SU1716401A1 - Microobject analyzer in liquid canal and method of its tuning - Google Patents

Abstract

The invention relates to biophysical instrumentation, allows for the implementation of a statistical analysis of cell populations by flow cytometry and can be used in biology, biotechnology, and medicine. The purpose of the invention is to expand the class of objects under study. To do this, from each micro-object in the zone of measurement of the flow of the suspension, fluorescence signals are recorded in the reverse direction of the rays through a high-aperture lens micro-lens 6 with a central screen

Description

The invention relates to technical physics and biophysical instrumentation, aimed at creating a flow cytometry method for solving scientific problems and applied problems in various areas of biology, biotechnology, and medicine, in particular, can be used in the early diagnosis of diseases, screening various substances, and studying cell proliferation. and the effects on the body of various physico-chemical factors.

A known analyzer of micro-objects in a fluid channel, containing an optical system for illuminating and collecting a fluorescence flux of micro-objects with a field diaphragm and a high-aperture lens micro-lens, as well as a system for transferring a suspension of micro-objects, made in the form of a chamber with a horizontal transparent glass in which there is a nozzle with a hydrodynamic focus, is set. horizontal directions or at an angle to the optical axis and connected by a hose to the capacity of the buffer liquid, and the feed channel with spenzii microscopic mounted on the center of the nozzle hydrodynamic focusing and associated with the vessel for the slurry of microscopic.

A known method of controlling these analyzers involves forming a measurement zone when illuminated through a high-aperture lens micro lens, forming a suspension duct in the measurement zone when it is hydrodynamically focused using a buffer liquid, and then recording the fluorescence signals of the micro-objects in the reverse path of the rays through the micro lens.

The disadvantages of the known analyzers of micro-objects in the fluid flow and, accordingly, the methods of their control are, firstly, the inability to

control over the size of each micro-object under study, for example, by recording light scattering signals in small angles; secondly, there is no possibility of orientation of the measurement zone relative to the flow channel of the suspension and tunable modes of hydrodynamic focusing of the flow channel are not possible, which would allow setting parameters such as the measurement area and the dimensions of the duct section in it.

Closest to the invention is a micro-object analyzer in a fluid flow, containing a first optical

a system of dark-field illumination and fluorescence flux collection of micro-objects with a field diaphragm and a high-aperture lens micro-lens with central shielding, a second optical system for collecting streams of light scattered forward by micro-objects, and a system for transferring the suspension of micro-objects to a transparent glass with a suspension supply channel connected to the vessel for suspension of micro-objects and installed in the center of the nozzle of hydrodynamic focusing, which is connected with a hose to the capacity of buffer fluid,

The control method of this analyzer is to form a measurement zone under dark field illumination by centrally shielding a high-aperture lens micro-lens, forming a slurry channel in the zone

measuring with its hydrodynamic focusing using a buffer liquid, minimizing the background scattered radiation level with the subsequent recording of the fluorescence signals of the micro-objects in the reverse path of the rays through the micro-lens and recording the signals of the forward-scattered light by the micro-objects through the optical system.

The main disadvantages of this analyzer and its control method are: the complexity of the fluid transfer system, which requires special devices to form both suspension and buffer fluid under pressure from the ducts, and this makes it difficult to accurately set the pressure difference on the flow of these fluids to ensure constant flow rate micro-objects with respect to the measurement zone, the laminar duct and adjustment of the dimensions of the cross section of the duct suspension in the measurement zone, i.e. all those parameters, the fulfillment of which is extremely necessary for maximum system resolution and ensuring the minimum coefficient of peak variation in the histograms of the distributions of the microobjects under study; the lack of orientation of the measurement zone relative to the flow of the suspension and the complexity of the system for recording the signals of light scattered forward by micro-objects as a result of the use of a two-step

imaging the measurement zone, which caused the presence of a large number of optical elements and a second recording light sensor; lack of free access to the suspension during the experiment; the possibility of sedimentation of micro-objects in suspension during the measurement process when it is supplied under pressure.

These drawbacks are due to the fact that the liquid transfer system is of a closed type under pressure, and the measurement zone is located in the free space between the micro-lens and the transparent glass, on the outer surface of which a suspension duct is applied. In addition, the well-known analyzer does not provide for the rotation of the field diaphragm around the optical axis, and also an optical system with a photomultiplier tube based on a telescope tuned to an image at infinity is used to collect light scattering streams.

The purpose of the invention is to expand the class of objects under study and simplify the analyzer.

The drawing shows a diagram of the proposed analyzer.

The analyzer of micro-objects in the fluid channel contains the first optical system 1 for dark-field illumination and collection of the fluorescence flux of micro-objects, which has a field diaphragm 2 with its means of moving 3 and turning 4, a beam-splitting plate 5 and a high-aperture lens micro objective 6 with central screening 7. For collection

0 streams of light scattered forward by micro-objects, the second optical system 8 with an opaque screen 9 and a monofilament light guide 10 with means of light protection 11 and displacement 12 are inserted into the analyzer. The analyzer also contains a camera 13 of a system for transferring a suspension of micro objects with vertical front 14 and rear 15 walls having two holes opposite each other along the axis

0 first 1 and second 8 optical systems. The opening of the front wall 14 is covered with transparent glass 16, and a second optical system 8 is placed in the opening of the rear wall 15 by means of the installation means 17

5 perpendicularly to transparent glass 16, which also has means of displacement 18 and a protective cone element 19 with an opening 20 for draining the liquid. In the upper part of the chamber 13, a nozzle 21 of hydrodynamic focusing with an air collector 22 is installed in the upper zone, which is connected by a hose 23 with a capacity of 24 buffer fluid. At the center of the nozzle 21, a slurry feed channel 25 is installed, associated with

5 with a vessel for suspension of micro-objects 26, which is equipped with an agitator 27 and is of an open type for adding additives to it during the experiment, for example, using a hand-type syringe 28. On

0 a hose 23 is installed in series with a device 29 for adjusting the supply of buffer fluid and an intermediate tank 30 with means for setting the liquid level of the sockets 31, 32 and its automatic sub-holding 33, 34. The means for setting the liquid level are, for example, in the form of a height-adjustable stand 31 and retractable fitting 32, and means for automatically maintaining the level of the liquid

0 is made, for example, in the form of a float 33, moved by the liquid level along the nozzle 32 and, thus, its inlet opening 34 with its pad 34.

five

In the lower part of the chamber 13, a liquid collection section 35 is made, connected via an outlet 36 with a liquid collection container 37, which is under vacuum.

The operation of a micro-object analyzer in a fluid flow according to the proposed method for its adjustment consists of seven steps.

First stage. By the first optical system 1, a limited field of view is formed on the surface of the transparent glass 16 facing the inside of camera 13. For this, the luminous flux from the illuminator passes through a rectangular field diaphragm 2, is reflected from the beam-splitting plate 5 and forms in the field of view a high-aperture lens microobjective 6 the entrance hatch or area of measurement of micro-objects, which is pre-positioned using the means 4 of rotation perpendicular to the direction of the intended flow of fluid from the nozzle 21. At the same time, t for location measurement zone 6 via a microscope objective in the reverse path of rays which have passed the plate 5 in the direction of flow of fluorescence or reflected light collection.

The second stage, carries out preliminary control of the micro-object suspension transfer system. To do this, set the intermediate tank 30 of the buffer liquid using the stand 31 so that the estimated level is higher than the level in the vessel for suspension of micro-objects 26. A container 24 of the buffer liquid is placed above the intermediate tank 30 with the possibility of its content being transferred by gravity. In this case, the entire system is filled with buffer liquid and air bubbles are removed through the air collector 22. Then the suspension supply channel 25 is connected to the micro-objects suspension vessel 26, in which the stirrer 27 is mixed and the liquid collection tank 37 is evacuated. by providing the vacuum inside the chamber 13 through the outlet 36 at the same time. A duct of buffer fluid is formed from the nozzle 21, which compresses the suspension duct located coaxially inside it estvl effect of hydrodynamic focusing. The suspension channel spreads over the surface of the transparent glass 16, passes the measurement zone and, together with the buffer liquid, enters the collection area 35, which is located at the minimum allowable angle to the transparent glass 16, thus allowing the free passage of the laminar flow of liquids.

The third stage. Adjust the optimal section parameters in the measurement zone.

duct suspension. For this, the following operations are performed alternately: control device 29 for adjusting the supply of buffer liquid;

level in intermediate tank 30 with the help of retractable fitting 32 and provide for automatic maintenance of the set level by moving the float 33 with the platform 34. Data

operations are performed until smooth adjustment of parameters of the slurry flow section is performed when the supply of buffer liquid is changed throughout the entire control range of the device 29.

Fourth stage. Orient the measurement zone relative to the flow of the suspension. To do this, turn the field diaphragm 2 by means of 4 turns, fix it, or when perpendicularly arranged

measuring zones to the suspension duct, or at a certain angle, thereby controlling the change in the duration of the signals recorded from each micro-object.

Fifth stage. The measurement zone and micro-lens 6 with central shielding 7 are mounted coaxially with respect to the optical axis. For this, the flow of the suspension channel and the buffer liquid is stopped, the liquid collection tank 37 is developed to evacuate and all the liquid from the chamber 13 is drained into it. Then, using the means 3 for moving perpendicular to the optical axis, the field diaphragm 2 is set so that the image through the micro-lens 6, located coaxially relative to the darkened light cone formed after the zone

measurements between the extreme inner rays of the dark-field illumination. The sixth stage. Provide control of the analyzer, thus achieving the registration of the fluorescence signal

optimal amplitude, duration and shape from each micro-object entering from the suspension into the measurement area from vessel 26 for suspension of micro-objects through channel 25. To do this, a vacuum is created in

the chamber 13 due to the recovery of the evacuation of the liquid collection tank 37, and the flow of the suspension and the buffer liquid is supplied to the measurement zone during its visualization, while controlling the flow rate

the buffer fluid using the device 29 to the optimal parameters of the cross section of the duct suspension. The position of the suspension flow channel is then adjusted by moving the chamber 13 relative to the measurement zone.

Seventh stage. Provide control of the analyzer, while achieving the registration of the required signal from the streams of light scattered in front of each micro-object. To do this, install a protective cone element 19 near the measurement zone at a minimum distance from the transparent glass 16, ensuring the free passage of the laminar flow of the suspension and the maximum possible overlapping of the rays of the light cone of the dark-field illumination without blocking the flow of the light scattering from the micro-objects under study. Then, the second optical system 8 is moved by means of the displacement means 18 perpendicular to the axis first, the opaque screen 9 is installed in the center of the dark area of the illumination, and then moved along the axis, collecting the stream of scattered light at the minimum (or required) angle a. In this case, taking into account the dimensions of the formed annular aperture diaphragm or changing them by correcting the light diameter of the second optical system 8 and / or the diameter of the screen 9, carry out a preliminary operation to minimize the background scattered radiation level. For this, the second optical system 8 is installed along the axis so that the size of the image of the measurement zone formed by it does not exceed the size of the input end of the monofilament light guide 10. The final minimization operation involves installing the light guide 10 using means of light protection 11 and moving 12 in the plane of forming the image of the measurement zone in the direction of receiving the light scattering fluxes from the second optical system 8. To register light scattering signals that are more likely to correlate with the size m (volume) of the microobjects under study, correcting the location along the axis of the second optical system 8, the monofilament light guide 10 relative to the measurement zone and each other to the optimum ratio of the intensities of the light fluxes at the minimum (and maximum scattering angles. This ratio is established depending on on the type of micro-objects under study and their sizes.

The use of the proposed micro-object analyzer in the fluid flow and the method of its adjustment for statistical analysis of cell populations using flow cytometry showed that the simplified design of the analyzer, based on evacuation of the measurement area

for supplying micro-objects and using fewer opto-mechanical elements, provided the convenience of control during the experiments and allowed

expand compared to the known functionality. At the same time, the laminar flow rate of the microobject suspension through the measurement zone is constant, the signal-to-noise ratio is increased when recording fluorescence signals and light scattering, a wide range of adjustment of the size of the flow section of the suspension in the measurement zone is realized, and the possibility of orientation of the measurement zone relative to the direction of suspension flow is realized , which increases the resolution of the system and reduces the coefficient of variation of the peaks in the histogram of the distributions of the studied

0 micro-objects to a value of no more than 1%. In addition, free access to the suspension under study during the experiment, the possibility of pipetting, mixing, i.e. implemented

5 advantages, allowing to obtain statistically reliable results when analyzing the minimum volumes of suspensions. The advantages of the invention allowed the creation of a flow cytometry method with

0 using specific fluorescent probes and solve a number of biomedical tasks based on the realized possibility of quantification, changes in the membranes and lymphoid genome

5 cells and cells cultured under the action of various physicochemical factors (glucocorticoids, ionizing radiation, etc.).

Claims (2)

Invention Formula 0 1. Analyzer of micro-objects in the fluid flow, containing the first optical system of dark-field illumination and collection of fluorescence from micro-objects with a field diaphragm and high-aperture 5 lens micro-lens with central shielding, a second optical system for collecting streams of light scattered forward by micro-objects, as well as a system for transferring a suspension of micro-objects to a transparent glass with a suspension supply channel connected to the vessel for suspension of micro-objects and installed in the center of the hydrodynamic focusing nozzle, which is connected main hose 5 of the buffer fluid, characterized in that, in order to expand the class of objects to be investigated by ensuring simultaneous registration of fluorescence fluxes of micro-objects and light scattered forward by micro-objects, as well as simplifying the analyzer, it further comprises means for mounting and moving the second optical system, an opaque central screen of the second optical system installed with the possibility of forming an annular aperture diaphragm, a monofilament light guide with means of light shielding and movement installed for receiving light fluxes from the second optical system with the possibility of forming its input end of the image of the field diaphragm, the intermediate capacity of the buffer fluid with the means of setting the level the device for adjusting the supply of buffer fluid to the hydrodynamic focusing nozzle and the means for rotating the field diaphragm, made rectangular in shape, while the system for transferring the suspension of micro-objects is made in the form of a vacuum chamber, the internal cavity of which is limited to transparent glass. to install the second optical system on the optical axis perpendicular to the transparent glass, with a protective cone inside the vacuumized chamber the element made with an opening for draining the liquid in the evacuated chamber is installed with the possibility of feeding the suspension at an angle to the surface of the transparent glass of the suspension supply channel with a hydrodynamic focusing nozzle, in the upper zone of the air collector in relation to the outlet opening, and section of liquid collection at an angle to the transparent glass, connected through an outlet fitting to a liquid collection container under vacuum, protective cone element of the mouth with a free flow passage through the transparent glass, the intermediate tank and the buffer supply adjustment device are installed in series between the main tank and the hydrodynamic focusing nozzle, while the means for setting the liquid level are arranged to supply the buffer liquid from the main tank to the intermediate one wherein the liquid level is higher than the level in the vessel of the microobject suspension, and the vessel of the microobject suspension is open and equipped with a stirrer. 2. A method for setting up a micro-object analyzer in a fluid flow, consisting in forming a measurement zone under dark field illumination by central shielding a high-aperture lens microobjective, forming a suspension flow in the measurement zone when it is hydrodynamically focused using a buffer liquid, minimizing the level of background scattered radiation with subsequent registering the fluorescence signals from the micro-objects in the reverse course of the rays through the micro-lens and 0 registering signals scattered forward by micro-objects of light through an optical system, characterized in that, in order to expand the class of objects under investigation by providing simultaneous recording of fluorescence flows of micro-objects and scattered forward by micro-objects of light, as well as simplifying the analyzer, the level of the buffer fluid is adjusted relative to suspension level 0 ensure the supply of liquid and suspension to the measurement area under vacuum, provide automatic maintenance of the established level of liquid, regulate the supply of buffer liquid to 5 installation in the area of measuring the constant path of micro-objects, then orient the area of measurement relative to the flow of the suspension and the buffer fluid by turning it 0 around the optical axis, stop the flow of the suspension duct and the buffer fluid and deactivate the measurement zone, then establish a measurement zone and microobjects with central shielding coaxially relative to the optical axis, restore the vacuum of the measurement zone and the flow of the suspension duct and buffer fluid into it and move the duct relative to the measurement zone, before the registration of fluorescence signals from micro-objects with the same amount of the measured substance, which coincide in amplitude, lasts form and form, after which the central shielding of the optical system is carried out, forming an annular aperture diaphragm inside it, located inside the dark illumination zone coaxially with a micro lens, while minimizing 0 the background scattered radiation level is performed by imaging an optical system measuring zone at the input end of a monofilament fiber, alternating movement of the monofilament fiber and the optical system before recording the signal from the light scattered forward by microobjects, which corresponds to the minimum coefficient of variation of the measured parameters micro objects with the minimum scattering angle and the maximum value of the ratio between the intensities of the light fluxes at the minimum and maximum scattering angles.