JP4594810B2 - Method for controlling position of particles in sample liquid and particle measuring apparatus - Google Patents

Description

  The present invention relates to a method for controlling the position of particles contained in a fluid, and in particular, a sample solution is continuously flowed through a narrow channel, and a laser beam is irradiated from the side to measure particles contained in the sample solution. The present invention relates to a particle position control method and a particle measuring apparatus suitable for flow cytometry.

  In recent years, an analysis technique called flow cytometry has been widely used in the medical field, the environmental field, the industrial field, and the like. In flow cytometry, particles such as chemical substances, cells labeled with fluorescent substances, proteins, bacteria, etc. are included in the sample solution and continuously supplied to the sample flow path of the flow cell, and the laser beam is emitted from the side. This is a technique for detecting the scattered light and the fluorescence emitted from the particles, and analyzing the number and characteristics of the particles.

As is well known, the intensity of the laser beam has a Gaussian distribution. For this reason, the intensity of the scattered light and the intensity of the fluorescence that are detected differ depending on whether the particles pass through the central part of the laser beam or the peripheral part. For this reason, in order to increase the reliability of measurement, it is desirable to control the particles contained in the sample liquid so as to pass through a certain position of the laser beam, particularly the central portion having the largest energy. Therefore, the laser beam is divided into two beams, these are collimated and incident on the flow cell, the deviation of the sample flow is detected from the detection signal based on each beam, and the position of the sample nozzle is adjusted by the adjustment knob provided on the nozzle holder. A method of adjusting the sample flow so that it passes through the center of one of the beams has been proposed (Patent Document 1).
JP 7-318478 A

  In the position adjustment method described in Patent Document 1, it is necessary to form the sample nozzle so as to be movable and to provide an adjustment knob on the sample nozzle. For this reason, a measuring apparatus becomes complicated and expensive. Moreover, the operator must adjust the position of the sample nozzle by rotating the adjustment knob by hand, which is troublesome and takes time to adjust. In addition, it is difficult to cope with temporary and sudden positional deviations due to pressure fluctuations on the sample liquid or sheath liquid supply side.

The present invention has been made to solve the above-mentioned drawbacks of the prior art, and has an object to automatically adjust the position of particles contained in a sample solution.
Another object of the present invention is to be able to easily cope with temporary and sudden displacement.

  In order to achieve the above object, a method for controlling the position of particles in a sample liquid according to the present invention is a method for controlling the position of particles in a sample liquid flowing in a flow cell by being wrapped in a sheath liquid, the flow cell An excitation laser in which a trap beam consisting of a laser beam is incident along the flow path into the sample flow path, the particles supplied to the flow cell are captured by the trap beam, and irradiated from the side of the sample flow path It is characterized by being guided to the center of the beam. The trap beam is preferably laser light in the red or infrared region. The wavelength of the trap beam is preferably different from the wavelength of the excitation laser beam.

  And the particle | grain measuring apparatus based on this invention for implementing said position control method is the sample which supplies the sample liquid to the flow cell which provided the sample flow path through which the sample liquid wrapped in the sheath liquid flows, and the said flow cell A liquid supply section; a sheath liquid supply section that supplies the sheath liquid around the sample liquid supplied by the sample liquid supply section; a head section that is attached to the flow cell; and the sample flow path via the head section And a trap beam light source for entering a trap beam consisting of a laser beam along the flow path.

  The surface of the head portion on which the trap beam is incident may be a plane formed orthogonal to the optical path of the trap beam. Further, the sample liquid supply unit is provided so as to be inclined with respect to the axis of the sample flow path, and the tip thereof is disposed close to the trap beam incident on the sample flow path. Then, the sheath liquid supply part is configured as a shell with a first supply part provided around the sample liquid supply part and a second supply part provided on the opposite side of the sample liquid supply part of the trap beam incident on the sample flow path. Good.

  The intensity (energy) of the laser beam has a Gaussian distribution. Therefore, the present invention as described above receives a force that is pulled back to the center by the radiation pressure when the particles deviate from the center of the trap beam. Thus, the particles are trapped at the center of the trap beam. Therefore, if the central portion of the trap beam is incident on the sample flow path so as to pass through the central portion of the excitation laser beam, the particles can surely pass through the central portion of the excitation laser beam. For this reason, the dispersion | variation in the measurement of particle | grains can be made small, a measurement precision can improve and reliability can be improved.

  When the trap beam is formed by laser light in the red or infrared region, the energy of the laser light absorbed by the particles is small, and damage to the particles can be reduced. Also, if the wavelength of the trap beam is different from the wavelength of the excitation laser beam, it is easy to identify whether the laser light reflected by the particles or the fluorescence emitted from the particles is due to the excitation laser beam or the trap beam. be able to.

  By making the surface on which the trap beam is incident a plane orthogonal to the optical path of the trap beam, the trap beam is not refracted, so that the trap beam can be easily and surely incident on the sample channel of the flow cell. If the sample liquid supply unit is provided to be inclined with respect to the axis of the sample channel, it is possible to prevent the trap beam from entering the sample channel. When the tip of the sample liquid supply unit is disposed close to the trap beam, particles in the sample liquid can be easily and reliably discharged into the trap beam. In addition, the sheath liquid supply unit includes a first supply unit provided around the sample liquid supply unit and a second supply unit provided on the opposite side of the sample liquid supply unit of the trap beam incident on the sample flow path. In addition, the sample liquid supplied (discharged) from the sheath liquid and the sample liquid supply unit can be prevented from flowing unevenly, and the particles in the sample liquid can be reliably trapped in the trap beam.

Preferred embodiments of a method for controlling the position of particles in a sample liquid and a particle measuring apparatus according to the present invention will be described in detail with reference to the accompanying drawings.
FIG. 2 is a block diagram showing an outline of the particle measuring apparatus according to the embodiment of the present invention. In FIG. 2, the particle measuring apparatus 10 constitutes a flow cytometer, and includes a laminar sheath flow forming unit 12 that forms a flow of a sample liquid wrapped in a sheath liquid. In the case of the embodiment, the laminar sheath flow forming part 12 is composed of a flow cell 14 and a head part 16 connected to the flow cell 14 in a watertight manner. The flow cell 14 and the head portion 16 are formed of a transparent body such as quartz glass.

  The flow cell 14 is provided with a fine sample flow path in the vertical direction at the center, and a sample liquid (laminar sheath flow) wrapped in a sheath liquid flows through the sample flow path. And the head part 16 has a sample liquid supply part and a sheath liquid supply part so that a detail may be mentioned later. The sheath liquid supply section supplies a sheath liquid around the sample liquid supplied from the sample liquid supply section, and forms a laminar sheath flow that flows into the sample flow path of the flow cell 14. A sample solution supply pipe 18 is connected to the sample solution supply unit of the head unit 16, and the sample solution 22 is supplied from the sample solution supply source 20. In addition, the tip of the sheath liquid supply pipe 24 is connected to the sheath liquid supply section of the head section 16, and the sheath liquid 28 is supplied from the sheath liquid supply source 26.

  The sample liquid 22 contains particles such as cells, proteins, and microorganisms. These particles are labeled with an appropriate fluorescent substance according to their properties and structure. The particle measuring apparatus 10 includes an excitation laser light source 30 for exciting the fluorescent material. The excitation laser beam 32 emitted from the excitation laser light source 30 is irradiated to the flow cell 14 through an optical system 34 including a reflecting mirror and a lens. The excitation laser beam 32 is incident perpendicularly to the sample channel from the side of the sample channel of the flow cell 14, and the center of the beam passes through the center of the cross section of the sample channel.

  Further, a condenser lens 38 constituting an optical system 36 is disposed on the side of the flow cell 14. The condensing lens 38 has an optical axis orthogonal to the optical path of the excitation laser beam 32, and condenses the fluorescence 40 emitted from the fluorescent material of particles excited by the excitation laser beam 32. The fluorescence 40 collected by the condenser lens 38 is guided to the optical sensor 42 by the optical system 36. The optical sensor 42 returns the incident fluorescence 40 to an electrical signal corresponding to its intensity and inputs it to the arithmetic unit 44. Although not shown, a lens that collects the scattered light of the excitation laser beam 32 scattered by the particles is disposed at an appropriate position on the side of the flow cell 14, and the scattered light collected by the lens. A light intensity signal is input to the arithmetic unit 44.

  Further, the particle measuring apparatus 10 includes a trap beam light source 46. The trap beam light source 46 emits a trap beam 48 made of laser light in the red or infrared region. The trap beam 48 enters the laminar sheath flow forming unit 12 from above the head unit 16 via the optical system 50. The trap beam 48 enters the sample flow path of the flow cell 14 along the flow path and passes through the central portion of the excitation laser beam 32, and is included in the sample liquid 22 as will be described in detail later. The trapped particles are trapped and guided to the center of the excitation laser beam 32.

  In the flow cell 14, as shown in FIG. 1, the idealized basic concept of the laminar sheath flow forming unit 12 is provided with sample flow channels 52 in a straight line in the vertical direction. In the case of the embodiment, the sample channel 52 is formed in a rectangular shape. In the flow cell 14, a sample liquid introduction part 54 communicating with the sample flow path 52 is formed on the upper part of the sample flow path 52. The sample liquid introduction part 54 is gradually expanded upward. And the lower end of the confluence | merging part 56 provided in the head part 16 is connected to the upper end of the sample liquid introducing | transducing part 54. FIG. The merging portion 56 expands upward, and the upper portion serves as a sheath liquid supply portion 58.

  The sheath liquid supply unit 58 includes a first supply unit 60 having a large diameter and a second supply unit 62 having a smaller diameter than the first supply unit 60. The first supply unit 60 and the second supply unit 62 include an inclined channel and a horizontal channel orthogonal to the side surface of the head unit 16. The inclined flow path is formed so that the center side of the head portion 16 is low and increases toward the outside, and the outer end communicates with the horizontal flow path. The head portion 16 has a nozzle insertion hole 64 formed in the upper part of the horizontal flow path of the first supply portion 60. The nozzle insertion hole 64 communicates with the first supply unit 60, and the axis coincides with the axis of the inclined flow path of the first supply unit 60. A sample liquid supply nozzle 66 serving as a sample liquid supply unit is inserted into the nozzle insertion hole 64. That is, the sample liquid supply nozzle 66 is disposed at the center of the first supply unit 60.

  The sample liquid supply nozzle 66 is inserted so that its tip is close to the trap beam 48 incident on the sample flow path 52. The particles contained in the sample liquid 22 supplied (discharged) into the head portion 16 from the tip of the sample liquid supply nozzle 66 are discharged or flown into the trap beam 48. The trap beam It flows into the sample flow path 52 of the flow cell 14 while being captured by 48. Further, when the sample liquid supply nozzle 66 is inserted into a predetermined position, the axis of the second supply part 62 of the sheath liquid supply part 58 substantially coincides with the tip of the sample liquid supply nozzle 66.

  Note that the trap beam 48 incident on the sample flow path 52 is configured such that the center of the optical path passes through the center of the excitation laser beam 32. In addition, a beam incident part 68 is formed between the first supply part 60 and the second supply part 62 constituting the sheath liquid supply part 58 immediately above the sample flow path 52. The beam incident portion 68 is formed of a plane orthogonal to the optical path of the trap beam 48 so that the trap beam 48 is not refracted, and the trap beam 48 can be easily and surely incident on the sample channel 52. .

  In the particle measuring apparatus 10 according to the embodiment as described above, the sample liquid 22 containing particles such as cells and proteins to be measured is supplied from the sample liquid supply source 20 to the sample liquid supply pipe 18 and the sample liquid supply nozzle 66. Is supplied into the head portion 16 of the laminar sheath flow forming portion 12. Further, the sheath liquid 28 from the sheath liquid supply source 26 passes through the sheath liquid supply pipe 24, the first supply section 60 of the sheath liquid supply section 58, and the second supply section 62, and the sample liquid supply nozzle 66 in the head section 16. Is supplied to the periphery of the sample liquid 22 discharged. The sheath liquid 28 supplied to the head section 16 encloses the sample liquid 22 to form a laminar sheath float, flows into the sample liquid introduction section 54 of the flow cell 14 through the merging section 56, and further flows through the sample liquid flow path 52. Flow down.

  The particles to be measured contained in the sample liquid 22 are discharged from the sample liquid supply nozzle 66 into the trap beam 48. The particles flow into the sample channel 52 while being trapped (captured) at the center of the trap beam 48 by the radiation pressure (light pressure) of the trap beam 48 formed of a laser beam. Since the center of the trap beam 48 passes through the center of the excitation laser beam 32, the particles are trapped by the trap beam 48 so as to pass through the center of the excitation laser beam 32. FIG. 3 is a diagram for explaining the principle of trapping particles by the trap beam 48.

  The trap beam 48 formed of a laser beam has a Gaussian distribution of intensity (energy) as shown by a curve 70 in FIG. Now, it is assumed that the trap beam 48 passes through a particle 72 whose particle center O is deviated from the beam center a. Consider a case in which the light beam 48a having the highest energy at the center of the trap beam 48 and the light beam 48b having a low energy at the peripheral portion are transmitted through the particle 72.

  In general, the particle 72 has a higher refractive index than the surrounding medium (sample liquid) made of physiological saline or the like. For this reason, when the light ray 48 a enters the particle 72 at the point A, it is refracted so as to approach the normal line of the particle 72. Further, when the light ray 48 a exits the particle 72 at the point B, it is refracted away from the normal line of the particle 72. Similarly, when the light ray 48b is incident on the particle 72 at the point C, the light ray 48b is refracted in a direction approaching the normal line, and when it exits from the point D, it is refracted in a direction away from the normal line.

の運動量ｐを有しており、

のエネルギーＥを有する。 For light, λ is the wavelength, ν is the frequency, c is the speed of light, and h is the Planck's constant.

Has a momentum p of

Having an energy E of

As described above, the light ray 48a enters the particle 72 from the point A and exits from the point B, so that it is refracted to change its traveling direction and its momentum changes. Thereby, the particle 72 receives the radiation pressures F _aA and F _ab due to the change in the momentum of the light beam 48a. The radiation pressures F _aA and F _aB are forces in the normal direction at the points A and _B of the particle 72. This is due to the following reason.

である。 As shown in FIG. 4, the medium 1 (refractive index n ₁ ) and the medium 2 (refractive index n ₂ ) are in contact with each other at the plane Z, and light 80 enters the medium 2 from the medium ₁ at an incident angle θ _1. Suppose that the light is refracted at a refraction angle θ ₂ . If the momentum (vector) of the light 80 incident on the medium 2 from the medium 1 is p ₁ ↑ and the momentum after refraction is p ₂ ↑, the change in momentum Δp ↑ due to the refraction of the light 80 is

It is.

となる。ただし、各ベクトルの向きは、図４に示した平面Ｚの法線８２を基準にして反時計方向に測った角度で示している。また、ｖ_１、ｖ_２は、対応する速度ベクトルの大きさを示し、ｅは自然対数の底、ｊは虚数単位である。そして、オイラーの公式により、

であるので、Δｐ↑を複素数形式で表現すると、

となる。 Assuming that the speed at which the light 80 travels through the medium 1 is v ₁ ↑ and the speed at which the light 80 travels through the medium 2 is v ₁ ↑, Δp ↑ is

It becomes. However, the direction of each vector is indicated by an angle measured counterclockwise with respect to the normal line 82 of the plane Z shown in FIG. V ₁ and v ₂ indicate the magnitudes of the corresponding velocity vectors, e is the base of the natural logarithm, and j is the imaginary unit. And according to Euler's formula,

Therefore, if Δp ↑ is expressed in complex number form,

It becomes.

となる。数式８のカッコ内は、スネルの法則によりｎ_１ｓｉｎθ_１＝ｎ_２ｓｉｎθ_２であるから、Δｐ_Ｐは０となる。すなわち、境界面と平行な方向の運動量は、常に保存される。したがって、境界面に働く運動量の変化分Δｐ↑による力は、平面Ｚに垂直な成分を有し、図４にＦとして示した力（放射圧）が境界面に作用する。 The refractive index n is 1 / v = n / c because n = c / v, where v is the speed of light in the medium. Therefore, the component (real part) Δp _V perpendicular to the plane Z of Δp ↑ and the parallel component (imaginary part) Δp _P are

It becomes. In the parentheses of Expression 8, n ₁ sin θ ₁ = n ₂ sin θ ₂ is obtained according to Snell's law, and Δp _P is 0. That is, the momentum in the direction parallel to the boundary surface is always preserved. Therefore, the force due to the momentum change Δp ↑ acting on the boundary surface has a component perpendicular to the plane Z, and the force (radiation pressure) shown as F in FIG. 4 acts on the boundary surface.

Such a radiation pressure is generated on the incident side of the light beam 48 to the particle 72 and on the emission side of the particle 72 when transmitted through the particle 72 like the trap beam (light beam) 48 shown in FIG. Therefore, at the center O of the particle 72, the combined radiation pressure (force) F _a of the sum of the radiation pressures F _aA and F _aB due to the light beam 48a and the combined radiation pressure F _a of the sum of the radiation pressures F _bC and F _bD of the light beam 48b. _b acts. These radiation pressures are based on the momentum of individual photons, and are obtained by multiplying the radiation pressure F by each photon by the number of photons. Since the light beam 48a having a large energy has more photons incident on the particle 72 than the light beam 48b having a small energy, the combined radiation pressure F _a greater than the combined radiation pressure F _b generated by the light beam 48b is given to the particle 72. For this reason, the particle 72 receives a force that moves in the direction of the beam center a of the trap beam 48 when the particle center O deviates from the center a of the trap beam 48. Therefore, the particles 72 are held (trapped) at the center of the trap beam 48.

  Since the center of the trap beam 48 passes through the center of the excitation laser beam 32, the particle 72 is guided by the trap beam 48 and reliably passes through the center of the excitation laser beam 32. For this reason, the dispersion | variation in particle | grain measurement can be made small, a measurement precision can improve and reliability can be improved. Moreover, in the embodiment, since the trap beam 48 is formed by a laser beam in the red or infrared region, even if the particle 72 is a protein or a cell, the energy absorption of the trap beam 48 by the particle 72 is small, The damage by the trap beam 48 is hardly received.

  Note that the wavelength of the trap beam 48 is different from the wavelength of the excitation laser beam 32. Thereby, when a filter is interposed on the detection side (light receiving side), it is easily identified whether the laser light scattered by the particles 72 or the fluorescence 40 emitted by the particles 72 is due to the excitation laser beam 32. be able to. In the embodiment, the sheath liquid supply unit 58 is provided on the side opposite to the sample liquid supply nozzle 66 of the first supply unit 60 provided around the sample liquid supply nozzle 66 and the trap beam 48 incident on the sample flow path 52. Since the second supply unit 62 is provided, it is possible to prevent the sheath liquid 28 and the sample liquid 22 from flowing unevenly, and the particles 72 in the sample liquid can be reliably trapped in the trap beam 48. it can.

  FIG. 5 schematically shows a specific example of the laminar sheath flow forming portion 12, wherein (1) is a plan view and (2) is a cross-sectional view taken along the line AA of (1). In FIG. 5, the laminar sheath flow forming unit 12 according to this embodiment has a head unit 16 and a flow cell 14 formed in a circular shape in plan view, for example. A first supply part 60 and a second supply part 62 constituting a sheath liquid supply part 58 for supplying the sheath liquid 22 into the head part 16 are provided on the circumferential surface in the diameter direction of the head part 16. is there. These supply parts 60 and 62 have supply holes 90 formed in the peripheral wall of the head part 16. A tube holder 92 holding the flexible sheath liquid supply tube 24 can be concentrically screwed to the outer peripheral side of the head portion of the supply hole 90.

  The head portion 16 has an inclined surface 94 that is appropriately inclined on a part of the upper surface. The inclined surface 94 is provided with a screw portion to which the nozzle holder 96 can be screwed at a position corresponding to the axis connecting the first supply portion 60 and the second supply portion 62. Then, by screwing the nozzle holder 96 onto the threaded portion, the tip of the sample liquid supply nozzle 66 held by the nozzle holder 96 can be inserted into the central portion of the head portion 16 through the nozzle insertion hole 64. It has become.

  FIG. 6 schematically shows another specific example of the laminar sheath flow forming portion 12. FIG. 6A is a plan view of the laminar sheath flow forming portion 12, and FIG. 6B is a longitudinal sectional view along the B-O line, the C-O line, and the E-O line on the left side of the center line. The right side of the center line is a cross-sectional view along the line D-O.

  The main difference between the specific example shown in FIG. 6 and the specific example shown in FIG. 5 is that three sheath liquid supply sections are provided. These supply portions are provided at equiangular intervals with respect to the center of the circular head portion 16, that is, at intervals of 120 ° with respect to the center O. Two of them located on both sides of the nozzle holder 96 constitute first supply parts 60a and 60b. The nozzle holder 96 is screwed between the first supply parts 60a and 60b. Therefore, the other supply part that becomes the second supply part 62 is located in the diameter direction of the head part 16 with respect to the nozzle holder 96.

  In the example of FIG. 6 configured as described above, the sheath liquid supply section is oriented at an interval of 120 ° with respect to the center of the head section 16 and around the sample liquid supplied from the sample liquid supply nozzle 66. The sheath liquid can be supplied isotropically and uniformly, and a laminar flow by the sheath liquid can be reliably formed. In addition, the arrangement | positioning space | interval of the 1st supply parts 60a and 60b and the 2nd supply part 62 is not limited to the example shown in FIG. 6, It can select suitably.

It is sectional drawing of the laminar sheath flow formation part which concerns on embodiment. It is a schematic block diagram of the particle | grain measuring apparatus which concerns on embodiment. It is a figure explaining the principle which traps the particle | grains by the trap beam which concerns on embodiment. It is explanatory drawing of the radiation pressure by light. It is explanatory drawing which showed typically the specific example of the laminar sheath flow formation part. It is explanatory drawing which showed typically the other specific example of the laminar sheath flow formation part.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ......... Particle measuring apparatus, 12 ......... Laminer sheath flow forming part, 14 ......... Flow cell, 16 ......... Head part, 22 ......... Sample liquid, 28 ...... Sheath liquid, 32 ......... Excitation Laser beam 48... Trap beam 52... Sample flow path 58... Sheath liquid supply section 60... First supply section 62. Liquid supply part (sample liquid supply nozzle), 72... Particles.

Claims (7)

A method of controlling the position of particles in a sample liquid flowing in a flow cell wrapped in a sheath liquid,
A trap beam consisting of a laser beam is incident along the flow path into the sample flow path of the flow cell, and the particles supplied to the flow cell are captured by the trap beam and irradiated from the side of the sample flow path. A method for controlling the position of particles in a sample liquid, characterized by guiding the central portion of an excitation laser beam. In the method for controlling the position of particles in the sample liquid according to claim 1,
The method of controlling the position of particles in a sample liquid, wherein the trap beam is laser light in a red or infrared region. In the method for controlling the position of particles in the sample liquid according to claim 1 or 2,
The trap beam has a wavelength different from that of the excitation laser beam. A flow cell provided with a sample flow path through which a sample liquid wrapped in a sheath liquid flows;
A sample liquid supply section that supplies the sample liquid to the flow cell; and a head section that includes a sheath liquid supply section that supplies the sheath liquid around the sample liquid supplied by the sample liquid supply section, and is attached to the flow cell;
A trap beam light source for injecting a trap beam consisting of a laser beam along the flow path into the sample flow path through the head portion;
A particle measuring apparatus comprising: In the particle measuring device according to claim 4,
The particle measuring apparatus according to claim 1, wherein a surface of the head unit on which the trap beam is incident is a plane formed orthogonal to an optical path of the trap beam. In the particle measuring device according to claim 4 or 5,
The particle measurement apparatus according to claim 1, wherein the sample liquid supply unit is provided so as to be inclined with respect to the axis of the sample flow path, and a tip thereof is disposed close to the trap beam incident on the sample flow path. In the particle | grain measuring apparatus in any one of Claim 4 thru | or 6,
The sheath liquid supply section includes a first supply section provided around the sample liquid supply section, and a second supply section provided on the opposite side of the sample liquid supply section of the trap beam incident on the sample flow path. A particle measuring apparatus comprising:

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