BG67480B1 - Device for differential counting of microparticles in biological liquids - Google Patents

Abstract

The invention relates to a device for differential counting of biological cells, suspended in liquid, which can be used as a fluorescent automated cell counting system and can find application in the counting and differentiation of microparticles in biological liquids such as milk, blood, urine, including differentiation of cells of other particles, as well as of different types of cells in raw milk samples. The device comprises a housing, in which is located a lens is below which is housed a chip with microfluidic cameras, in which is placed a sample of biological liquid to be tested, a CCD or a CMOS camera, a counting part and an axial displacement mechanism for the sample is positioned, at which an illuminator is directed to the sample and at least one liquid lens (7) is located in the lens (2). The chip (4) is designed as a CD-based chip of a round shape, on which are formed microfluidic cameras (19), located on the periphery of the chip (4) connected to a motor (22), in which the illuminator (3) is at least one and is positioned at an angle, and the counting part is designed as a microprocessor (23), connected with a generator (8), controlling the liquid lenses (7).

Description

The present invention relates to a device for counting biological cells suspended in liquid, which can be used as a fluorescent automated cell counting system and can find application in counting and differentiating microparticles in biological fluids such as milk, blood, urine, including distinguishing cells from other particles as well as different cell types in raw milk samples.

Prior art

Among many parameters determining the quality of milk, bacterial count (BCC) and somatic cell count (SCC) are of extreme importance, as they have a significant impact on the quality of milk products, while at the same time providing up-to-date information on the health status of the the cows.

Total somatic cell count (SCC) is a recognized indicator of cow health and milk quality. Somatic cells are white blood cells known as leukocytes (lymphocytes, granulocytes, monocytes and macrophages) and originate in the animal's udder, and their number depends on the cellular immune defense. The number of these cells can increase in response to bacterial infections that can cause mastitis. It is for this reason that the somatic cell count, differential count and identification of leukocytes in milk is essential for the diagnosis of mastitis. SCC is an indicator of the presence of infection and inflammation of the dairy cow's mammary gland, called mastitis.

Normal milk from healthy cows has somatic cells less than 100,000 cells/ml, values higher than 300,000 cells/ml indicate infected cows, and values above 1,000,000 cells/ml are typical of sick cows. The limit value for human consumption or for further processing into food products for human consumption is below 400,000 cells/ml in Europe, but may be higher in the USA. In general, cows with an SCC higher than 300,000 cells/ml are considered infected (Smith, KL, in Standards for somatic cells in milk: physiological and regulatory (1996), International Federation of Dairy Mastitis, Bulletin September, p. 7 ).

In order to evaluate the quality of milk, it is necessary to use devices that allow a reliable and accurate somatic cell count to be performed. In recent years, portable automatic analyzers, in particular fluorescence imaging cytometers, have been developed based on a direct microscopic fluorescence method for counting somatic cells, the better known devices for such analysis being the ADAM SCC, using digital technology for counting and analysis purposes ; NucleoCounter SCC 100 Chemometec; DeLaval's DCC; and flow cytometry-based fluorescence devices: Somacount 150 Bentley instruments; Delta Instruments' SomascopeTM, Foss Electric's FossoMatic 4000TM).

The mentioned automatic flow analyzers are based on monochrome fluorescence and determine only the total number of cells. In these, the flow sample passes through a very small diameter, allowing only one cell to pass through and the count applied (Gunasekera T. S. et al. 2003, Feng W. et al. 2004). In the mentioned analyzers, fluorescent dyes are used as markers: ethidium bromide, propidium iodide and DAPI (Gonzalo B. et al 2004, Wallen CA et al 1982).

Samples are taken from individual animals on farms and sent to centralized laboratories equipped with flow cytometers to enumerate total somatic cells.

Expensive analyzes are mainly related to currently available technologies and automatic analyzers that are constructed based on optical or electrical measurements.

Apparatus are known in which optical measurements are performed, for which purpose it is necessary to initially label the cells before the analysis, which is why these apparatus usually use expensive chemical reagents or are designed for single use.

Apparatus are also known which work on the principle of electrical measurements, in which the result is obtained by measuring resistance, where the presence of other cellular milk particles interferes and for this reason they must be removed by applying several centrifugation steps. Precisely because of this, such devices cannot be used directly, on site, at the milk producer, and many efforts are made to simplify the methods and reduce the costs of analysis.

Electronic instruments such as so-called Coulter counters are known and used in practice, which can count cells and determine their size. Because raw milk contains many particles, mostly fat globules of a size similar to leukocytes (5-10 μm), these instruments do not provide a reliable cell count because these particles outnumber somatic cells and greatly complicate accurate measurement. Therefore, these particles must be separated from somatic cells before measurement. This is achieved by centrifugation, in which the cells are pelleted and the fat globules are separated from the supernatant.

A significant number of methods have been developed for the differential determination of cells using specific antibodies, which, however, have application in the field of human medicine, in particular in the processing of blood samples. These methods give much better selectivity and sensitivity of measurement, which is due to a specific antibody-antigen immune reaction. Herzenberg and Loken developed a method for counting cells using monoclonal antibodies (Herzenberg Loken et al. 1976 & 1977) which is called two-color immunofluorescence.

Numerous apparatus and patents based on image cytometry are known for counting and differentiating microparticles in biological fluids, such as milk, blood, urine, etc. For example, patent US 6919960 B2. These apparatuses use microfluidic cameras or slides, eg US patent 4513438 for the sample and a fluorescence microscope to capture the images via a CCD or CMOS sensor of the microparticles. With the help of processing devices, image analysis, counting and differentiation of microparticles in the examined sample is carried out, for example patent US 4513438 A

When using multiple illuminators, it is usually necessary to periodically change the emission filter in the lens axis to ensure that the required wavelength passes to the sensor. The filter units can be changed manually or by means of a mechanical device that changes the filters in the lens, and which is most often electronically controlled, as described and shown in figure 7 in patent publication US 9046489 B2.

In addition, some of these devices capture only one image of the sample, resulting in a low coefficient of variation. The coefficient of variation, according to the Poisson distribution, depends on the number of particles counted in the sample. For samples with low particle content, below 10000 per ml of blood, (e.g. from AIDS patients) or for highly diluted samples, capturing one, two or three images on different locations of the sample, (as described in US 20120223260 and US 20120314092 ) is not sufficient to obtain acceptable coefficients of variation. Therefore, in order to obtain a measurement with a higher degree of accuracy, it is appropriate to carry out imaging of a larger number of fields from the sample, and for this purpose the chip with the examined sample must be moved.

Known devices use optical systems that have a depth of focus of 20-100 μm, where if the sample falls outside this range, capturing an in-focus image is not possible. At the same time, with the optimal depth of the microfluidic chamber within 50 μm and less, it is necessary to use a focusing system. Depending on the specific implementation of the device, it is possible to use manual or mechanical focusing systems, the latter using micromotors or manual systems. A disadvantage of mechanical focusing systems providing the required micron precision is their high cost.

It is known that the coefficient of variation depends, according to the Poisson distribution, on the number of particles counted. At high magnifications, for example 10x, the scanned sample volume in front of the objective is insufficient to reach the required coefficient of variation. In this situation, it is necessary to increase the number of captured fields. This results in an unacceptable increase in analysis time. On the other hand, in morphological analysis of cells and/or two-color differential counting of leukocytes, for example in blood or milk, an optical magnification of 10 or higher should be used.

When precise counting with a low coefficient of variation is required, without differentiation, a magnification of 1 to 4 times is appropriate. Commonly known systems use interchangeable fixed zoom optical lenses or expensive mechanical zoom systems.

Patent publication US 20120223260 is known, relating to a method and system for determining particles in a liquid sample, in which only one field of the sample is imaged, and in such systems there is practically no need for autofocusing the image. If the sample needs to be moved during examination, this automatically requires focusing the image. When moving the sample chip by XY table, it is mandatory to provide image focusing due to the fact that when moving the sample chip, there is an accumulation of error along the Z axis, which is formed by XY table moving error and deformation error of the plastic microfluidic chips, with the error most often exceeding 100 μm along the Z-axis, which in turn leads to inaccuracy in cell counting.

An example of an XY table for moving successive sub-regions of the sample in front of the microscope objective is contained in patent publication US 7327901B2.

Patent publication US 7411680 B2 (CN 100520374 C) is known, in which a microparticle counting device is described, consisting of: a light source; a chip containing a sample with microparticles; a microscope lens, a microfluidic camera, a capturing CCD camera, a counting part, and a mechanism for moving the chip position. The movement of the chip with the sample is done by means of a table which is capable of being moved along the XY axes by means of a screw or other mechanism. A disadvantage of the described device is the insufficient accuracy of counting microparticles, which is due to the absence of a technical possibility to focus the image, as a result of which an error is formed when moving the table along XY, which is further increased by the deformation of the plastic microfluidic chips. wherein said errors accumulate as the Z-axis error, which often exceeds 100 micrometers in the Z-axis.

By means of said screw mechanism, the position of the chip is moved to predetermined coordinates, at a predetermined time interval, so that an area of the chip that is in close proximity to the imaged area can be shifted to the point where the light falls from the light source and is in the field of view of the lens. In this way, individual subregions in the chip are imaged once, sequentially, counting the number of microparticles in each (subzone) subregion and subsequently summed to calculate the total number of microparticles in a given sample.

The described method of sequentially observing or capturing only one picture and subsequently calculating the number of microparticles in the individual consecutive fields and in the sample is generally known and disclosed in detail in the accepted and implemented as a mandatory standard ISO 133666 methods for using a Neubayer camera.

Such a method of photographing and counting microparticles is also disclosed in patent publication RU 2147123 C1, in particular in a first independent claim, where the operations that are carried out for the purpose of enumerating macroparticles contained in a photographed field are described in sequence, as part of the main claim with a priority date of 1998 and in the description of patent US 4513438 A "Automated microscopy system and method for locating and re-locating objects in an image".

The described methods of photographing and counting the particles, besides being known, are also associated with disadvantages, which are mainly expressed in insufficient accuracy of the counting of microparticles due to the following: as already stated, the method of capturing single consecutive images of the sample does not provides the necessary conditions for accurate enumeration of microparticles, since in the absence of a mechanical focus system, the single captured image, when using a mechanism for moving polymer chips along XY, is not in focus, and therefore does not allow for high-quality and accurate enumeration. To this basic inaccuracy it is correct to add the inherent deviations from the focal plane - over 100 pm, as a result of which the enumeration and analysis of the microparticles, especially in solution, is greatly hindered or compromised in terms of enumeration accuracy.

Furthermore, when counting microparticles in liquid media with different refractive indices and a fixed mechanical focal length, it was found that the focus changes as the media changes. As a result, in consecutive single captured fields from the sample, some of them are "out of focus", making enumeration difficult or compromised. For example, when counting somatic cells in whole milk, the refractive index is 1.460 at 633 nm, and when counting brewer's yeast in aqueous solution, the refractive index is 1.340.

Another drawback of enumeration when using the known devices and applying the methods already described is related to the type of fluorescent dyes used. When simultaneously using two or more fluorescent dyes with different emission (eg FITC-530 nm for the first dye and Propidium Iodide - 620 nm for the second dye) to label the biological microparticles in liquid or dried monolayer samples, if the particles glow with a wavelength 530 nm is in focus, then those 620 nm wavelengths are out of focus due to chromatic aberrations in the lens, and as a result cell enumeration in a large proportion of single imaged sequential fields of the sample will be compromised for counting.

In another variant implementation of the described apparatus, more images are captured, as a microfluidic camera with the examined sample is moved in front of the microscope lens, and for the purposes of capturing a larger volume of sample, the movement is performed by means of a movable XY table.

Carrying out movement of the sample with microparticles by means of movement along an XY table is also known from patent RU 2147123 C1 and from patent US 4513438.

The known device for counting microparticles from patent US 7411680 B2 (CN 100520374 C) has a number of design flaws that do not allow to achieve accurate measurement and enumeration of microparticles due to the following: even with a very precise implementation of the mechanisms for moving the chip, the surface the XY table has a deviation from the lens of more than 10 pm, which together with the technological curvature of the microfluidic chip determines a deviation that typically exceeds 50 pm. Such deviation from the objective plane results in an image that is not «in focus» and affects the software counting of microparticles, significantly reducing the precision. No image focusing mechanism is provided in the device discussed here.

Another disadvantage of the known device described in US 7411680 (CN 100520374 C) is related to the mutual arrangement of some of the essential elements, namely that the illuminator is located in front of the objective and behind the microfluidic chamber. Such a mutual arrangement of the illuminator in relation to the other elements redefines a disadvantage such that the device must necessarily use a microfluidic chip composed of two transparent surfaces.

Illumination of the sample is of particular importance for the activation of the fluorochromes in the sample. The patent publications US 7411680 B2, US 6970246 B2, US 20110211058 A1 show different methods of illuminating the sample with lamps or LEDs, and the patent US 2015/0053872 A1 describes an LED illuminator with increased power by multiplying several LED crystals. Also described is a method for achieving uniform illumination of the object by placing two arrays of microlenses and a polarizing filter between them. Such illuminators have the disadvantage that it is very difficult to focus the light on the object in a spot of less than 4 mm. At higher objective magnifications, for example above 5x, the field of view is less than 1 mm and much of the light is not used to activate the fluorochromes in the sample. Laser illuminators can focus light into much smaller spots, eg 100 pm, and collimate the light with a single aspherical lens to the required diameter, eg 5 mm. Thus, all the energy of the light source can be used to activate the fluorochrome dye in the field of view of the fluorescence microscope. The large selection of wavelengths and powers of modern diode lasers allows choosing the most suitable wavelength for the selected fluorochrome. Laser diodes have a very narrow emission band, of only a few nanometers, which allows them to be used without an expensive, thin-film absorption filter, thereby further reducing the cost of the illuminator compared to LED-based systems. Laser illuminators also have one major drawback, making it difficult to use them as illuminators in imaging fluorescence microscopy systems, namely the "speckle pattern" effect. On the plane of the sample, the illumination spot is not homogeneous. At magnification, bright and dark spots are noticed, and uneven illumination leads to a reduced emission of objects or their parts caught in the spots with low illumination.

It is worth noting that for the purpose of obtaining a quality image with this illuminator and lens configuration, a microfluidic chamber that is transparent to the length of the excitation illuminator must be used. The use of microfluidic chambers filled with two transparent surfaces significantly increases the cost of the consumable, since the bonding of said transparent surfaces requires expensive, complicated and unreliable bonding methods, usually by bonding with liquid or dry adhesives, as described in patent publications US 20040145805 A1, US 20120223260 A1, US 7842157 B2 and US8808642 B2.

Another drawback of the known device for counting microparticles is related again to the already described mutual arrangement of the elements, as well as to their type. For example, when a part of the emitted light hits the plane of the lens with an angle of 90 ^o , correspondingly it also hits the filter of a CCD or CMOS matrix, the signal-to-noise ratio drastically decreases. Taking into account the fact that some of the fluorescent dyes, such as the widely used propidium iodide, have a high intrinsic fluorescence in solution, this predetermines poor quality of the capture, accordingly, it greatly complicates the subsequent software processing of the image.

Known differential counting devices are also used in sample analysis and counting where precise enumeration of malarial plasmodia growing inside or attached to red blood cells is required when a fluorescent microscope is used in the device. In such cases, fixation of the red blood cells is desirable, especially for long CMOS camera exposure times.

Patent publication US 10093957 B2 is known, in which a method is described for forming a monolayer of erythrocytes at the bottom of a microfluidic chamber by means of gravitational sedimentation of erythrocytes at a speed of 1 pm/s. This sedimentation rate is not sufficient especially for microfluidic chambers with a thickness greater than 100 pm and leads to a delay in counting at high sample numbers.

The design of the device is additionally complicated, related to the XY table used to move the examined sample. To ensure the passage of light through the XY table and the chip, the entire imaged area under the microfluidic chip must be freed. In the case of the need for lighting with several illuminators, with different wavelengths, such relocation is particularly difficult to implement from a technical point of view, including due to the need to achieve a compact design of the device

Technical essence of the invention

The state of the art in the subject area described so far shows a need to offer a wide-spectrum device for differential counting of microparticles in biological fluids, which provides a high degree of accuracy of counting microparticles, as well as detailed analysis of various samples containing biological microparticles - milk, fermentable fluids, blood, semen, etc., including differential counting of biological microparticles, such as somatic cells in milk, leukocytes in blood, body cells, algae and yeast. The device must be technological, built from cheap and accessible elements, the mutual arrangement of which allows optimizing the qualities of the optical system in terms of autofocus and image magnification, which is essential in the capture, counting and analysis of different types and composition of biological liquids, as well as to use a chip whose displacement ensures precise positioning of the sample relative to the lens and imaging cameras.

The task is solved with a device for differential counting of microparticles in biological fluids, consisting of a housing in which they are located: a lens, under which is placed a chip with microfluidic cameras, in which a sample of biological fluid to be studied is placed, capturing A CCD or CMOS camera, a counting part, and a sample moving mechanism where an illuminator is directed at the sample.

According to the invention, an optical filter and at least one liquid lens are located in the lens, and the chip is implemented as a CD-based chip, with a circular shape, on which microfluidic chambers are formed, located on the periphery of the chip, which is directly connected to a drive motor , wherein the illuminator is at least one and is located above the plane in which the CD-based chip is located, being oriented at an angle to provide an illumination angle with minimum reflection, and the counting part is implemented as a microprocessor connected and with a generator driving the liquid lenses.

According to a preferred variant embodiment of the device according to the invention, the optical system includes three fluorescent thin-film filters, located one after the other in the axis of the optical microscope, each of them connected to a stepper motor, ensuring rotation of the fluorescent thin-film filters from 0-90 ^o .

According to a variant embodiment of the device, two liquid lenses are located one below the other in the upper part of the lens, and one liquid lens is located in the lower end of the lens, wherein an optical filter is installed between the upper liquid lenses and the lower liquid lens and a lens comprising two achromatic lenses.

Preferably, the optical filter is a single-band or multi-band optical filter.

The liquid lens is connected to a generator with a variable step voltage of 0-70 V and a frequency of 1 kHz.

The illuminator consists of a light source, a collimating lens, a band optical filter, and a focusing lens, which are placed in series in front of each other.

It is preferable to use an LED, for example Luxeon, or a diode laser light source as the light source.

According to a variant embodiment of the device, a laser illuminator positioned at an angle to the sample is used as a light source.

It is preferable to use a rotating diffuser made in the form of a disk located in the path of the coherent laser radiation, the diffuser being located before or after the collimating aspherical lens.

According to a preferred embodiment, the CD-based chip consists of an upper part made of PMMA polymer with an optically transparent surface and a lower, opaque part made of black ABS polymer. The two elements are shaped and connected by laser welding so that after welding them together, with a laser wavelength of 800-1050 nm, microfluidic channels 15 with a height of 50 pm are formed in the space between them, in which a sample mixture is placed from biological fluid, fluorescent dyes or fluorescently labeled antibodies.

According to a variant embodiment, the CD-based chip consists of two parts, respectively an upper one with a transparent surface and a lower one with an opaque surface, whereby mixing chambers are formed between the surfaces of said two parts. Said mixing chambers are filled with a sample, a test subject, and dry fluorescent dyes and/or fluorescently labeled antibodies, wherein the mixing chambers are connected via hydrophobic channels to a microfluidic chamber, and the CD-based chip is mounted to a rotating axis connected to a drive motor .

The drive motor is preferably a stepper motor and is connected directly to the CD based chip.

According to another variant embodiment of the CD-based chip, sodium nitrite or sodium dithionite is added to the examined blood sample, whereby the CD-based microfluidic chamber 4 with the diluted blood sample is placed on a neodymium magnet 37 (Fig. 16).

The device also works efficiently when using a rectangular-shaped chip composed of two parts, an upper transparent part and a lower opaque part, respectively. The two elements are shaped and connected by laser welding in such a way that, after joining them, 50 μm high chambers are formed in the space between them, into which a mixture of biological fluid sample, fluorescent dyes or fluorescently labeled antibodies is pipetted. The rectangular chip is placed on a movable XY stage located below the illuminators and the objective, in which a multiband optical filter and liquid lens are mounted.

The device for differential counting of microparticles in biological fluids, according to the invention, is distinguished by a compact design, and the mutual arrangement of the elements allows accurate measurement and analysis of the microparticles in the processed samples. High-quality and low-cost elements are used, resulting in an apparatus that provides technical capabilities for obtaining quality images when capturing and processing samples. To improve the quality of the image, liquid lenses are used, with small dimensions, which are located in the objective of the microscope in such a way as to provide simultaneous focusing and magnification of the image.

At the same time, the control of the liquid lens allows the autofocus time to be on the order of milliseconds. The size of the liquid lens also allows it to be incorporated in the construction of mobile and handheld devices for measuring small particles.

In the proposed device, a fundamentally new and different model chip with CD-based microfluidic cameras is used, the positioning of which in front of the lens for capturing an image depends only on the accuracy of the microfluidic camera, which in turn automatically leads to a reduction in the requirements for the autofocus system. At the same time, the positioning of the microfluidic camera in front of the lens is greatly simplified - just by rotating the CD-based chip, only in certain cases requiring a minimal movement along the X-axis, which can be done with affordable technical means, for example: a NEMA8 mini stepper motor for the rotary axis and mini-servo motor DS-5001HV along the X axis, this way of moving the X axis can provide up to 10mm of movement.

When implementing the variant embodiment using three rotating sequentially located thin-film filters, conditions are created to achieve regulated transmission in the range from 440 nm to 660 nm, with a step of 2 nm. Such discrete bandpass tuning of the fluorescence emission allows fine-tuning of the signal-to-noise ratio of the cell image in a multicolor fluorescent labeling system with different emission wavelength fluorochromes and several LEDs or laser emitters with different wavelengths, which is more in contrast to using a suboptimal emission filter for the selected fluorochrome. Contrast imaging facilitates software analysis of fluorescence images.

The performance of the differential counting device is further improved by using a laser illuminator positioned at an angle to the sample. With a correctly selected rotation speed of the rotating diffuser, a high homogeneity of illumination is ensured.

Explanation of the attached figures

Next, an exemplary embodiment of a device for differential counting of microparticles in biological fluids will be described, which is presented with the help of the accompanying drawings, where:

Figure 1 - appearance of the device according to the invention;

Figure 2 - schematic diagram of a device according to the invention, with a partial view of a CD-based chip with microfluidic chambers;

Figure 3 - schematic of the device with a partial view of a CD-based chip, with mixing and microfluidic chambers;

Figure 4 - diagram of the device according to the invention with a rectangular chip, with an upper transparent surface and a lower opaque surface.

Figure 5 - axonometric view of a CD-based chip with microfluidic chambers; Figure 6 is a cross-sectional view of the CD-based chip of Figure 5;

Figure 7 is an axonometric view of a variant embodiment of a CD-based chip with microfluidic chambers;

Figure 7a is an axonometric view of the CD-based chip of Figure 7 with the sample positioned as the stepper motor rotates;

Figure 7b - axonometric view of a CD-based chip with filled microfluidic chambers for capturing the sample;

Figure 8 is a schematic front view of the device, with a liquid autofocus lens mounted in the lens;

Figure 9 - schematic diagram of a device according to the invention, with two additional liquid lenses, suitable for simultaneous scanning of a large sample volume of 10 µl, i.e. samples with < 10000 cells per ml and differential morphological measurement;

Figure 10a - exemplary embodiment of a device according to the invention, using a membrane-penetrating dye and applicable for counting live/dead cells in a sample;

Figure 10b - exemplary embodiment of a device according to the invention, using BO-PR03 and FITC dye and applicable for counting live/dead cells in a sample;

Figure 11 - an exemplary embodiment of a device applicable to the differential counting of somatic cells in milk, with one illuminator and a dichroic color-separating mirror in the objective;

Figure 12 - presents a general view of a "Hand Held" variant of a device according to the invention;

Figure 13 - schematically represents the device of Figure 12;

Figure 14a, b, c - presents a graphical representation of the results of the sample examination and their visualization on the display of the device;

Figure 15 - represents the 3D sequential capture plane;

Figure 16 - represents a variant embodiment of a device according to the invention, using a CD chip placed on a neodymium magnet;

Figure 17 - presents a variant implementation of the optical system of a device according to the invention, with three fluorescent thin-film filters;

Figure 18 - represents an illuminated field image of a water sample with fluorescein, without a rotating diffuser;

Figure 19 - represents an image of an illuminated field of a water sample with fluorescein, after passing laser light through a rotating diffuser;

Figure 20 - presents an image of an illuminated field of a water sample with fluorescein, after the passage of laser light.

List of positions

- hull

- lens

- illuminator

- microfluidic chip (CD based chip) ’ - rectangular microfluidic chip

- sample of biological material

- optical filter

- liquid lens (another 7', 7")

- alternating voltage generator

- light source

- collimating lens

- optical bandpass filter

- focusing lens

- upper transparent chip element

- bottom, opaque chip element

- microfluidic channels

- fluorescent dyes (more dry fluorine, dyes)

- mixing chamber

- hydrophobic channels

- microfluidic chambers

- additional opening

- rotating axis

- drive motor (drive stepper motor)

- microprocessor device

- dichroic mirror

- power module

- display

- externally based minicomputer

- mobile communication devices

- mini PC

- laser light emitter

- stepper motor

- a disc-shaped diffuser

- collimating aspheric lens

- filter stepper motor

- thin film fluorescent filters

- erythrocytes in the blood sample

- Neodymium magnet

Examples of implementation of the invention

An exemplary embodiment of the device according to the invention is presented in more detail, and the described design solution does not limit the use of other technical elements that have a similar structural design and equivalent functional impact, where the overall composition of the device will preserve the idea and spirit of the invention.

The device for differential counting of microparticles in biological fluids consists of a housing 1, in which a vertically oriented microscope with an objective 2 is located one below the other, on the side of which is mounted a system of two illuminators 3, located at an angle, above the plane in which it is located microfluidic chip 4 containing a sample 5 of biological material. The microfluidic chip 4 is implemented as a CD-based chip, and it is possible to use a rectangular chip 4'.

The lens 2 is filled with an optical filter 6, preferably a single-band thin-layer filter, and a liquid lens 7 located in its lower part, powered and controlled by an alternating voltage generator 8, preferably from 0-70 V and a frequency of 1 kHz. Through the built-in liquid lens 7 controlled by the generator 8, the lens 2 performs autofocus with a depth of 1 mm, and the autofocus time is very short, within milliseconds.

In a variant embodiment of the device, two liquid lenses 7', 7" (fig. 9) are additionally provided in the lens 2, located one under the other in the upper part of the lens 2. Such a design solution of the lens 2, with a total of three liquid lenses 7, 7', 7” provides the possibility of variably adjustable magnification and autofocus in the range of 1 to 10 times, for example 2 times magnification (Fig. 9) and 10 times magnification (Fig. 9) and is suitable to be applied in devices at counting and differentiating samples with low cell counts per ml.

As already stated above, the illuminators 3 are located laterally on the lens 2 and are oriented so as to provide an illumination angle providing minimal reflection of the excitation light, for example close to Brewster's angle. Each illuminator 3 consists of a sequentially located light source 9 type LED for example Luxeon and/or diode laser 30, Mitsubishi 658 nm collimating lens 10 and/or aspherical lens 33, optical bandpass filter 11 and focusing lens 12.

The described arrangement of the device allows the placement of more than one illuminator, and it is possible to alternatively use diode laser light sources, such as are necessary and suitable for multi-color fluorescence imaging, for example, for differential counting of biological microparticles, such as somatic cells in milk, leukocytes in the blood, body cells and yeast. The arrangement of the illuminator 3 above the plane in which the CD-based chip 4 is located ensures that a large part of the light is directed and falls through the upper transparent element 13 of the microfluidic CD chip 4 onto the sample 5 and only a very small part of that reflected from the surface of the upper element 13 for light to pass through the liquid lens 7, through the lens 2 and enter the imaging cameras 2a CCD or CMOS1 sensor.

In one variant embodiment of the device, it is appropriate to use a laser illuminator that is positioned at an angle to the sample under investigation. Laser illuminators can focus the light into much smaller spots, for example 100 µm, and collimate the light with a single aspherical lens 33 (Fig. 16) to the required diameter, for example 5 mm. The efficiency of the emitted laser light is greatly improved if it is provided to pass through a rotating diffuser 32 made in the form of a disc and located before or after the collimating aspherical lens 33 (Fig. 17/). It is preferable that the diffuser is made of Luminit PET film, on which a diffraction relief is formed, diffusing the passing light at a different angle from 2 to 100 ^o .

The diffuser is mounted on the axis of a miniature DC or stepper motor 31, for example a Nidec 2 phase 4 Wire micro stepper motor. The rotation speed is chosen according to the diameter of the diffuser and the exposure speed of the CMOS camera (2a) used to form the image, in this case a speed of 700 rpm is suitable. Figure 18 shows an image of the illuminated field of a fluorescein water sample without using a rotating diffuser, and Figure 19 shows an image of the same field after passing the laser light through a rotating diffuser.

The high homogeneity of the illuminance after passing the laser light through the rotating diffuser is clearly visible.

A CD-based microfluidic chip 4 is located under the illuminator 3, which contains a sample 5 with microparticles to be examined.

According to a preferred embodiment of the device, the chip 4 consists of two parts, respectively an upper element with a transparent surface 13, made of PMMA polymer, with an optically transparent surface and a lower element 14, preferably opaque, made of black ABS polymer. The two elements 13 and 14 are connected to each other by laser welding on the two surfaces, preferably with a laser wavelength of 1050 nm and an axially applied pressing force. The two elements 13 and 14 are shaped so that after they are connected by welding, microfluidic channels 15 with a height of up to 50 pm are formed in the space between the two elements, into which a premixed sample 5 containing fluorescent dyes 16, for example dry fluorescent dyes, is pipetted and/or fluorescently labeled antibodies. The described CD chip 4 is suitable for the analysis of various samples containing biological microparticles - milk, fermentable liquids, blood, semen, etc.

In another alternative embodiment of the device for differential counting, a chip 4 can be used, the design of which is presented in more detail in Figures 7, 7a, 7b. The chip 4 is a CD-based chip composed of two elements, respectively, with an upper surface 13 and a lower surface 14, and mixing chambers 17 containing dry fluorescent dyes, into which the sample 5 is pipetted, are formed between their surfaces. The mixing chambers 17 are connected by hydrophobic channels 18 with a microfluidic chamber 19 for capturing a sample of the sample 5. The microfluidic chamber 19 is filled with an additional hole 20 designed to release the air contained in the chamber 19 when it is filled with the liquid sample 5 pushed out by centrifugal forces.

The CD-based chip 4 is placed on a rotating axis 21 (Fig. 9) and is connected to a drive motor 22, preferably a stepper motor, the rotation of which moves the sample sample 5 in front of the lens surface 2. The stepper motor 22 is connected with a microprocessor device 23, in whose memory are entered data and coordinates determining the displacement of the sample 5, and the recorded image of the sample 5 is also recorded in it.

In a variant embodiment of the device for differential counting, a chip 4' (Fig. 4) can be used, with a rectangular shape, composed of two parts, respectively an upper transparent part 13' and a lower opaque part 14', and between their surfaces are formed microfluidic chambers containing dry fluorescent dyes 16 into which the sample 5 is pipetted.

The rectangular chip 4' is positioned and moved to image the sample via an XY table which is located below the illuminators 3 and the objective lens 2, in which the multi-band optical filter 6 and liquid lens 7 are mounted.

For the purpose of more precise enumeration, depending on the samples being processed, for example milk, it is sometimes necessary to initially separate the fat globules and somatic cells in the sample by centrifugation. For this purpose, it is suitable to use as a drive motor a miniature stepper motor, for example type P310 Disc Magnet High Speed Step Motor, whose technical characteristics define them as high-speed stepper motors, with a rotation speed of up to 20000 rpm, compared to conventional stepper motors that reach a rotation speed of up to 1000 rpm. The CD based chip 4 is mounted on the stepper motor rotor 22, with the opaque surface 14 on its upper side. Together with the protective opaque board, ambient light is isolated from the lens and thus the device can be implemented without a protective top cover. In such a configuration of the differential counting device, when analyzing milk samples, the sample is initially centrifuged at over 10000 rpm, resulting in the separation of fat globules and somatic cells, after which the stepper motor is switched to a lower speed , for example 200-300 rpm, where the sample is positioned in front of the objective to image the supernatant.

Figures 10a, 10b show an exemplary embodiment of a device for differential counting of cells in biological fluids, the construction of which corresponds to the composition of the device described so far, which is suitable to be used for counting live and dead cells in one sample of biological fluids containing cells. In the lens 2, a two-band thin-layer optical filter 6 is used, preferably Semrock FF01-527/645-25 type, and on the side of the lens 2, at an angle, there are two illuminators 3, with a LED LUXEON 475 nm light source (fig. 10a) and optical band filter 11, with parameters 470/30, as well as alternatively light source 9 type LED LUXEON 565 nm, with optical band filter 11, with parameters 560/30 (fig. 10b), for two-color dyeing.

In sample 5, membrane-penetrating dye 16 FITC staining DNA of live and dead cells 5a (Fig. 10a) 10 and BO-PR03 dye 16 DNA of dead cells only 5b (Fig. 10b) were used.

Figure 16 shows one embodiment of the device for differential counting of cells in a biological sample, for example a blood sample, and for the purpose of successfully enumerating malarial plasmodia with the fluorescence microscope, developing inside the red blood cells or attached to their surface, it is necessary to form a monolayer on the bottom of the microfluidic chip 4. Fixation of the red blood cells on the bottom is desirable especially for longer exposure times of the CMOS camera 2a. Acceleration of erythrocyte sedimentation 36 occurs by converting erythrocytes into paramagnetic cells. This is done by oxidizing their cellular oxyhemoglobin to methemoglobin with sodium nitrite or, when the cells are treated with isotonic sodium dithionite, the hemoglobin is converted to a deoxygenated reduced form and the cells become paramagnetic.

When the sample 5 in the mixing chamber 17 is blood, sodium nitrite or sodium dithionite is added, whereby the CD-based microfluidic chamber 4 with the diluted blood sample is placed on a neodymium magnet 37.

The cells are treated with sodium nitrite, the hemoglobin is oxidized to methemoglobin, and the cells become paramagnetic. The CD-based microfluidic chamber 4 with the diluted blood sample is placed on a neodymium magnet 37, which accelerates the sedimentation of the erythrocytes 36 to the bottom of the microfluidic chamber 4 and they form a monolayer. In normal red blood cells, hemoglobin is in an oxidized form, contains Fe ²⁺ ions, and the red cells are diamagnetic. Treatment of red blood cells is easily performed by mixing the blood sample with 5 mM NaNO2 solution at a ratio of 1:40 (v/v) to oxidize the hemoglobin in the red blood cells and convert it to a paramagnetic form. In this NaNO2 treatment, ferrous ions (Fe ²⁺ ) in hemoglobin are converted to ferrous (Fe ³⁺ ) ions. Paramagnetic cells can be precipitated by a magnetic field. Upon application of a high gradient magnetic field created by the neodymium magnet 37, erythrocytes from the upper layers of diluted 1:5 blood located at a maximum distance of 50 μm from the bottom surface of the microfluidic chamber made of transparent material 14 (PMMA) move at a speed of 3 -4 μm/s to the bottom of the chamber and form a monolayer. Reaching the bottom of the microfluidic chamber 14, the positively charged paramagnetic erythrocytes form an ionic bond with the negative surface charge of the polymer, as a result of which they remain fixed at the bottom of the microfluidic chamber even after removing the magnetic field.

Figure 11 shows an exemplary embodiment of a device which is suitable to be used for differential counting of somatic cells in milk and is particularly advantageous in cases where the cells are moved by Brownian motion in the liquid. The device is particularly configured for use in studies where the ratio of neutrophils to lymphocytes in milk or blood is determined. The lens 2 is provided with an optical filter 6 located in its upper part and a liquid lens 7 located in the lower part of the lens 2. Near the optical filter 6 and below it, in the axis of the lens 2, a dichroic color separator is mounted at an angle mirror 24, and a second camera 2a, for example a CCD or CMOS sensor, is arranged perpendicular to the lens axis 2. An illuminator 3 equipped with a light source with a wavelength of 675 nm, type LED LUXEON 675, is located on the side of the lens 2. In the CD chip 4, a fluorescent dye acredine orange is used, which stains DNA and RNA of living and dead cells 5a, using only one excitation wavelength and the emissions for DNA and RNA being at different wavelengths. When 5b cells from sample 5 are illuminated at a wavelength of 675 nm, the DNA-bound acredine orange dye in the nucleus has a green emission of 530 nm, and the RNA-bound RNA in the cytoplasm of 5b cells emits at at the wave 620 nm with red light.

Thus, with the device configuration described above, without the use of expensive movable mechanical devices, two images of the sample at different wavelengths can be taken simultaneously. Subsequently, the images are combined by the processing unit 23, and through the use of the software, on the basis of the intensity and the ratio of the green and red emission, cell differentiation is carried out.

Depending on the type of biological fluids examined, as well as the need for rapid and accurate examination, it is possible to realize a "Hand Held" variant of the differential counting device according to the invention, which is essentially an imaging cytometer, as shown in Figures 12 , 13. In the lower part of the housing 1, a power module 25 of the device is located, which is filled with battery power. In the housing 1 there is also a lens 2, type SUNEX-M12x0.5, and it is suitable to use a liquid lens type Arctic 16F, with a magnification of X4, which allows to configure a reversed lens 2, for a CCD or CMOS camera with autofocus. This lens is specially designed with the ability to collect light from significant angles, containing up to 6 aspherical glass miniature lenses, not shown in the figures, allowing to work with megapixel sensors, with large diagonals, resulting in the lens 2 having a spatial resolution ability around 2 pm, with minimal aberrations.

The differential counting device according to the invention can also be implemented with an improved optical system, presented in more detail in Figure 17. According to this alternative embodiment, instead of the conventional emission filter, three fluorescent, thin-film filters are mounted in series in the axis of the optical microscope 35', 35”, 35'”, preferably VersaChrome Edge™ Tunable Longpass Filters, each of the filters being coupled to a stepper motor 34, wherein the filters 35', 35”, 35”' can be rotated from 0 to 90 ^o . Replacing the emission filter with tunable filters allows to achieve a better adjustment of the passband, for example with a step of 2 nm. Thus, for example, it was found that when rotating from 0 to 60 ^o , the transmission band moves to the blue spectrum, and at an angle of 60 ^o , the transmission front is 12% smaller than that at 0 ^o . That is, at 0 ^o the emission filter will pass green light above 505 nm, and at 60 ^o blue light after 442 nm. Thus, by rotating the fluorescent thin-film filters 35' - 35'”, in the range from 0 to 60 ^o , the transmission band can be adjusted with a step of 2 nm.

The described handheld device (cytometer) can work in autonomous mode, with the autofocus provided by the liquid lens 7, the positioning, the control of the laser diode illuminator 30 Mitsubishi 658 nm, filled with an optical bandpass filter 6, aspherical collimating lens 33, Nidec 2 phase 4 wire micro srepper motor and are controlled by the microprocessor device 23, and after processing the images, the result is displayed on the display 26. The cytometer is also equipped with standard communication terminals (USB), not shown in the figures, (fig. 12), through which the device can be connected to an externally based minicomputer 27, with a specially developed program for managing the operation of the device, including graphic processing and visualization of the results of the analysis of the processed samples 5. Through the described interfaces, the device can be controlled remotely, through mobile communication devices (smartphone, tablet, based on Windows or Android) on which insta lira control program (Fig. 14 a, b, c,), with all information available online, in a cloud service. The software of the device allows processing of the images received from the CMOS or CCD cameras, in which the number of biological microparticles (somatic cells in milk, leukocytes in blood, body cells, red blood cells, and yeast) is determined - in ml, as well as an assessment of the size of the particles and their graphical distribution is carried out. Using the method of differential counting and size estimation, the number of local maxima in image intensity can also be determined, as well as image binarization through local histogram analysis. The latter is done in order to increase the signal-to-noise ratio by eliminating background irregularities throughout the image.

The differential counting device according to the invention is used as follows: the CD-based chip 4 is prepared, with a sample of the sample 5 to be tested. With the actuation of the stepper motor 22, the sample 5 is mixed with the fluorescent dyes, and when the revolutions of the stepper motor 22 are increased to 500-1000 rpm, a centrifugal force acts on the sample 5 mixed with the fluorescent dyes 16. As a result, the liquid sample 5 (Fig. 7a) overcomes the resistance of the hydrophobic channel 18 (Fig. 7a) and falls into the microfluidic chamber 19 (Fig. 7b), where the air displaced by the displacement of the sample 5 is released through the additional hole 20. Sequentially, by rotating the stepper motor 22, the sample 5 is moved in front of the lens 2, according to precisely defined coordinates previously entered into the memory of the microprocessor device 23, and the position for capturing the image of the sample 5 is not shifted or deviated relative to the lens 2.

In this case, the distance of the microparticles from the sample 5 to the lens 2 depends to a large extent on the manufacturing accuracy of the microfluidic chamber 19, as a result of which the requirements for the autofocus system are significantly reduced. At the same time, the signal-to-noise ratio, crucial for the detection and software enumeration of faintly luminescent microparticles from the sample, is dramatically increased. For the purpose of accurate enumeration of the microparticles in a sub-region, more than one image is taken, selecting one of them that is best focused and enumeration is performed on this selected image, after which displacement and similar acquisition of the next sub-area, and the images selected for counting are saved to the device's memory.

As already stated, particle counting from single captured images of consecutive fields of a sample moved by an XY or rotating axis in front of the lens axis is performed under the conditions of a significantly more accurate way of scanning a sample moved by an XY table or moved by a rotating axis in front of the lens axis, with subsequent enumeration and analysis of a sample containing microparticles, which we set out in a sequence: the introduction of a high-speed liquid lens in the main optical axis allows the sample contained in the microfluidic channels of CD-based microfluidic chambers or conventional rectangular microfluidic chambers to be placed in the carrier of the device performing the displacement, representing an XY displacement or a directly coupled axis of a stepper motor. In this position, according to predetermined coordinates in front of the axis of the lens 2, positioning of the first analyzed field of the sample 5 is carried out, during which a series of photos is taken, using one emission illuminator, for example, 5 photos above the preset during the calibration of the device image plane (Fig. 15) and 5 pictures below the expected image plane, and it is recommended to choose a distance between the planes of 0.05 mm, thus scanning the Z-axis image at equidistant intervals (Fig. 15).

The resulting photos are processed by the microprocessor device, selecting one photo in which the microparticles are "in focus". The particles are then enumerated and/or analyzed morphologically. If the analysis reveals that the image(s) is compromised, for example by air bubbles occupying a large part of the image, the imaged field is excluded from subsequent counting or analysis, and the result is recorded in the device's memory.

Simultaneously, when using two fluorescent dyes with different emission wavelengths, for example FITC-530 and TO-PRO3-660 nm, first a series of 5 pictures is taken with the LED emitter at 470 nm or, laser diode emitter 470 nm, the focused particle image with 530 nm emission is located and is memorized for subsequent enumeration.

The same is then done with a 630 nm LED emitter, at the end of which two field images of the sample are obtained and stored in the processing memory. After the capture of said pictures is completed, the moving mechanism moves the sample to the next capture field, the procedure being repeated until the end of the preset movement sequence for scanning the sample. After the scan is completed, the average number of microparticles in the sample is calculated using a formula, for example the ISO 13366 formula, by dividing the sum of the number of particles in the individual fields of the sample by the number of fields enumerated and calculated per 1 ml, for example by counting somatic cells in milk.

Application of the invention

An alternative mobile embodiment of the cytometer is suitable for use by farmers directly on the farm, for example for the purpose of detecting preclinical mastitis, by counting somatic cells in milk.

Another application of the mobile version of the device is in telemedicine, in particular in emergency medical care, when it is necessary to count and differentiate leukocytes in the blood on the spot, at the patient. Due to the low cost, battery power and the possibility of use by unqualified personnel, it is particularly convenient for application in countries where a large number of research and diagnosis of diseases, such as AIDS, are carried out. Such an apparatus is especially necessary for use in field conditions, as it can scan a blood sample of up to 10 μΐ, and in such blood tests, leukocytes decrease to 500 pcs/ml. The results can be monitored remotely by a specialist located anywhere on earth, with the ability to quickly and accurately determine a diagnosis. The device can also be used in blood collection centers, where blood is usually collected first and then sent for testing in centralized laboratories. When taking 20 μΐ. blood from the donor's finger, the sample can be tested on the spot, thus immediately assessing whether a patient is suitable as a blood donor.

Claims (16)

Patent claims 1. A device for differential counting of microparticles in biological fluids, consisting of a housing in which a lens is located, under which a chip with microfluidic cameras is placed, in which a sample of biological fluid to be examined is placed, capturing CCD or CMOS a camera, a counting part and a mechanism for axial movement of the sample, in which an illuminator is directed to the sample, in particle counting or in differential counting, characterized in that an optical filter (6) and the most a small liquid lens (7), and the chip (4) is implemented as a CD-based chip, with a circular shape, on which microfluidic chambers (19) are formed, located on the periphery of the chip (4), which is directly connected to a drive motor (22), wherein the illuminator (3) is at least one and is located above the plane in which the CD-based chip (4) is located, being oriented at an angle, and the counting part is implemented as a microprocessor (23), connected to a generator (8) controlling liquid those lenses (7). 2. Device according to claim 1, characterized in that two liquid lenses (7, 7') are located one under the other in the upper part of the objective (2), and one liquid lens is located in the lower end of the objective (2) (7), where between the upper liquid lenses (7', 7”) and the lower liquid lens (7) the optical filter (6) and the lens (2) are mounted. 3. Device according to claim 1, characterized in that the optical filter (6) is a multi-band filter. 4. Device according to claim 1, characterized in that the liquid lens (7) is powered by a generator (8) with a variable step voltage of 0-70 V and a frequency of 1 kHz. 5. A device according to claim 1, characterized in that the illuminator (3) consists of a light source (9), a collimating lens (10) and an optical bandpass filter (11) and a focusing lens (12) located in series with each other . 6. Device according to claim 5, characterized in that the light source (9) is an LED or diode laser light source. 7. Device according to claim 6, characterized in that the light source is a laser whose emitted light is directed to a rotating diffuser (32) mounted on the axis of a stepping motor (31) and made in the form of a disk with surface diffraction relief , wherein the diffuser (32) is located before or after the collimating aspherical lens (33). 8. Device according to claim 7, characterized in that the diffuser is made of Luminit PET foil, and the diffraction relief diffuses the passing light at an angle of 2 - 100 ^o . 9. Device according to claim 1, characterized in that the CD-based chip (4) consists of an upper element (13) made of PMMA polymer with an optically transparent surface and a lower opaque element (14) made of black ABS polymer, and the two elements (13) and (14) are shaped in such a way that microfluidic channels (15) with a height of 50 μm are formed in the space between the two elements, in which a mixture of sample (5) and fluorescent dyes ( 16). 10. Device according to claim 1, characterized in that a rectangular chip (4') is used, with a rectangular shape, composed of two parts, respectively an upper transparent part (13') and a lower opaque part (14'), as between their surfaces are shaped chambers containing dry fluorescent dyes (16) in which a sample of biological fluid (5) is placed, whereby the rectangular chip (4') is placed on a movable XY table located below the illuminators (3) and the objective ( 2), in which a multi-band optical filter (6) and a liquid lens (7) are mounted. 11. Device according to claim 1, characterized in that a rectangular chip (4') is used, with a rectangular shape, composed of two parts, respectively an upper transparent part (13') and a lower opaque part (14'), as between their surfaces are shaped chambers containing dry fluorescent dyes (16) into which the sample (5) is pipetted, whereby the rectangular chip (4') is placed on a movable XY table located below the illuminators (3) and the objective (2), in which three fluorescent thin-film filters (35', 35”, 35'”) are mounted in series, each connected to a stepper motor (34) and a liquid lens (7). 12. Device according to claim 1, characterized in that a CD-based chip (4) is used, composed of two parts, respectively an upper transparent part (13') and a lower opaque part (14'), and between their surfaces are formed chambers containing dry fluorescent dyes (16) into which the sample (5) is pipetted, wherein a CD-based chip (4') is placed on a stepper motor located below or above the illuminators (3) and the objective (2) in which three fluorescent thin-film filters (35', 35”, 35”') are mounted in series, each connected to a stepper motor (34) and a liquid lens (7). 13. Device according to claim 1, characterized in that the CD-based chip (4) consists of two parts, respectively with an upper transparent surface (13) and a lower part with an opaque surface (14), such that the two elements (13) and (14) are shaped so that after joining them by welding, with a laser wavelength of 800-1050 nm, mixing chambers (17) filled with sample (5) and dry fluorescent dyes (16) are formed in the space between the two elements ), wherein the mixing chambers (17) are connected via hydrophobic channels (18) to a microfluidic chamber 19, and the CD-based chip (4) is mounted to a rotating axis (21) connected to a drive motor (22). 14. Device according to claim 9, characterized in that the sample in the CD-based microfluidic chamber (4) is placed on a neodymium magnet (37). Device according to claim 1 and 4, characterized in that the drive motor is a stepper motor (22) connected directly to the CD-based chip (4). Device according to claims 1 and 4, characterized in that the drive motor is a high-speed disc stepper motor (22), the CD-based chip (4) being mounted on the rotor of the stepper motor (22), with the opaque surfaces (14). on its upper side.