claims 1 to 20, characterized in that the optical instrument (4) is a magnifying glass.

22.- Counting system according to any one of claims 1 to 20, wherein the optical instrument (4) is a microscope.

23.- Counting system according to claim 22, characterized in that it comprises a mechanism that automatically focuses the microscope.