CN109580440B - Method for rapidly counting and measuring microparticles in liquid sample - Google Patents
Method for rapidly counting and measuring microparticles in liquid sample Download PDFInfo
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- CN109580440B CN109580440B CN201811220630.4A CN201811220630A CN109580440B CN 109580440 B CN109580440 B CN 109580440B CN 201811220630 A CN201811220630 A CN 201811220630A CN 109580440 B CN109580440 B CN 109580440B
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- 239000011859 microparticle Substances 0.000 title claims abstract description 64
- 239000007788 liquid Substances 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 39
- 238000003756 stirring Methods 0.000 claims abstract description 71
- 238000006073 displacement reaction Methods 0.000 claims abstract description 44
- 238000001228 spectrum Methods 0.000 claims abstract description 16
- 230000003287 optical effect Effects 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims description 19
- 238000005086 pumping Methods 0.000 claims description 17
- 239000007850 fluorescent dye Substances 0.000 claims description 5
- -1 polytetrafluoroethylene Polymers 0.000 claims description 4
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 4
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 4
- 229910001220 stainless steel Inorganic materials 0.000 claims description 4
- 239000010935 stainless steel Substances 0.000 claims description 4
- 239000003086 colorant Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 abstract description 2
- 238000010586 diagram Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 5
- 238000001917 fluorescence detection Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 2
- 230000000737 periodic effect Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001208 nuclear magnetic resonance pulse sequence Methods 0.000 description 1
- 230000000886 photobiology Effects 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
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- 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
- G01N15/06—Investigating concentration of particle suspensions
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- 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
- G01N15/06—Investigating concentration of particle suspensions
- G01N15/075—Investigating concentration of particle suspensions by optical means
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Abstract
The invention relates to the field of material engineering, in particular to a method for rapidly counting and measuring microparticles in a liquid sample, wherein an ultrafast spectrum measuring device for microparticles comprises a displacement table I, a rotary motor, a rotary shaft, a magnet, a displacement table II, a sample cavity, a stirring sheet, a lens I, a beam splitter, a laser, an optical filter, a lens II, a small hole, a lens III, a detector and a computer.
Description
Technical Field
The invention relates to the field of material engineering, in particular to a method for rapidly counting and measuring microparticles in a liquid sample for researching a low-concentration sample by adopting an ultrafast spectrum method.
Background
The ultrafast spectrum is a method for researching sample characteristics by adopting ultrafast laser, can obtain a time resolution spectrum with femto-second resolution, is generally used for researching ultrafast process in photochemistry or photobiology, needs to emit focused laser to a part region in a sample in experiments, and the laser repetition rate of a laser system in the prior art can reach the kHz order, so that the exchange speed between the part which is excited by the laser and the part which is not excited in the sample is required to be fast enough, the influence on an experiment result caused by repeated excitation of the sample by the laser is avoided, and certain prior art adopts a circulating pump method to exchange the part in the sample, so that the needed sample quantity is large, mechanical noise is introduced, and even the sample is damaged; other prior art adopts a stirrer rotating at a constant speed to exchange samples in different areas, but in some sample cavities with smaller volumes, the stirring range is smaller, namely, samples around a stirring sheet can be only stirred and mixed, and the samples can generate periodical refraction to laser, so that periodical noise is introduced into an ultrafast spectrum, and the result of the ultrafast spectrum is influenced. In some applications, the number of particles in the liquid needs to be counted, the counting measurement in the prior art takes a long time, and only a sample with a large number of particles in a unit volume of liquid can be measured, and the problem can be solved by the method for rapidly counting and measuring the microparticles in the liquid sample.
Disclosure of Invention
In order to solve the problems, the method adopts a special sample cavity and a novel stirring method, so that samples in different areas in the sample cavity can be quickly exchanged when an ultrafast spectrum experiment is carried out, in addition, the method of rotating the sample cavity and combining fluorescence detection is adopted to carry out high-precision measurement on the number of microparticles in liquid, no sample loss is caused, and the measurement process is simple.
The technical scheme adopted by the invention is as follows:
the ultra-fast spectrum measuring device for the micro-particles comprises a displacement table I, a rotary motor, a rotary shaft, a magnet, a displacement table II, a sample cavity, a stirring sheet, a lens I, a beam splitter, a laser, an optical filter, a lens II, a small hole, a lens III, a detector and a computer, xyz is a three-dimensional space coordinate system, the rotary motor is fixed on the displacement table I, the laser, the beam splitter, the lens I and the sample cavity sequentially form an incident light path, the sample cavity, the lens I, the beam splitter, the optical filter, the lens II, the small hole, the lens III and the detector sequentially form an emergent light path, the laser can emit continuous laser and laser pulses with adjustable wavelength, the laser pulses comprise pumping pulses and detection pulses, the pumping pulses and the detection pulses have different powers, frequencies and durations, and the time interval between the detection pulses and the pumping pulses can be adjusted; the magnet is connected with a rotary motor through a rotary shaft, the rotary shaft is parallel to a z coordinate axis, the rotary motor can drive the magnet to rotate, the sample cavity is fixed on a displacement table II, the displacement table II can move in three dimensions, the displacement table II can drive the sample cavity to rotate around a y-direction central line of the sample cavity, the upper half part of the sample cavity is a cuboid cavity, the lower half part of the sample cavity is a cylindrical barrel with an axis along the y direction, a grid mesh is arranged between the upper half part and the lower half part, the cuboid cavity is 30 mm in height, 10 mm in length and 2 mm in width, the cylindrical barrel is 20 mm in height and 10 mm in diameter on the bottom surface, the y-direction central line of the cuboid cavity is collinear with the axis of the cylindrical barrel, a stirring piece is made of polytetrafluoroethylene wrapped outside a magnetic stainless steel rod, the diameter of the stirring piece is 1.2 mm, the length of the stirring piece is 13 mm, the stirring piece is positioned on the upper half part of the sample cavity, and the grid mesh can prevent the stirring piece from falling into the cylindrical barrel; the rotary motor, the laser and the detector are respectively connected with the computer through cables, and the time difference between the laser pulse emitted by the laser and the rotation angle of the rotary motor can be adjusted through the computer; the size of the small hole can be adjusted, and the detection resolution of the detector can be adjusted according to the microparticles to be detected with different sizes.
The principle of the stirring blade for enabling sample parts in different areas in the sample cavity to be rapidly exchanged is as follows:
the width direction of the cuboid cavity in the upper half part of the sample cavity is parallel to the z axis, the axis extension line of the rotating shaft is located in the center of the cuboid cavity, the rotating motor is started to enable the magnet to rotate around the axis of the rotating shaft at a constant speed, the rotating speed is typically 0.5 revolution/second, due to the action of the magnetic moment of the magnet, the stirring piece rotates along with the magnet, the stirring piece rotates until the two ends are in contact with the inner wall of the cuboid cavity and limited, the magnet continues to rotate, the stirring piece still receives the action of the magnetic moment of the magnet, but the position of the stirring piece is unchanged, when the magnet rotates to a certain angle, the stirring piece is reversely acted by the magnetic moment of the magnet, the stirring piece can be quickly turned over in the reverse direction of the rotation of the magnet, the stirring piece is turned over until the two ends are in contact with the inner wall of the cuboid cavity and limited, in the process of uniformly rotating the magnet for one circle, the stirring piece rotates at a time along with the rotating direction of the magnet at a constant speed, the stirring piece rotates at a fast speed again, and the sample in the vicinity of the stirring piece is quickly exchanged with the sample in the other parts of the sample cavity. The technical advantages are: the stirring piece can not introduce noise along with the slow rotation process of the magnet rotation direction, and mechanical noise in a sample can be effectively prevented from entering a spectrum by adjusting the time interval between the laser pulse and the rapid overturning process of the stirring piece in the opposite direction of the magnet rotation, and periodic refraction of laser in the sample is also prevented.
The principle of counting and measuring microparticles in a liquid sample is as follows:
the liquid sample containing the microparticles to be detected is placed in a sample cavity, and fluorescent dye is added into the liquid sample according to the types of the microparticles to be detected, so that the surface of the microparticles can emit fluorescence under the irradiation of laser with specific wavelength, the laser emitted by the laser irradiates the sample, so that the microparticles emit fluorescence, and the fluorescence is recorded by a detector through an emergent light path; after the laser emitted by the laser device is deflected by the beam splitter, the laser is focused into a cylindrical barrel at the lower half part of the sample cavity by the lens I, the displacement table II drives the sample cavity to rotate around the central line of the sample cavity in the y direction, and microparticles suspended in the liquid sample move together with the solvent and emit fluorescence through the focus of the laser in the sample cavity; the part of the volume in which the fluorescence emitted by the microparticles in the part of the volume irradiated by the laser can be detected by the detector, without rotation of the sample chamber, is defined as the detectable volume, and the method of estimating the detectable volume is as follows: fixing a fluorescent reference capable of emitting fluorescent light into the sample cavity, enabling the detector to recognize that the laser beam enters the sample cavity along the negative z direction, and recording the fluorescent signal with the intensity of the fluorescent signal of the detector when the sample cavity is fixedWherein w is 0 For the beam waist diameter of the laser beam in the x-direction, z 0 For the beam waist diameter of the laser beam along the z direction, the displacement platform II is adjusted to enable the sample cavity to translate along the x direction, the detector records the fluorescent signal of a fluorescent reference object, and the data is fitted by adopting two-dimensional Gaussian distribution to obtain w 0 Adjusting the displacement platform II to enable the sample cavity to translate along the z direction, recording fluorescent signals of a fluorescent reference object by a detector, and fitting data by adopting two-dimensional Gaussian distribution to obtain z 0 Fitting the obtained data by adopting a pair of data, and estimating the detectable volume, wherein a typical value is 0.5 nanoliter; the detector continuously records the intensity of fluorescence emitted by the microparticles in the detectable volume within a period of time T to obtain signal data with different intensities, and calculatesAnd processing the signal data by a machine to obtain the number of microparticles in the detectable volume in the T time, and combining the rotating speed of the sample cavity to obtain the number of microparticles in the unit liquid volume.
The ultra-fast spectrum experiment steps of the microparticles are as follows:
step 1, placing a liquid sample containing microparticles to be detected in a sample cavity;
step 2, adjusting the displacement platform II to enable the width direction of a cuboid cavity at the upper half part of the sample cavity to be parallel to a z coordinate axis, and adjusting the displacement platform I to enable an axis extension line of a rotating shaft to be positioned at the center of the cuboid cavity at the upper half part of the sample cavity;
step 3, adjusting the positions of the lens I, the beam splitter, the laser, the optical filter, the lens II, the small hole, the lens III and the detector, so that laser emitted by the laser is focused into a cuboid cavity at the upper half part of a sample cavity by the lens I after being deflected by the beam splitter, and light reflected by the sample sequentially passes through the lens I, the beam splitter, the optical filter, the lens II, the small hole and the lens III and then enters the detector;
step 4, starting a rotary motor to enable the magnet to rotate at a constant speed around the axis of the rotary shaft, wherein the rotating speed is typically 0.5 r/s, so that the stirring sheet stirs the sample;
step 5, the laser periodically emits a pumping pulse and a detection pulse sequence, and the pumping pulse and the detection pulse are periodically incident into the sample;
step 6, according to the rotation angle of the rotary motor corresponding to the position of the stirring sheet, adjusting the interval time between the pumping pulse and the detection pulse sent by the laser through the computer, so that the stirring sheet can rapidly turn over in the opposite direction of the rotation of the magnet, and the pumping pulse and the detection pulse are incident into the time gap of the sample;
and 7, recording light emitted from the sample by the detector and generating corresponding data, and analyzing the data by the computer to obtain an ultrafast spectrum of the sample.
The method for rapidly counting and measuring the microparticles in the liquid sample comprises the following steps:
step one, placing a liquid sample containing microparticles to be detected in a sample cavity;
adding a fluorescent coloring agent into the liquid sample according to the types of the microparticles to be detected, so that the surfaces of the microparticles can emit fluorescence under the irradiation of laser with corresponding wavelength;
step three, adjusting the positions of a lens I, a beam splitter, a laser, an optical filter, a lens II, a small hole, a lens III and a detector, so that laser emitted by the laser is focused into a cylindrical barrel at the lower half part of a sample cavity by the lens I after being deflected by the beam splitter, and light reflected by the sample sequentially passes through the lens I, the beam splitter, the optical filter, the lens II, the small hole and the lens III and then enters the detector;
step four, the displacement platform II drives the sample cavity to rotate around the central line of the sample cavity in the y direction, and the rotating speed range is 50 to 400 revolutions per minute;
step five, the continuous laser emitted by the laser is incident on the sample in the sample cavity, and the wavelength of the continuous laser is determined according to the type of fluorescent dye added in the liquid sample;
step six, adjusting the size of the small hole so that the detector can clearly record the fluorescence emitted by the single micro-particles;
and seventhly, recording light emitted from the sample by the detector and generating corresponding data, and after analyzing the data by the computer, obtaining the quantity information of the to-be-detected micro-particle sample in the detectable volume of the liquid sample, wherein the minimum value of the quantity of micro-particles in the detectable volume of the liquid is 50 per milliliter for the micro-particles with the diameter of 2 micrometers by combining the rotating speed of the sample cavity.
The beneficial effects of the invention are as follows:
the method of the invention adopts a special sample cavity and combines a fluorescence detection method, can carry out ultra-fast spectrum experiment and micro-particle counting measurement, has no sample loss and simple experimental steps.
Drawings
The following is further described in connection with the figures of the present invention:
FIG. 1 is a schematic illustration of the present invention; FIG. 2 is an enlarged schematic side view of a sample chamber; FIG. 3 is a top view of FIG. 2;
FIG. 4 is one of the schematic diagrams of the stirring blade rotating with the magnet;
FIG. 5 is a second schematic view of the stirring blade rotating with the magnet;
FIG. 6 is a third schematic illustration of the rotation of the stirring blade with the magnet;
FIG. 7 is a schematic diagram showing the rotation of the stirring blade with the magnet;
FIG. 8 is a fifth schematic illustration of the rotation of the stirring blade with the magnet;
FIG. 9 is a schematic diagram showing a rotation process of the stirring blade with the magnet.
In the figure, 1, displacement stage I,2, rotary motor, 3, rotary shaft, 4, magnet, 5, displacement stage II,6, sample cavity, 7, stirring sheet, 8, lens I,9, beam splitter, 10, laser, 11, optical filter, 12, lens II,13, aperture, 14, lens III,15, detector.
Detailed Description
As shown in FIG. 1, the xyz is a three-dimensional space coordinate system and comprises a displacement table I (1), a rotary motor (2), a rotary shaft (3), a magnet (4), a displacement table II (5), a sample cavity (6), a stirring piece (7), a lens I (8), a beam splitter (9), a laser (10), an optical filter (11), a lens II (12), a small hole (13), a lens III (14), a detector (15) and a computer, wherein the rotary motor (2) is fixed on the displacement table I (1), the magnet (4) is connected with the rotary motor (2) through the rotary shaft (3), the rotary shaft (3) is parallel to the z coordinate axis, the displacement table I (1) can move three-dimensionally, the rotary motor (2) can drive the magnet (4) to rotate, the sample cavity (6) is fixed on the displacement table II (5), the displacement table II (5) can move in three dimensions, the displacement table II (5) can drive the sample cavity (6) to rotate around the central line of the y direction of the sample cavity (6), the upper half part of the sample cavity (6) is a rectangular cavity, the lower half part is a cylindrical barrel with an axis along the y direction, a grid is arranged between the upper half part and the lower half part, the rectangular cavity is 30 mm in height, 10 mm in length and 2 mm in width, the cylindrical barrel is 20 mm in height and 10 mm in bottom surface diameter, the Y-direction center line of the rectangular cavity is collinear with the axis of the cylindrical barrel, the stirring sheet (7) is formed by wrapping polytetrafluoroethylene outside a magnetic stainless steel rod, the diameter of the stirring sheet (7) is 1.2 mm, the length of the stirring sheet is 13 mm, the stirring sheet (7) is positioned at the upper half part of the sample cavity (6), the grid can prevent the stirring sheet (7) from falling into the cylindrical barrel, the laser (10), the beam splitter (9), the lens I (8) and the sample cavity (6) sequentially form an incident light path, the sample cavity (6), the lens I (8), the beam splitter (9), the optical filter (11), the lens II (12), the small hole (13), the lens III (14) and the detector (15) sequentially form an emergent light path, the laser (10) can emit continuous laser with adjustable wavelength and laser pulses, the laser pulses comprise pumping pulses and detection pulses, the pumping pulses and the detection pulses have different power, the frequency and the duration, the time interval between the detection pulses and the pumping pulses can be adjusted, and the rotation angle of the rotation motor (2), the detector (10) and the detector (15) can be respectively connected with a computer (10) through the rotation motor (10); the size of the small hole (13) can be adjusted, and the detection resolution of the detector (15) can be adjusted according to the microparticles to be detected with different sizes.
Fig. 2 is an enlarged schematic side view of a sample cavity, fig. 3 is a top view of fig. 2, an upper half part of a sample cavity (6) is a cuboid cavity, a lower half part of the sample cavity is a cylindrical barrel with an axis along a y direction, and a y direction central line of the cuboid cavity is collinear with an axis of the cylindrical barrel.
Fig. 4 is a schematic diagram of a rotation process of the stirring plate along with the magnet, fig. 5 is a schematic diagram of a second rotation process of the stirring plate along with the magnet, fig. 6 is a schematic diagram of a third rotation process of the stirring plate along with the magnet, fig. 7 is a schematic diagram of a fourth rotation process of the stirring plate along with the magnet, fig. 8 is a schematic diagram of a fifth rotation process of the stirring plate along with the magnet, fig. 9 is a schematic diagram of a sixth rotation process of the stirring plate along with the magnet, an arrow in the diagram is a rotation direction, the rotating motor (2) can drive the magnet (4) to rotate, a magnetic pole position of the magnet (4) is changed, and the stirring plate (7) rotates along with the magnet (4).
The ultra-fast spectrum measuring device for the micro-particles comprises a displacement table I (1), a rotary motor (2), a rotary shaft (3), a magnet (4), a displacement table II (5), a sample cavity (6), a stirring sheet (7), a lens I (8), a beam splitter (9), a laser (10), a light filter (11), a lens II (12), a small hole (13), a lens III (14), a detector (15) and a computer, xyz is a three-dimensional space coordinate system, the rotary motor (2) is fixed on the displacement table I (1), the laser (10), the beam splitter (9), the lens I (8) and the sample cavity (6) sequentially form an incident light path, the sample cavity (6), the lens I (8), the beam splitter (9), the light filter (11), the lens II (12), the small hole (13), the lens III (14) and the detector (15) sequentially form an emergent light path, the laser (10) can emit continuous laser and laser pulses with adjustable wavelengths, the laser pulses comprise pumping pulses and detection pulses, the pumping pulses and the detection pulses have different power, the frequencies and the duration pulses can be adjusted, and the time intervals between the detection pulses and the pumping pulses can be adjusted; the magnet (4) is connected with the rotary motor (2) through the rotary shaft (3), the rotary shaft (3) is parallel to the z coordinate axis, the displacement table I (1) can move in three dimensions, the rotary motor (2) can drive the magnet (4) to rotate, the sample cavity (6) is fixed on the displacement table II (5), the displacement table II (5) can move in three dimensions, the displacement table II (5) can drive the sample cavity (6) to rotate around the y-direction central line of the sample cavity (6), the upper half part of the sample cavity (6) is a cuboid cavity, the lower half part is a cylindrical barrel with an axis along the y-direction, a grid mesh is arranged between the upper half part and the lower half part, the cuboid cavity is 30 mm in height, 10 mm in length and 2 mm in width, the cylindrical barrel is 20 mm in height, the bottom surface diameter is 10 mm, the y-direction central line of the cuboid cavity is collinear with the axis of the cylindrical barrel, the stirring piece (7) is made of polytetrafluoroethylene wrapped by a magnetic stainless steel rod, the diameter of the stirring piece (7) is 1.2 mm, the length is 13 mm, the stirring piece (7) can be prevented from falling into the cylindrical barrel (6); the rotary motor (2), the laser (10) and the detector (15) are respectively connected with a computer through cables, and the time difference between the laser pulse emitted by the laser (10) and the rotation angle of the rotary motor (2) can be adjusted through the computer; the size of the small hole (13) can be adjusted, and the detection resolution of the detector (15) can be adjusted according to the microparticles to be detected with different sizes.
The stirring blade (7) enables the sample parts in different areas in the sample cavity (6) to be rapidly exchanged according to the principle that:
according to fig. 1, the displacement table II (5) is adjusted to enable the width direction of a cuboid cavity at the upper half part of the sample cavity (6) to be parallel to the z axis, the displacement table I (1) is adjusted to enable an axis extension line of the rotating shaft (3) to be located at the center of the cuboid cavity, the rotating motor (2) is started to enable the magnet (4) to rotate at a constant speed around the axis of the rotating shaft (3), the rotating speed is typically 0.5 r/s, according to fig. 4 and 5, due to the action of magnetic moment of the magnet (4), the stirring sheet (7) rotates along with the magnet (4), according to fig. 6, the stirring sheet (7) rotates until two ends are contacted with the inner wall of the cuboid cavity and are limited, according to fig. 7, at the moment, the magnet (4) continues to rotate, the stirring piece (7) still receives the magnetic moment action of the magnet (4), but the position of the stirring piece (7) is unchanged, as shown in fig. 8, when the magnet (4) rotates to a certain angle, the stirring piece (7) receives the magnetic moment of the magnet (4) reversely, at the moment, the stirring piece (7) can be quickly turned over in the reverse direction of the rotation of the magnet (4), as shown in fig. 9, the stirring piece (7) is turned over until the two ends are contacted with the inner wall of the cuboid cavity and limited, in the process of uniformly rotating the magnet for one circle, the stirring piece (7) rotates twice, once the stirring piece (7) rotates slowly along with the rotation direction of the magnet (4), and once again the stirring piece (7) is quickly turned over in the reverse direction of the rotation of the magnet (4), the rapid overturning process of the stirring blade (7) enables the samples in the area near the stirring blade (7) to be rapidly exchanged with the samples in other parts of the sample cavity (6). The technical advantages are: the stirring piece (7) can not introduce noise along with the slow rotation process of the rotation direction of the magnet (4), and mechanical noise in a sample can be effectively prevented from entering a spectrum by adjusting the time interval between the laser pulse and the rapid overturning process of the stirring piece (7) to the opposite direction of the rotation of the magnet (4), and periodic refraction of laser in the sample is also prevented.
The principle of counting and measuring microparticles in a liquid sample is as follows:
a liquid sample containing microparticles to be detected is placed in a sample cavity (6), a fluorescent dye is added into the liquid sample according to the types of the microparticles to be detected, so that the surface of the microparticles can emit fluorescence under the irradiation of laser with specific wavelength, the laser emitted by a laser (10) irradiates the sample to enable the microparticles to emit fluorescence, and the fluorescence is recorded by a detector (15) through an emergent light path; after the laser emitted by the laser (10) is deflected by the beam splitter (9), the laser is focused to the cylinder at the lower half part of the sample cavity (6) by the lens I (8)Wherein the displacement table II (5) drives the sample cavity (6) to rotate around the central line of the sample cavity (6) in the y direction, and microparticles suspended in the liquid sample move together with the solvent and emit fluorescence through the focus of laser in the sample cavity (6); in the case where the sample chamber (6) is not rotated, the part of the volume in which the fluorescence emitted by the microparticles in the part of the volume irradiated by the laser can be detected by the detector (15) is defined as a detectable volume, and the method of estimating the detectable volume is as follows: fixing a fluorescent reference capable of emitting fluorescent light in the sample cavity (6) and enabling the detector (15) to identify that the laser beam is incident on the sample cavity (6) along the negative z direction, wherein when the sample cavity is fixed, the intensity of a fluorescent signal recorded by the detector (15) is thatWherein w is 0 For the beam waist diameter of the laser beam in the x-direction, z 0 For the beam waist diameter of the laser beam along the z direction, the displacement table II (5) is adjusted to enable the sample cavity (6) to translate along the x direction, the detector (15) records the fluorescence signal of the fluorescence reference object, and the two-dimensional Gaussian distribution is adopted to fit the data to obtain w 0 Adjusting the displacement table II (5) to enable the sample cavity (6) to translate along the z direction, recording fluorescent signals of a fluorescent reference object by the detector (15), and fitting data by adopting two-dimensional Gaussian distribution to obtain z 0 Fitting the obtained data by adopting a pair of data, and estimating the detectable volume, wherein a typical value is 0.5 nanoliter; the detector (15) continuously records the intensity of fluorescence emitted by the microparticles in the detectable volume within a period of time T to obtain signal data with different intensities, and the computer processes the signal data to obtain the number of microparticles in the detectable volume within the period of time T and combines the rotating speed of the sample cavity (6) to obtain the number of microparticles in the unit liquid volume.
The method for rapidly counting and measuring the microparticles in the liquid sample comprises the following steps:
step one, placing a liquid sample containing microparticles to be detected in a sample cavity (6);
adding a fluorescent coloring agent into the liquid sample according to the types of the microparticles to be detected, so that the surfaces of the microparticles can emit fluorescence under the irradiation of laser with corresponding wavelength;
step three, adjusting the positions of a lens I (8), a beam splitter (9), a laser (10), an optical filter (11), a lens II (12), an aperture (13), a lens III (14) and a detector (15), so that laser emitted by the laser (10) is focused by the lens I (8) into a cylindrical barrel at the lower half part of a sample cavity (6) after being deflected by the beam splitter (9), and light reflected by a sample sequentially passes through the lens I (8), the beam splitter (9), the optical filter (11), the lens II (12), the aperture (13) and the lens III (14) and then enters the detector (15);
step four, the displacement table II (5) drives the sample cavity (6) to rotate around the central line of the sample cavity (6) in the y direction, and the rotating speed range is 50 to 400 revolutions per minute;
step five, the continuous laser emitted by the laser (10) is incident on the sample in the sample cavity (6), and the wavelength of the continuous laser is determined according to the type of fluorescent dye added in the liquid sample;
step six, the size of the small hole (13) is adjusted, so that the detector (15) can clearly record the fluorescence emitted by single microparticles;
and seventhly, the detector (15) records light emitted from the sample and generates corresponding data, after the data are analyzed by a computer, the quantity information of the micro-particle sample to be detected in the detectable volume of the liquid sample is obtained, and the quantity of the micro-particles in the unit liquid volume can be obtained by combining the rotating speed of the sample cavity (6), and for the micro-particles with the diameter of 2 micrometers, the minimum value of the quantity of the micro-particles in the unit liquid volume which can be detected is 50/milliliter.
The method adopts a special sample cavity and a stirring method, is suitable for ultrafast spectrum experiments, and adopts a method of rotating the sample cavity and combining fluorescence detection to count and measure microparticles in liquid, so that no sample loss exists and the measurement time is short.
Claims (1)
1. The ultra-fast spectrum measuring device for the micro-particles in the liquid sample comprises a displacement table I (1), a rotary motor (2), a rotary shaft (3), a magnet (4), a displacement table II (5), a sample cavity (6), a stirring sheet (7), a lens I (8), a beam splitter (9), a laser (10), a light filter (11), a lens II (12), a small hole (13), a lens III (14), a detector (15) and a computer, xyz is a three-dimensional space coordinate system, the rotary motor (2) is fixed on the displacement table I (1), the laser (10), the beam splitter (9), the lens I (8) and the sample cavity (6) sequentially form an incident light path, the sample cavity (6), the lens I (8), the light splitter (9), the light filter (11), the lens II (12), the small hole (13), the lens III (14) and the detector (15) sequentially form an emergent light path, the laser (10) can emit continuous laser and laser pulses with adjustable wavelength, the laser pulses comprise pumping pulses and detection pulses, and the pumping pulses and the detection pulses have different pumping pulses and the duration and the pumping pulses can be adjusted with different pulse frequency and the duration; the magnet (4) is connected with the rotary motor (2) through the rotary shaft (3), the rotary shaft (3) is parallel to the z coordinate axis, the displacement table I (1) can move in three dimensions, the rotary motor (2) can drive the magnet (4) to rotate, the sample cavity (6) is fixed on the displacement table II (5), the displacement table II (5) can move in three dimensions, the displacement table II (5) can drive the sample cavity (6) to rotate around the y-direction central line of the sample cavity (6), the upper half part of the sample cavity (6) is a cuboid cavity, the lower half part is a cylindrical barrel with an axis along the y-direction, a grid mesh is arranged between the upper half part and the lower half part, the cuboid cavity is 30 mm in height, 10 mm in length and 2 mm in width, the cylindrical barrel is 20 mm in height, the bottom surface diameter is 10 mm, the y-direction central line of the cuboid cavity is collinear with the axis of the cylindrical barrel, the stirring piece (7) is made of polytetrafluoroethylene wrapped by a magnetic stainless steel rod, the diameter of the stirring piece (7) is 1.2 mm, the length is 13 mm, the stirring piece (7) can be prevented from falling into the cylindrical barrel (6); the rotary motor (2), the laser (10) and the detector (15) are respectively connected with a computer through cables, and the time difference between the laser pulse emitted by the laser (10) and the rotation angle of the rotary motor (2) can be adjusted through the computer; the size of the small hole (13) can be adjusted, the detection resolution of the detector (15) can be adjusted according to the micro-particles to be detected with different sizes,
the method is characterized in that: the method for rapidly counting and measuring the microparticles in the liquid sample comprises the following steps:
step one, placing a liquid sample containing microparticles to be detected in a sample cavity (6);
adding a fluorescent coloring agent into the liquid sample according to the types of the microparticles to be detected, so that the surfaces of the microparticles can emit fluorescence under the irradiation of laser with corresponding wavelength;
step three, adjusting the positions of a lens I (8), a beam splitter (9), a laser (10), an optical filter (11), a lens II (12), an aperture (13), a lens III (14) and a detector (15), so that laser emitted by the laser (10) is focused by the lens I (8) into a cylindrical barrel at the lower half part of a sample cavity (6) after being deflected by the beam splitter (9), and light reflected by a sample sequentially passes through the lens I (8), the beam splitter (9), the optical filter (11), the lens II (12), the aperture (13) and the lens III (14) and then enters the detector (15);
step four, the displacement table II (5) drives the sample cavity (6) to rotate around the central line of the sample cavity (6) in the y direction, and the rotating speed range is 50 to 400 revolutions per minute;
step five, the continuous laser emitted by the laser (10) is incident on the sample in the sample cavity (6), and the wavelength of the continuous laser is determined according to the type of fluorescent dye added in the liquid sample;
step six, the size of the small hole (13) is adjusted, so that the detector (15) can clearly record the fluorescence emitted by single microparticles;
and seventhly, the detector (15) records light emitted from the sample and generates corresponding data, after the data are analyzed by a computer, the quantity information of the micro-particle sample to be detected in the detectable volume of the liquid sample is obtained, and the quantity of the micro-particles in the unit liquid volume can be obtained by combining the rotating speed of the sample cavity (6), and for the micro-particles with the diameter of 2 micrometers, the minimum value of the quantity of the micro-particles in the unit liquid volume which can be detected is 50/milliliter.
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