RU2569053C2 - Scanning cytometer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of examination and analysis of biological materials and deals with method for counting biological objects in sample and scanning cytometer on its basis. To realise counting of biological objects sample is placed in rectangular capillary, made with possibility of reflecting fluorescence signal from upper surface of rectangular capillary. Additional collection of fluorescence signal is provided by means of parabolic reflector, in focus of which analysed zone of capillary is placed, and replacement of analysed zones with biological objects is realised by moving capillary along its axis. Detected light flow is divided by wavelength and directed on first and second photomultipliers, output signals of which are proportional to light flow and fluorescence signal. From photomultipliers output signals are transmitted to amplitude and phase transformers for simultaneous processing and following multiplication of arrays of data, obtained from outlets of amplitude and phase transformers of signals in microprocessor.

EFFECT: technical result consists in increase of accuracy and simplification of measurement method, as well as in reduction of dimensions and increase of device mobility.

13 cl, 4 dwg

Description

Technical field

The present invention relates to the field of scanning cytometers using fluorescence detection from objects that are irradiated with intense and modulated light. The device can be used in medicine, environmental control and life support facilities, in space research.

State of the art

One of the most common cell counting methods currently used is flow cytometry (PC). The advantages of the PC include the possibility of studying a large number of cells (over 100,000) during one analysis, high sensitivity, which allows the detection of bacterial cells in an amount of 10-100 in 1 ml of liquid and ensuring a high counting rate.

Flow cytometers are known for use in medical and biological applications. The most widely represented are cytometers, which are based on the detection of fluorescence from fluorochromes associated with measurement objects.

One of the main disadvantages of the PC method is the complexity and high cost of its technical implementation. The apparatus for the PC includes a measuring cell in the form of a capillary with a cross section of 100-300 μm, a special design to ensure a laminar flow of fluid flowing through the cell, a system for supplying a sample to the apparatus, a dosing system for a fluorescent dye, a pump to ensure a constant speed of fluid movement, a washing system measuring path that prevents cross-contamination - infection of the measured samples with previous samples, an optical system with a laser, receiver, photomultiplier, optical filters.

US Pat. No. 5,270,548 describes a flow cytometer using a phase detector to count in a stream of cells labeled with fluorescent inks with overlapping emission spectra but with different fluorescence lifetimes. However, due to the design features of flow cytometers, at each instant of time, the light source illuminates the region in which no more than one cell can be located, and the phase detector captures the signal from one cell [1].

U.S. Patent Application No. US20130327957 describes a microjet type flow cytometer. The rectangular channel inside the removable polymer transparent chip is illuminated by a laser beam. The relative position of the chip and the laser beam is mechanically adjusted to achieve a better signal to noise ratio. The cytometer has two radiation detectors, one of which receives noise, and the other receives a useful fluorescence signal [2].

Known US patent US 5009503, which uses a microscope with a motorized two-coordinate table for counting microorganisms, on which there are cylindrical glass capillaries with an agarized nutrient medium with microbial cells or colonies. The capillaries are illuminated by a laser beam that is precisely guided by a system of moving mirrors along the axis of the capillary. When the table moves, the lens automatically focuses. A sensor or photomultiplier sequentially detects the light reflected from each cell. This system is notable for its complexity, high cost, low scanning speed, long and laborious sample preparation. The use of a cylindrical capillary complicates the optical scheme [3].

In the international application No. WO 9702482 for counting leukocyte cells stained with a fluorescent dye interlinked to DNA or RNA, the prepared sample is placed in a glass or acrylic capillary of rectangular cross section with an internal cross section of 200-400 μm by 1-3 mm and a length that precisely provides the necessary sample volume. The sample is illuminated with a focused laser beam, the capillary is scanned in two directions, and fluorescence from individual cells is successively recorded using a photomultiplier [4].

The disadvantage of the described method and device is the scanning of the capillary in two directions, as well as the need for its accurate positioning in relation to the lens. This design feature, as well as the use of a laser, makes the device more expensive.

A variety of electronic circuits are known for detecting fluorescence radiation using the amplitude method. For example, in a flow cytometer described in Japanese Patent No. JPH 046440, light from a moving colored particle falling into the laser-illuminated region is detected several times in succession, each time in a new photodetector cell made in the form of a CCD line. Writing to cells is clocked from the generator. The total charge accumulated in the photodetector is recorded as a signal from one cell, and a special circuit ensures that the signal from different cells is not mixed [5].

Known flow cytometer described in US patent No. 8415627, which is designed to analyze a wide range of objects, such as proteins, cells, DNA, RNA or enzymes [6]. The device can simultaneously operate at several frequencies of the exciting signal and distinguish simultaneously two or more different biological substances. The excitation circuit of a light source, which is used as a laser, can have several radiation sources with different frequencies. In addition, the laser control circuit has three oscillators, one of which is a clock, the other oscillator determines the modulation frequency, and the third oscillator generates a reference signal to determine the fluorescence relaxation time. In this case, the modulation signal and the reference signal are synchronized with each other. The signal processing unit extracts information about the phase delay of fluorescence and receives a signal that is obtained from the side scattered light. Moreover, such processing is carried out in different signal processing channels on different wave ranges. To increase the accuracy of determining the fluorescence relaxation time, the frequency of the modulation signal differs from the frequency of the reference signal, and the analyzing device additionally contains devices for converting the real and imaginary parts of the signal, and the analysis requires digitization of these data and their storage in the analyzing device. A complex signal conversion scheme is caused by the task of simultaneously identifying several types of biological objects.

There is a need to develop simpler portable compact cytometers for the tasks of quickly detecting one of different types of biological objects in the environment or in places where people are located, such as public places (hospitals, metro, sports facilities, shops, etc.). For this purpose, portable cytometers with low current consumption, simplicity of sample preparation, small dimensions and weight and with the ability to transfer data via the Internet or mobile communications with databases, which include the results of scanning multiple objects from each other, can be used more effectively. in order to determine the degree of infection of water bodies, or air, or residential and other premises. Small dimensions and weight, simplicity and structural strength and the use of a capillary in the device allow it to be used in biological research in space. To create small-sized scanning cytometers, it is imperative to use new technical solutions to improve the accuracy and reproducibility of measurements.

The technical task of this invention is to expand the arsenal of cytometers with high accuracy, small dimensions, high mobility and ease of obtaining results to obtain a large data array in the study region.

Another technical problem is to increase the signal-to-noise ratio, which leads to an increase in the dynamic range and resolution of the device.

SUMMARY OF THE INVENTION

One of the aspects of the invention is a method for counting biological objects in a sample, which comprises: preliminary selection of samples with biological objects, mixing with a fluorescent dye and incubation, illumination of the working area with the studied object to form a fluorescent glow of the studied objects, separation of light fluxes, their detection, processing and analysis of the number of detected biological particles. The solution with biological objects is placed in a rectangular capillary configured to reflect the fluorescence signal from the upper surface of the rectangular capillary. An additional collection of the fluorescence signal is provided using a parabolic reflector, in the focus of which the studied area of the capillary is set. The change of the studied zones with biological objects is carried out by moving the capillary along its axis. The detected light flux is separated by the wavelength and sent to the first and second photomultipliers, the output signals of which are proportional to the incident light flux and the fluorescence signal. The signals are fed to the amplitude and phase converters for the simultaneous processing and subsequent multiplication of the data arrays obtained from the amplitude and phase signal converters. The data are pre-collected, digitized and stored in the microprocessor memory, while before starting the main processing algorithm, a calibration is carried out at which the signal from individual biological objects is recognized and the interval is recorded at which individual objects are recorded, and the number of objects is determined as the sum of signals in the interval.

Another aspect of the invention relates to a device for measuring the number of biological objects in a sample. The device comprises: a light flux forming unit, which includes a light source and optical elements for forming a narrow light flux, a capillary attachment unit with an object to be studied, a detection unit, an electronic signal conversion unit, a microprocessor, a display, a modulator. At the same time, the capillary attachment unit contains a parabolic reflector equipped with a rectangular cutout, into which a rectangular capillary with a test sample is mounted, with the possibility of moving the working zones of the capillary relative to the light flux using a stepper motor. The upper surface of the capillary has a mirror coating that reflects the fluorescence signal at the input of the detection unit. The detection unit contains an optical part, which includes the first and second beam splitters, band-pass optical filters, the first and second photomultipliers, the outputs of which are connected to the inputs of the electronic node. The electronic unit contains amplitude and phase converters and the first and second ADCs, while the inputs of the amplitude and phase converters are connected to the outputs of the first and second photomultipliers, the outputs of the converters are connected to the inputs of the first and second ADCs, the outputs of which are connected to the microprocessor. The microprocessor provides the ability to process the data array, control the modulator and display data.

Brief Description of the Drawings

Figure 1. Structural diagram of the formation of the light flux and its transmission to the capillary mount.

Figure 2. Section view of a parabolic reflector and capillary.

Figure 3. Structural diagram explaining the separation of the light flux.

Figure 4. The block diagram of the electronic unit and its connection with the microprocessor, display, modulator and capillary movement unit.

Description of the invention

In the considered method of detecting biological objects, such as microorganisms, cells, viruses, proteins, enzymes, RNA, DNA, the accuracy of measuring the number of objects is increased due to the synergistic effect of hardware and software solutions. At the same time, capillaries are used at the hardware level with the possibility of amplifying the fluorescence signal by at least 1.5-2 times. At the software level, the signals from the amplitude and phase converters are processed and the signal-to-noise ratio is increased by multiplying the data arrays obtained from the amplitude and phase converters previously collected, digitized, and stored in the microprocessor memory.

The steps of the method include the following steps. A biological sample, for example, containing microbial cells, is preliminarily taken into a test tube and, after the addition of a fluorescent dye, the solution is mixed and incubated. A part of the incubation solution is taken into the capillary, which is configured to reflect the fluorescent signal, and set it on the displacement unit with the possibility of sequential illumination of the working areas of the capillary. To illuminate the capillary, a narrow light beam is formed, for which the light flux from the light source is focused by a collimator lens into a parallel beam and passed through a multilayer band-pass optical filter. Then, using a slit diaphragm, a narrow beam is formed, incident on the capillary with the sample, causing a fluorescent glow of dye-stained cells. In this case, fluorescence radiation is collected at the input window of the second photomultiplier, while the fluorescence signal is formed due to the synergistic action of the reflecting surface located on the upper side of the surface of the rectangular capillary and the reflecting surface of the parabolic mirror.

A parallel light beam at the output of a parabolic mirror is focused by collimator lenses into a parallel light beam and fed through two beam splitters to two photomultipliers. In this case, the light incident on the first photomultiplier is preliminarily transmitted through a selective beam splitter made in the form of a flat mirror, and then the light passes through a band-pass optical filter that transmits only the light flux from the light source and does not pass the fluorescent glow, which causes the appearance of the first photomultiplier amplitude signal proportional to the luminous flux (hereinafter reference signal). Additionally, a second beam splitter and a second optical filter are used, passing only the fluorescence to the input of the second photomultiplier, from the output of which a useful signal about the fluorescence of biological objects is removed.

The useful signal and the reference signal from the outputs of the photomultipliers are fed to the inputs of the amplitude and phase signal converters and, through analog-to-digital converters, the data are transferred to the microprocessor memory, in which the signals from the amplitude and phase converters are processed at the software level. At the same time, the signal-to-noise ratio is increased due to the multiplication of data arrays obtained from the amplitude and phase converters.

A device for measuring the number of biological objects in a sample, made to implement the method, contains:

a light flux forming unit, which includes: a light source 1, a collimator lens 2, a first band-pass optical filter 3, a slit diaphragm 4, a cylindrical lens 5;

a capillary mounting unit 26, which comprises: a parabolic reflector 7 provided with a rectangular cutout 8, into which a capillary 6 with a sample is mounted, an object stage 21 for moving the capillary 6 using a stepper motor 22;

detection unit, which contains: the optical part, which includes the first and second beam splitters, made in the form of flat mirrors 10 and 13, band-pass optical filters 11 and 14, the first and second photomultipliers 9 and 12;

an electronic node that contains: amplitude 15 and phase 16 channels, the first and second ADCs 17 and 18, a microprocessor 19, a display 20, a modulator 23.

The device operates as follows.

In a test tube with a volume of at least 100 μl containing microbial cells in an amount of 10 ¹ to 10 ⁸ per ml, a fluorescent dye, for example, SYTO 9 from Invitrogen, attached to nucleic acids, is added. The mixture is stirred manually or in a mixer and incubated for 1-30 min at a temperature of 15 to 25 ° C, then part of a sample of liquid with a dye is taken into a capillary in an amount of 20-200 μl for subsequent scanning of a capillary with a sample taken. For scanning, a narrow luminous flux is formed and the fluorescence intensity is recorded and the number of microbial cells is calculated by the formula.

To form the luminous flux, the source light from a lamp, LED or laser with a wavelength of 200 to 780 nm is used. The luminous flux is focused by a collimator lens 2 into a parallel beam with a diameter of 1-3 mm, and then the luminous flux is passed through an optical filter 3, then a narrow beam is formed using a slit diaphragm 4 with a width of 5-20 μm, which is then further focused to compensate for the diffraction expansion of the cylindrical beam lens 5 with a focal length of 1 to 3 mm.

A narrow beam of light falls on a rectangular capillary with sample 6 made of quartz or silicate glass with a hole height of 1 mm to 1.5 mm and a width of 0.5 mm to 1 mm, a length of 30-150 mm and a wall thickness of 0, 1 mm to 1 mm. The luminous flux of the source causes the luminescence of biological particles or cells in the sample stained with fluorescent dye. The capillary is in focus of the parabolic reflector 7 with a rectangular cutout 8, designed to install the capillary inside the reflector.

To increase the amount of fluorescence light collected, the upper wall of the capillary has a mirror coating 25, for example, in the form of an aluminum film deposited by vacuum deposition.

A parallel light beam at the output of a parabolic reflector with a diameter of 7-20 mm is focused by collimator lenses 27 into a parallel light beam with a diameter of 1-5 mm at the input windows of two photomultipliers.

The light incident on the first photomultiplier 9 is preliminarily transmitted through a selective beam splitter in the form of a flat mirror 10 located at an angle of 45 degrees to the direction of the beam, and a band-pass optical filter 11, which transmits only the source’s luminescence in the range from 200 to 780 nm and does not transmit fluorescence , and causes the output of the first photomultiplier reference signal.

The light incident on the second photomultiplier 12 is preliminarily transmitted through two beam splitters made in the form of flat mirrors 10 and 13, and a band-pass optical filter 14, which transmits only fluorescence in the range from 390 to 810 nm and does not transmit light, causes an output to appear a second photomultiplier of the useful fluorescence signal.

The reference and useful signals from the outputs of the first and second photomultipliers 9 and 12 are fed to the inputs of the electronic node.

Increasing the resolution allows to reduce the likelihood of errors in cell counting, for example, while registering in the range from 1 to 10 cells. To implement the technical task, the electronic circuit of the device has an amplitude 15 and phase 16 signal converters.

The inputs of the amplitude converter receive signals from the outputs of the first and second photomultipliers 9 and 12. The amplitude converter generates a signal proportional to the ratio of the useful fluorescence signal and the reference signal.

The phase converter generates a signal proportional to the phase difference of the modulated fluorescence signals and the reference signal.

The operation of the phase Converter occurs as follows [7]. The reference and useful signals from the outputs of the first and second photomultipliers 9 and 12, which are multiplied, are fed to the input, and then the resulting signal is passed through a low-pass filter. A constant component proportional to the phase shift of the input signals remains at the detector output. With a sinusoidal shape of the reference and useful signals, the operation of the phase detector can be illustrated as follows:

It can be seen that the output signal of the phase detector is independent of the modulation frequency of the reference signal. If there is no phase shift between the reference and the useful signal, the output signal is 0. If there is a phase shift between the signals at the detector input, the output signal has a constant value proportional to this shift.

However, the output signal also depends on the amplitude of the reference signal, which leads to increased noise at the output of the phase detector. For example, a random increase in voltage at the light source will lead to an increase in the reference and useful signals, since this increases the brightness of the light source and the illumination of stained cells, which in turn causes an increase in the fluorescence of the cells. The amplitudes of the reference and useful signals will increase and multiplicatively increase the output signal of the phase detector.

The presence of an amplitude converter, at the output of which a signal is generated proportional to the ratio of the reference and useful signals, allows you to compensate for noise in the reference channel. To do this, the operation algorithm of the device provides for the operation of multiplying data arrays from the amplitude and phase channels previously collected, digitized and stored in the microprocessor's memory. It can be seen from the formulas that in this case, not only does the dependence of the output signal on the reference signal disappear, but its dependence on the amplitude of the useful signal also quadratically increases. All this leads to an increase in the signal-to-noise ratio and the resolution of the detector, which allows more accurate calculation of the number of simultaneously detected cells:

The light from the source 1 has amplitude modulation, which is provided by the modulator 23, under the control of the microprocessor 19. The modulation frequency is from 3 to 30 MHz. The modulation coefficient is from 0.1 to 0.7. The fluorescence glow also acquires amplitude modulation. The fluorescent glow does not occur immediately after the light enters the fluorochrome, but with some delay (for SYTO 9, the delay is about 6 ns), so the phase of the registration signal lags behind the phase of the illumination signal. Thus, the appearance of fluorescence leads to a change in the signals at the outputs of the amplitude and phase converters.

The signals of the amplitude and phase converter are digitized using two ADCs 17 and 18 and fed to the inputs of the microprocessor 19. The results of the processing of the data array are displayed on the display 20.

The capillary with the test sample is fixed on a single-coordinate table 21, which is driven by a stepper motor 22. Thus, the capillary is progressively moved in increments of 0.5-5 μm in front of the slit diaphragm 4, as a result of which the beam from the source sequentially scans the entire working area of the capillary 6.

The determination of the number of cells is performed according to the following algorithm.

и

. После полного прохождения капилляра в памяти микропроцессора формируются одномерные массивы сигналов

и

длиной k. Затем происходит поэлементное перемножение этих массивов и формируется массив U_вых. По данным этого массива осуществляется калибровка, которая заключается в распознавании сигналов от единичных клеток. При этом фиксируется интервал, на котором происходит регистрация единичной клетки (от j до j+m), и определяется сумма сигналов на этом интервале (e).The coordinate table is set to the extreme position, then the translational movement of the table is carried out with the selected step and number of steps (n), and at each step (i), fluorescence is measured, while the microprocessor is recorded in the memory

and

. After complete passage of the capillary, one-dimensional arrays of signals are formed in the microprocessor memory

and

length k. Then there is an elementwise multiplication of these arrays and an array of U _{out is formed} . According to the data of this array, calibration is carried out, which consists in the recognition of signals from single cells. In this case, the interval at which a single cell is registered (from j to j + m) is fixed, and the sum of the signals in this interval (e) is determined.

Calibration is successful if the sample contains no more than 10 ⁴ -10 ⁵ CFU / ml. If the calibration is successful, then the number of cells is determined by the following formula:

If the sample contains a large concentration of cells, the device determines the required degree of dilution of the sample and displays the appropriate message about it, after which the sample must be diluted the appropriate number of times and run the device again with the diluted sample.

Industrial applicability

The scanning cytometer is easy to maintain, has small dimensions and weight.

Literature

1. STEINKAMP JOHN A. Phase-sensitive flow cytometer. US patent No. US 5270548 (1993-12-14).

2. AYLIFFE HAROLD E. FLUORESCENCE FLOW CYTOMETRY US patent applic. No. US 2013327957 (2013-12-12).

3. MURPHY JR MARTIN J, STUBBS JACK B. Automated capillary scanning system. US 5009503 (1991-04-23).

4. WOO SAM L BAER THOMAS M. VOLUMETRIC CELL QUANTIFICATION METHOD AND SYSTEM, PCT appl No. WO9702482 (1997-01-23).

6. YAMAZAKIISAO, OKI HIROSHI PARTICLE ANALYZER. JP Patent No. JPH 046440 (1992-01-10).

6. DOI KYOUJI, NAKADA SHIGEYUKI FLUORESCENCE DETECTION DEVICE USING INTENSITY-MODULATED LASER LIGHT AND FLUORESCENCE DETECTION METHOD. US Patent No. 8415627 (2013-04-09).

7. I.N. Sinitsyn, P.V. Gorbunov THE EXPERIENCE IN THE DEVELOPING SUBOPTIMAL ALGORITHM OF ANALYSIS, MODELING AND INTERPRETING SIGNALS IN OUTER SCANNING ANALYZER OF MICROORGANISMS. The 11th International Conference on Pattern Recognition and Image Analysis: New Information Technologies (PRIA-11-2013). Samara, September 23-28, 2013. Conference Proceedings (Vol.I-II), Volume II, Samara: IPSI RAS, 2013.410 p. (377-786 pp.).

Claims (13)

1. A method for counting biological objects in a sample, containing preliminary selection of samples with biological objects, mixing with a fluorescent dye and incubation, illumination of the working area with the studied object to form a fluorescent glow of the studied objects, separation of light fluxes, their detection, processing and analysis of the number of detected biological particles, characterized in that the solution with biological objects is placed in a rectangular capillary configured to reflect the fluo signal escence from the upper surface of a rectangular capillary, where an additional collection of the fluorescence signal is provided using a parabolic reflector, the focus of which sets the studied zone of the capillary, while changing the studied zones with biological objects is carried out by moving the capillary along its axis, while the detected light flux is divided along wavelength and sent to the first and second photomultipliers, the output signals of which are proportional to the luminous flux and the fluorescence signal, and on amplitude and phase converters for simultaneous processing and subsequent multiplication of data arrays obtained from amplitude and phase signal converters, while the data is pre-collected, digitized and stored in the microprocessor memory, while before starting the main processing algorithm, a calibration is performed in which the signal from single biological objects and fix the interval at which registration of single objects takes place, and the number of objects is determined as the sum of signals n interval. 2. The method according to p. 1, characterized in that the biological objects are included in the group consisting of microorganisms, cells, viruses, proteins, enzymes, RNA, DNA. 3. The method according to p. 1, characterized in that the incubation is carried out at a temperature of from 15 to 25 ° C for 1-30 minutes 4. The method according to p. 1, characterized in that the volume of the capillary is selected in the range from 20 to 200 μl. 5. A device for measuring the number of biological objects in a sample, comprising a light flux forming unit, which includes a light source and optical elements for forming a narrow light flux, a capillary attachment unit with an object to be studied, a detection unit, an electronic signal conversion unit, a microprocessor, a display, a modulator, characterized in that the capillary mount includes a parabolic reflector equipped with a rectangular cutout, into which a rectangular capillary is installed with the second sample, with the possibility of moving the working area of the capillary relative to the light flux using a stepper motor, where the upper surface of the capillary has a mirror coating that additionally reflects the fluorescence signal at the input of the detection unit, the detection unit contains an optical part, which includes the first and second beam splitters, band-pass optical filters, the first and second photomultipliers, the outputs of which are connected to the inputs of the electronic node, which contains the amplitude and phase converters and the first and the second ADC, while the inputs of the amplitude and phase converters are connected to the outputs of the first and second photomultipliers, the outputs of the phase and amplitude converters are connected to the inputs of the first and second ADCs, the outputs of which are connected to a microprocessor that provides the ability to process the data array, control the modulator and output data to display. 6. The device according to p. 5, characterized in that the capillary is made of quartz or silicate glass with a hole height of 1 mm to 1.5 mm, a width of 0.5 mm to 1 mm, a length of 30-150 mm. 7. The device according to p. 6, characterized in that the capillary is made with a wall thickness of from 0.1 mm to 1 mm 8. The device according to p. 5, characterized in that the first and second beam splitters are made in the form of flat mirrors. 9. The device according to p. 5, characterized in that the first band-pass optical filter operates in the range from 200 to 780 nm. 10. The device according to p. 5, characterized in that the second band-pass optical filter passes in the range from 390 to 810 nm, depending on the type of fluorescent dye. 11. The device according to p. 5, characterized in that the parabolic reflector has a diameter of from 7 to 20 mm 12. The device according to p. 1, characterized in that the light source works with a wavelength of from 200 to 780 nm. 13. The device according to p. 1, characterized in that the modulation frequency is from 3 to 30 MHz, and the modulation coefficient from 0.1 to 0.7.

Images