WO2021081607A1 - Dispositif pour le comptage différentiel de microparticules dans des liquides biologiques - Google Patents
Dispositif pour le comptage différentiel de microparticules dans des liquides biologiques Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N35/00069—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1429—Signal processing
- G01N15/1433—Signal processing using image recognition
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N2035/00168—Manufacturing or preparing test elements
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
Definitions
- the current invention refers to a device for counting of biological cells, suspended in a liquid, which can be used as a fluorescent automated system for cell counting and can find application at counting and differentiating of micro particles in biological liquids such as milk, blood, urine, including differentiating of cells from other particles, and different types of cells in raw milk samples.
- the number of 3 ⁇ 4 the bacterial (BCC) and the number of somatic cells (SCC) are from a very high significance because they have big influence on the quality of milk products, since along with this they also give an actual information for the health condition of the 15 cows.
- the total number of somatic cells is a recognized indicator for the health of the cows and the quality of the milk.
- the somatic cells are white blood cells, known as leucocytes (lymphocytes, granulocytes, monocytes and macrophages) and they originate from the udder of the animal, and their number 20 depends on the cellular immune defense. The number of these cells may increase as a response of bacterial infections, which can cause mastitis. For this reason, the counting of somatic cells, the differential counting and identification of leucocytes in milk is very important for the mastitis diagnosis.
- SCC is an indicator for the presence of infection and inflammation of the mammary gland of milk cows, 25 called mastitis.
- the above mentioned flow automated analyzers are based on a monochrome fluorescence and determine only the total number of the cells. With them the flow sample passes through a very small diameter, which allows the passing only of one single cell and implementing the counting (Gunasekera T. S. etc. 2003, Feng W. etc. 2004).
- fluorescent dyes ethidium bromide, propidium iodide and DAPI (Gonzalo B. et AL 2004, Wallen CA et al 1982).
- the samples are taken from individual animals in farms and are sent to centralized laboratories, equipped with flow cytometers for counting the total number of somatic cells.
- a disadvantage of the described device is the insufficient precision of micro particle’s counting due to the lack of technical possibility of focusing the image, resulting in an error during XY stage moving, which is further increased by the deformation of the plastic microfluidic chips, wherein said errors accumulate as the error on the Z axis, which often exceeds 100 micrometers along the Z axis.
- the position of the chip is being moved to preliminary determined coordinates, through a preliminary determined period of time, so a separate field of the chip, which is situated close to the captured area, could be moved to the point where the light falls from the source of light and is in the range of the lens’ field of vision.
- the separate sub- areas in the chip are captured once, consecutively, as the number of micro particles in each sub-area is being calculated and then summed, so the total number of micro particles in a sample to be calculated.
- Another disadvantage of counting by using known devices and applying the methods described above is related to the type of fluorescent dyes used.
- two or more fluorescent dyes of different emission e.g., FITC-530 nm for the first dye and Propidium Iodide -620nm for the second dye
- FITC-530 nm for the first dye
- Propidium Iodide -620nm for the second dye
- the particles luminous at 530 nm wavelength are in focus
- those luminous at wavelengths 620 nm are not focused due to chromatic aberrations in the lens, and as a result, enumeration of the cells in most of the single-frame images of successive fields of the sample will be compromised for counting.
- the known device for counting of micro particles from the patent US7411680 (CN 100520374 C) has several constructive disadvantages, which do not allow to obtain an accurate measurement and counting of the micro particles because of the following: even at a very precise embodiment of the mechanisms for moving of the chip, the surface of the XY stage has a deviation from the lens over IOmhi, which along with the technical incurvation of the microfluidic chip determines a deviation, which usually exceeds 50 pm. Such deviation from the plane of the lens leads to an image, which is not focused and affects the software counting of the micro particles, which lowers the precision significantly. In the commented device there is no provided mechanism for focusing of the image.
- the illumination of the sample is of special importance for the activation of the fluorochromes in the sample.
- US 7411680B2 US6970246 B2, US20110211058 are shown different methods of illumination of the sample with lamps or LED
- US 2015/0053872 A1 LED illuminator with increased power by multiplying several LED crystals.
- a method for achieving uniform illumination of the object by placing two arrays of micro lenses and a polarization filter between them.
- Such illuminators have a disadvantage that it is very difficult to focus light on the object in a stain less than 4mm. In the case of higher lens increases, e.g.
- the observed field is less than 1 mm and most of the light is not used to activate the fluorochromes in the sample.
- Laser illuminators can focus the light in much smaller spots, e.g. 100 pm, and collims the beam with a single aspherical lens to the required diameter, e.g. 5 mm. Thus, all the energy of the light source can be used to activate the fluorochromatic dye in the sight of the fluorescent microscope.
- the large selection of wavelengths and capacities of modem diode lasers allows choosing the most suitable wavelength for the selected fluoropchrome.
- Laser diodes have a very narrow radiation strip, only a few nanometers, which allow to be used without expensive, thin-layer absorption filter, thus achieving additional fall in price of the illuminator in comparison with the LED systems.
- Laser illuminators also have one big disadvantage, hindering their use as illuminators in the imaging fluorescent microscopic systems, namely "speckle pattern" effect.
- the light stain is not homogeneous on the sample plane. When enlarged, a light and dark spot is noted, with uneven illumination resulting in reduced emission of objects or their parts falling into low-light spots.
- microfluidic camera which is transparent for the length of the excitation illuminator must be used.
- the use of microfluidic cameras, provided with two transparent surfaces significantly increases the value of the consumable, because expensive, complicated and unreliable bonding methods are used for bonding of the above mentioned transparent surfaces, usually by gluing with liquid or dry adhesives as described in patent publications US 20040145805, US20120223260, US 7842157 B2, and US8808642.
- Another disadvantage of the known device for counting of micro particles is also connected with the already described mutual disposal of the elements and their appearance as well. For example, when a part of the emitted light falls in the plane of the lens with an angle of 90 degrees, respectively it’s falling through the filter of CCD or CMOS matrix, the signal-noise ratio dramatically falls. Having in mind the fact that some of the fluorescent dyes, as for example the widespread Propidium Iodide, have a high own fluorescence in a solution, this predetermines a bad quality of capturing and respectively makes it difficult to make further software processing of the image.
- the known differential-count devices are also used in the analysis and counting of samples, which requires precise enumeration of malarial plasmodiis developing inside the red blood cells or glued to their surface when a fluorescent microscope is used in the device. In such cases, there is a need of fixation of red blood cells, especially in large times of exposure to the CMOS camera.
- a patent publication is known as US 10093957B2, in which a method of forming a monolayer of erythrocytes at the bottom of a microfluidic chamber is described by gravity sedimentation of erythrocytes at a rate of 1 pm /s. This precipitation rate is not sufficient especially in microfluidic chambers with a thickness of more than 100 pm and leads to a delay in counting in a high number of samples.
- Another task of the invention is to propose a device, which is technological, made from cheap and available elements, which mutual disposal allows an optimization of the qualities of the optical system in regard to the autofocusing and magnification of the image, which is of a great significance during the capturing, counting and analyzing of biological liquids of different types and composition and also to use a chip, the moving of which guarantees an exact positioning of the model of the sample relative to the lens and cameras for capturing.
- a device for a differential counting of micro particles in biological liquids which consists of housing in which are situated: lens, under which is located a chip with microfluidic cameras, in which there is a biological liquid as a subject for examination, a capturing CCD or CMOS camera, a counting part and mechanism for moving the sample, wherein there is an illuminator, directed to the sample.
- the chip is performed as a CD based chip, with a circular shape, on which microfluidic cameras are formed, located on the periphery of the chip, which is directly connected to a drive motor, at which the illuminator is at least one and is located above the plane, in which is located the CD based chip, being oriented as to provide an angle for illuminating with a minimal reflection, and the counting part is performed as a microprocessor, connected to a generator, which controls the liquid lens.
- the optical system includes three fluorescent thin-film filters, arranged consecutively one after another in the axis of the optical microscope, each connected to Stepper motor, ensuring rotation of fluorescent thin film filters of 0-90 degrees.
- two liquid lens are located one under another at the top of the lens, and at the bottom of the lens one liquid lens is located, wherein an optical filter and a lens of two achromatic lenses are fitted between the upper liquid lens and the lower liquid lens.
- the optical filter is single-lane or multi-lane optical filter.
- the liquid lens is connected to a generator with a variable stepwise voltage from 0-70 V and a frequency of 1kHz.
- the illuminator consists of consecutively located one in front of another source of light, collimating lens, tape optical filter, and focusing lens.
- a LED is used as a light source, for example Luxeon or a diode laser source of light.
- a laser illuminator located at an angle of the sample, is used.
- a rotating diffuser in the shape of a disc, located on the path of the coherent laser radiation, as the diffuser is located before or after the collimating aspherical lens.
- the CD based chip consists of upper part, performed from a PMMA polymer, with an optically transparent surface, and a lower, nontransparent part, performed from a black ABS polymer.
- the both parts are formed and connected by laser welding in such a way that 50 pm height microfluidic channels 15 are formed in the space between them, in which a mixture of sample, fluorescent dyes or fluorescently labeled antibodies is placed.
- the device can also work efficiently when using a chip with a rectangular shape, consisting of 2 parts, respectively upper transparent part and lower nontransparent part.
- the two parts are formed and connected by laser welding so that after coupling in the space between them 50pm cameras are formed in which a sample mixture, fluorescent dyes or fluorescently labeled antibodies are pipetted.
- the rectangular chip is placed on a moving XY stage, located under the illuminators and the lens, in which the multi-lane optical filter and the liquid lens are mounted.
- the CD based chip consists of two parts, respectively upper part with a transparent surface and lower part with nontransparent surface whereby mixing cameras are formed between the surfaces of the mentioned two parts.
- the mentioned mixing cameras are filled with a test sample and dry fluorescent dyes and/or fluorescently labeled antibodies wherein the mixing cameras are connected by hydrophobic channels to the microfluidic camera, while the CD based chip is mounted on a rotating axis connected to a drive motor .
- the drive motor is preferably a stepper motor and is directly connected to the CD-based chip.
- the CD-based chip sodium nitrite or sodium dithionite is added in the blood test sample, in which the CD-based Microfluid camera 4 with the diluted blood sample is placed on neodymium Magnet 37 (Fig. 16).
- the device for a differential counting of micro particles in biological liquids is characterized by a compact design, as the mutual disposal of the elements allows an accurate measurement and analysis of the micro particles in the processed samples. Elements with high quality and low value are being used, and as a result of this there is a device, which provides technical abilities for obtaining quality images while capturing and sample processing.
- liquid lenses with small sizes, which are located in the lens of the microscope in a way in which they ensure focusing and magnification of the image at the same time. Meanwhile the control of the liquid lens allows the time for autofocus to be in the range of milliseconds.
- the size of the liquid lens also allows it to be embedded in the construction of mobile and hand held devices for measurement of small particles.
- the proposed device uses a principally new and different model of the chip with CD based microfluidic cameras, which positioning in front of the lens for capturing of an image depends only on the accuracy of processing of the microfluidic camera, which in turn automatically leads to decreasing of the requirements towards the autofocusing system.
- the positioning of the microfluidic camera in front of the lens is significantly simplified - only by rotation of the CD based chip, as only in a few cases is needed a minimal moving on the X axis, which can be made with available technical means, for example: mini stepping motor NEMA8 for a rotating axis and mini-servo motor DS-5001HV on the axis X, as in this way the moving of the X axis can assure moving up to 10mm.
- the conditions are created to achieve a regulated omission in the range from 440 nm to 660 nm, with step 2 nm.
- Such discreet adjustment of the fluorescent emission’s stripe allows precise adjustment of the signal-to-noise ratio of the cell image in a multi-colour fluorescent marking system with different emitted in-length fluorochroms and several LED or laser emitters with different wavelengths, which is more contrasting compared to using the non-optimal emission filter for the selected fluoropchrome. Contrast image facilitates the software analysis of fluorescent images.
- the performance of the differential counting device is further improved by the use of a laser illuminator at an angle to the sample. A high homogeneity of illumination shall be ensured at the correct rotation speed of the rotating diffuser.
- Fig.2- a schematic diagram of the device according to the invention with a partial view of the CD based chip with microfluidic cameras;
- Fig.3- scheme of the device with a partial view of the CD based chip with mixing and microfluidic cameras
- Fig.4- scheme of the device according to the invention with a rectangular chip, with upper transparent surface and lower nontransparent surface.
- Fig.6- a cross-section of the CD based chip from Fig.5;
- Fig.7b axonometric view of the CD based chip with provided microfluidic cameras for capturing the sample
- Fig.9- a schematic diagram of the device according to the invention, with two additional liquid lenses, appropriate for a simultaneous scanning of a large sample volume of 10 m ⁇ , i.e. samples with ⁇ 10000 cells per ml and a differential morphological measurement;
- Fig.10a- exemplary embodiment of the device according to the invention using a membrane penetrating dye and applicable for counting of alive/dead cells in a single sample
- Fig.10b- exemplary embodiment of the device according to the invention using BO-PR03 and FITC dye and applicable for counting of alive/dead cells in a single sample
- Fig.ll- exemplary embodiment of the device applicable for a differential counting of somatic cells in milk, with one illuminator and a dichroic color reflection mirror in the lens;
- Fig.12 - presents general appearance of the “Hand Held” version of the device according to the invention
- Fig.13 - presents schematically the device from Fig.12;
- Fig.14a, b, c presents graphical representation of the results from the analysis of the sample and their visualization on the display of the device.
- Fig. 15 presents the plane for 3D consecutively capturing.
- Fig. 16 presents the variant of a device according to the invention using a CD chip affixed to the neodymium magnet.
- Fig 17 - Presents the variant of the optical system of a device according to the invention, with three fluorescent thin film filters
- Fig. 18 - presents an image of a lighted field on a water sample with fluorescein, without rotating diffuser
- Fig 19- presents an image of a lighted field on a water sample with Fluorescein after passing the laser light through a rotating diffuser
- Fig. 20 presents an image of a lighted field on a water sample with Fluorescein after passing the laser light
- the device for differential counting of micro particles in biological liquids consists of housing 1, in which are located, one under another, a vertically oriented microscope with lens 2, on the side of which is being mounted a system of two illuminators 3, located at angle, above the plane in which the microfluidic chip 4, containing sample 5 from biological material is located.
- the microfluidic chip 4 is a CD based chip, as it is possible a rectangular chip 4’ to be used also.
- the lens 2 is provided with an optical filter 6, preferably single-lane thin- layer filter and a liquid lens, located in its lower part 7, powered and controlled by a generator with variable voltage 8, preferably from 0-70 V and frequency 1 kHz.
- a generator with variable voltage 8 preferably from 0-70 V and frequency 1 kHz.
- the lens 2 is provided with two additional liquid lenses 7’, 7” (Fig.9), located one under another in the upper part of the lens 2.
- Such a constructive decision for the lens 2, with a total of 3 liquid lenses 7, 7’, 7” ensures a possibility for a variable, adjustable magnification and an autofocus in the range from 1 to 10 times, for example 2 times magnification (Fig.9) and 10 times magnification (Fig.9) and is appropriate to be applied in devices when counting and differentiating of samples with small number of cells in ml.
- the illuminators 3 are located on the side of the lens 2 and are oriented in a way they can ensure an angle for illuminating, which provides a minimal reflection of the excitation light, for example proximal to the angle of Houseer.
- Each illuminator 3 consists of consecutively located sources of light 9, type LED, for example Luxeon, and/or diode laser 30, Mitsubishi 658 nm collimating lens 10, and/or aspheric lens 33, optical tape filter 11 and focusing lens 12.
- the described structure of the device allows the placement of more than one illuminator, as alternatively it is also possible diode laser sources of light to be used as this is needed and is suitable for multicolor fluorescence capturing, for example in the differential counting of biological micro particles such as somatic cells in milk, leucocytes in the blood, body cells and yeasts.
- a laser illuminator which is positioned at an angle to the test sample. Laser illuminators can focus the light on smaller spots, e.g. 100 pm, and collims the light with a single Aspherical lens 33 (Fig.
- the diffuser is made of PET foil Luminit, on which are formed diffracted relef, diffusing the passing light at different angles from 2 to 100 degrees.
- the diffuser is mounted on the axis of miniature DC or stepper motor 31, e.g. NIDEC 2 Phase 4 Wire Micro stepper motor.
- the rotational speed is selected according to the diameter of the diffuser and the exposure speed of the CMOS camera (2a), which is used to form the image, in this case the appropriate speed is 700 rev/min.
- Fig. 18 An image of the illuminated field of a water sample with fluorescein without the use of a rotating diffuser is shown, and in Fig. 19 is shown an image of the same field after passing the laser light through a rotating diffuser.
- the high homogeneity of illumination is clearly visible after passing the laser light through the rotating diffuser.
- a CD based microfluidic chip 4 which contains a sample 5 with micro particles to be examined.
- the chip 4 consists of two parts, respectively upper part 13, provided from PMMA polymer, with an optically transparent surface, and a lower part 14, preferably nontransparent, provided from a black ABS polymer. Both parts 13 and 14 are connected with a laser welding of their surfaces, preferably with a wavelength of the laser 1050 nm and axially applied compressive force. The both parts 13 and 14 are formed in such a way that after welding together, microfluidic channels 15 of up to 50 pm are formed in the space between the two elements and a pre-mixed sample 5 containing fluorescent dyes 16, for example dry fluorescent dyes and/or fluorescently labeled antibodies are pipetted.
- fluorescent dyes for example dry fluorescent dyes and/or fluorescently labeled antibodies are pipetted.
- the described CD chip 4 is appropriate for analysis of different samples, containing biological micro particles — milk, fermentable liquids, blood, sperm etc.
- a chip 4’ (Fig.4), with a rectangular form, consisting of two parts, respectively upper transparent part 13’ and lower nontransparent part 14’, interconnected by laser welding, as between their surfaces are formed microfluidic cameras, which contain dry fluorescent dyes 16, in which the sample 5 is being pipetted.
- the rectangular chip 4’ is situated and is being moved for capturing of the sample through XY stage, which is located under the illuminators 3 and the lens 2, in which are mounted a multi-lane optical filter 6 and a liquid lens 7.
- a chip 4 which constructive embodiment is presented in details on Fig. 7, 7a, 7b.
- the chip 4 is a CD based chip, consisting of two parts, respectively upper 13 and lower 14, as between their surfaces are formed mixing cameras 17, which contain dry fluorescent dyes, in which the sample 5 is being pipetted.
- the mixing cameras 17 are connected by hydrophobic channels 18 with a microfluidic camera 19 for capturing a part of the sample 5.
- the microfluidic camera 19 is with an additional opening 20, designed to release the air contained in the camera 19, when it is provided with the pressurized by the centrifugal force liquid sample 5.
- the CD based chip 4 is placed on a rotating axis 21 and is connected with a driver motor 22, preferably a stepper motor, and when it rotates the sample 5 moves in front of the lens 2 plane.
- the stepper motor 22 is connected with a microprocessor device 23, in which memory are entered data and coordinates, which determine the moving of the sample 5, as in it is being saved the captured image of the sample 5.
- a drive to be used a miniature stepper motor, e.g.
- CD- based Chip 4 is mounted on the rotor of the stepper motor 22, with nontransparent surface 14 on its upper side. Together with the protective nontransparent board, the ambient light is insulated from the lens and thus the device can be executed without a top protection cover.
- the differential counting device in the case of a milk sample analysis, initially the sample is centrifugated by more than 10000 rev/min, resulting in a separated fat globules and somatic cells, after which the stepper motor switches to a lower speed, e.g. 200-300 rpm, where the sample is positioned in front of the lens to capture the supernatant.
- a lower speed e.g. 200-300 rpm
- FIG. 10a 10b is presented an exemplary embodiment of a device for differential counting of cells in biological liquids, which construction corresponds to the so far described structure of the device, which is appropriate to be used for counting of alive and dead cells in a single sample of biological liquids, containing cells.
- a tape filter 11 with parameters 470/30, and also an alternative light source 9 type LED LUXEON 565 nm, with an optical tape filter 11, with parameters 560/30 (Fig.lOb), for a bicolor dyeing.
- a membrane penetration dye 16 FITC which dyes DNA of alive and dead cells 5a (Fig.lOa) and 10 BO-PR03 dye 16 DNA only of dead cells 5b (Fig. 10b).
- Fig. 16 is presented one embodiment of the differential cell counting device in a biological sample such as a blood sample, for the purpose of successful enumeration with the fluorescent microscope of the malarial plasmodias developing inside the red blood cells or attached on their surface, it is necessary to form a monolayer at the bottom of the micro fluid chip 4.
- the fixation of the red blood cells at the bottom is desirable especially in the case of large exposure times of the CMOS camera 2a. Acceleration of erythrocyte sedimentation 36 is carried out by converting erythrocytes into paramagnetic cells.
- the cells are treated with sodium nitrite, the hemoglobin is oxidized to methaemoglobin and the cells become paragnetic.
- the CD-based microfluidic camera 4 with the diluted blood sample is put on neodymium magnet 37, which accelerates the erythrocyte sedimentation 36 to the bottom of the microfluidic camera 4 and they form a monolayer.
- neodymium magnet 37 which accelerates the erythrocyte sedimentation 36 to the bottom of the microfluidic camera 4 and they form a monolayer.
- hemoglobin In normal red blood cells, hemoglobin is in oxidized form, contains Fe 2+ ions and red cells are diamagnetic.
- red blood cells The treatment of red blood cells is carried out easily, with the blood sample mixed with a 5 mm solution of NaN0 2 in a ratio of 1 : 40 (V/V) to oxidize hemoglobin in red blood cells and convert it into a paramagnetic form.
- NaN0 2 Ferro-ions (Fe 2+ ) in hemoglobin are converted into ferry (Fe 3+ ) ions.
- Paramagnetic cells can be acceleratedly precipitated by a magnetic field.
- erythrocytes from the upper layers of diluted 1:5 blood located at a maximum of 50 pm from the lower surface of the microfluidic camera, made of transparent material 14 (PMMA) are moved at a speed of 3-4 pm/sec to the bottom of the camera and form a monolayer.
- PMMA transparent material 14
- Fig. 11 is presented an example embodiment of a device, which is appropriate to be used for a differential counting of somatic cells in milk and is very useful in the cases in which the cells are moving meanwhile from the Brown movement in the liquid.
- the device is configured especially for the use of analysis, in which the ratio between neutrophils and lymphocytes in milk or blood is being determined.
- the lens 2 is provided with one optical filter 6, located in its upper part, and one liquid lens 7, located in the lower part of the lens 2. Near to the optical filter 6 and under it, in the axis of the lens 2 is being mounted at angle a dichroic color separating mirror 24 and perpendicularly to the axis of the lens 2 is located the second camera 2a, for example CCD or CMOS sensor. On the side of the lens 2 is located one illuminator 3, provided with a light source with a wavelength of 675 nm, type LED LUXEON 675.
- a fluorescent dye acridine orange which dyes the DNA and RNA of the alive and dead cells 5 a, using only one excitation wavelength, and DNA and RNA emissions are of different wavelengths.
- the connected with DNA in the core “acredine orange” dye has a green emission 530 nm, and the connected with RNA in the cytoplasm of the cells emits with a wavelength of 620 nm - with a red light.
- a “Hand held” variant of the device for differential counting which is essentially an image cytometer, like it is shown on Fig. 12, 13.
- a power unit 25 of the device which is powered by a battery.
- a lens 2 type SUNEX-M12xO,5, as it is appropriate to be used a liquid lens type Arctic 16F, with a magnification of X4, which allows to be configured a reversed lens 2, for CCD or CMOS camera with an autofocus.
- This lens is specially designed with an ability to collect light from significantly big angles, as it contains up to 6 aspherical glass miniature lenses, not shown in the figures, which allows working with megapixel sensors with large diagonals, in a result of which the lens 2 has a spatial resolution capability around 2 pm with minimal aberrations.
- the differential counting device can also be performed with an improved optical system, in more detail presented in Fig. 17.
- an improved optical system in more detail presented in Fig. 17.
- in place of the conventional emission filter in the axis of the optical microscope are consecutively mounted three fluorescent, thin-film filter 35 ',35 ',35 ', preferably VersaChrome Edge TypeTM Tunable Longpass Filters, each filter is connected with a stepper motor 34, where the filters 35 ’,35 ',35 ' can be rotated from 0 to 90 degrees.
- Replacing the emission filter with adjustable filters allows better regulation of the pass-though band, for example with step 2 nm.
- the range of the pass-through band moves to the blue spectrum and at an angle of 60 degrees the pass-through front is 12% smaller than at 0 degrees. That is, at 0 degrees, the emission filter will pass the green light above 505 nm, and at 60 degrees the blue light after 442 nm.
- the fluorescent thin film filters 35 '-35 1 ' in the range from 0 to 60 degrees can be adjusted with step 2 nm.
- the described Hand held device can work in an autonomous mode, as the autofocusing, which is being provided through the liquid lens 7, the positioning, the control of the laser diode illuminator 30 Mitsubishi 658 nm, provided with an optical tape filter and aspheric collimating lens 33, Nidec 2 phase 4 wire micro stepper motor are controlled by the microprocessor device 23, as after the processing of the images the result is shown on a display 26.
- the cytometer is also provided with standard communication outputs (USB), not shown on the figures, (Fig.12), through which the device can be connected with an external based minicomputer 27, with a specially developed program for work control of the device, including the graphical processing and visualization of the results from the analysis of the processed samples 5.
- the device can be controlled remotely, via mobile communication devices (smart phone, tablet, based on Windows or Android), in which has been installed a controlling program (Fig. 14 a, b, c), as the whole information is available online in the cloud service.
- the software of the device allows a processing of the images, obtained from the capturing cameras CMOS or CCD, at which is being determined the number of biological micro particles (somatic cells in milk, leucocytes in the blood, body cells, red blood cells and yeasts) - in m ⁇ and is also made an assessment of the particles size and their graphical distribution.
- Using the method of the differential counting and assessment of the size can also be determined the number of local maximum of the image intensity through a local histogram analysis. The last is being done with the aim of increasing the signal/noise ratio through eliminating the background irregularities across the image.
- the device for differential counting according to the invention is used in the following way: the CD based chip 4 with the sample, which is to be analyzed 5, is being prepared. With the start of the stepper motor 22, the sample 5 is being mixed with the fluorescent dyes, as when increasing the speed of the stepper motor 22 to 500-1000 rpm, a centrifugal force is being applied on the mixed with the fluorescent dyes 16 sample 5. As a result, the liquid sample 5 /Fig. 7 a/ overcomes the resistance of the hydrophobic channel 18 (Fig. 7a) and goes into the microfluidic camera 19 (Fig. 7b), thereby through the additional opening 20 the air displaced by the movement of the sample 5 is released.
- the sample 5 is moving in front of the lens 2 at exactly determined coordinates, preliminary entered into the memory of the microprocessor device 23, as the position for capturing of the image of the sample 5 is not moving or deviating relatively to the lens 2.
- the distance between the micro particles from the sample 5 to the lens 2 to a large extent depends on the precision of making the microfluidic camera 19, in a result of which the requirements towards the autofocusing system are significantly decreased.
- the ratio between signal and noise which is critical for the detection and software counting of the low illuminating micro particles of the sample, is increased significantly.
- the counting of particles from single- captured images of the consecutive fields of the sample, moved by XY or a rotating axis in front of the lens axis is carried out at the conditions of a significantly more accurate way of scanning the sample, moved by the XY stage or moved by a rotating axis in front of the lens axis, with a subsequent enumeration and analyzing of a sample, containing micro particles, which we expose in the following sequence:
- the introduction of a high-velocity liquid lens into the main optical axis allows the sample contained in the microfluidic channels of CD based microfluidic chambers or conventional rectangular microfluidic cameras to be placed in the carrier of the movement device, being XY stage or directly coupled axis of a stepping motor.
- a positioning of the first analyzed field from the sample 5, at which series of images are being captured with the use of one emission illuminator, for example 5 images over the preliminary set during the calibration of the device plane where the image is (fig. 15) and 5 images under the expected plane of the image as it is recommended to choose a distance of 0.05 mm between the planes, as in this way the image on the axis Z is being scanned at equal intervals (fig. 15).
- the obtained images are being processed by a micro processing device, as one image on which the micro particles are focused (fig. 15) position 1 is being chosen. After that the particles are being counted and/or morphologically analyzed. If during the analysis it becomes clear that the image is being compromised for example by air bubbles, which occupy a very big part of the image, the captured field is excluded from the next counting or analysis and the result is saved in the memory of the device.
- FITC-530 and TO-PR03 -660 nm is firstly made a sequence of 5 images with the LED emitter at 470 nm, or laser diode emitter 470 nm, then the image with the focused particles with emission of 530 nm is found and saved for further counting. After that the same is being done with a LED emitter at 630 nm, as at the end of this stage are obtained two images from the sample, which are being saved in the memory for processing. After finishing the capturing of the mentioned images the moving mechanism moves the sample to the next field for capturing, as the procedure is repeated until the end of the preliminary set sequence of moving for scanning of the sample.
- the average number of micro particles in the sample is being calculated by a formulae, for example the formulae, referred to in ISO 13366, as the sum of the number of particles in the separate fields of the sample is divided by the number of counted fields and is calculated for 1 ml, for example at counting of somatic cells in milk.
- One version mobile embodiment of the cytometer is appropriate to be used by farmers directly in the farm, for example for the aims of finding sub-clinical mastitis by counting the somatic cells in milk.
- Another application of the mobile version of the device is in the telemedicine, especially in the emergency medical care, when it is necessary at place, at the patient, to be counted and differentiated the leucocytes in the blood. Thanks to the low price, battery power and the ability to be used by an unqualified staff, it is really convenient for an application in the countries, where a great amount of examinations and diagnosing diseases such as AIDS are being made.
- Such device is really necessary for use in field conditions, as far as it can scan a blood sample up to lOjpl, as in such examinations of blood the leucocytes decrease to 500 per ml.
- the results can be observed remotely by a specialist, who can be anywhere on Earth, with a possibility for a fast and accurate determination of the diagnosis.
- the device can also find application in the blood collection centers, where usually firstly the blood is being collected and then is being sent for examination in centralized laboratories.
- the sample When taking 20 m ⁇ of blood from the finger of the donor, the sample may be examined at place, and respectively can be immediately examined in order to assess whether the patient is suitable for a blood donor.
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Abstract
L'invention concerne un dispositif pour le comptage différentiel de cellules biologiques, en suspension dans un liquide, qui peut être utilisé comme un système de comptage de cellules automatisé fluorescent et peut être appliqué dans le comptage et la différenciation de microparticules dans des liquides biologiques tels que le lait, le sang, l'urine, comprenant la différenciation de cellules d'autres particules, ainsi que de différents types de cellules dans des échantillons de lait cru. Le dispositif est constitué d'un boîtier, dans lequel une lentille est située au-dessous de laquelle une puce ayant des caméras microfluidiques est logée, dans lequel un échantillon de liquide biologique à tester est placé, une caméra CCD ou CMOS, une partie de comptage et un mécanisme de déplacement axial pour l'échantillon est positionné, au niveau duquel un illuminateur est dirigé vers l'échantillon et au moins une lentille liquide (7) est située dans la lentille (2). La puce (4) est exécuté comme une puce à base de CD, ayant une forme ronde, sur laquelle sont formées des caméras microfluidiques (19), situées sur la périphérie de la puce (4) reliée au moteur (22), dans lequel l'illuminateur (3) est au moins un et est positionné au niveau d'un angle, et la partie de comptage est exécutée comme un microprocesseur (23), connecté à un générateur (8), commandant les lentilles liquides (7).
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BG113017A BG67480B1 (bg) | 2019-10-30 | 2019-10-30 | Устройство за диференциално броене на микрочастици в биологични течности |
BG113017 | 2019-10-30 |
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Cited By (3)
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CN113340894A (zh) * | 2021-05-28 | 2021-09-03 | 上海睿钰生物科技有限公司 | 一种非透明微粒的检测方法 |
CN114859443A (zh) * | 2022-04-24 | 2022-08-05 | 武汉大学 | 基于声学及微流控技术的液体可调微透镜阵列 |
CN116046647A (zh) * | 2023-01-28 | 2023-05-02 | 深圳安侣医学科技有限公司 | 血液成像分析系统和方法 |
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