JP2019033688A - Optical trapping device, sample sorting device, and method of trapping sample by irradiation with light - Google Patents

Abstract

To provide an optical trapping device with which the diameter of illumination light is adjusted to a value suited for obtaining a desired optical trapping force.SOLUTION: An optical trapping device according to the present invention comprises: a light source; a beam diameter adjusting optical system for adjusting the beam diameter of the light from the light source; an objective lens for projecting the beam diameter-adjusted light onto a sample; a storage device which stores beam diameter information indicating the value of a beam diameter which gives an optical trapping force with respect to combinations of sample sizes and the number of apertures of the objective lens; and a processor which refers to the beam diameter information stored in the storage device to acquire the value of a beam diameter corresponding to a combination of the sample size of the sample to be trapped and the number of apertures of the objective lens used in the optical trapping device, and controls the beam diameter adjusting optical system to adjust the light beam diameter to the acquired beam diameter.SELECTED DRAWING: Figure 4

Description

  The present disclosure relates to an optical trap device, a sample sorting device, and a method for irradiating and capturing light on a sample.

  Conventionally, an optical trap (also called optical tweezers) has been used when capturing and manipulating fine particles in a medium. This light trap is a technique for supplementing an object by utilizing a reaction against a change in momentum obtained by refraction when light passes through an object (fine particle) having a refractive index different from that of the surrounding medium.

  An apparatus for moving a minute object using such an optical trap technology has been developed. For example, Patent Document 1 discloses that an optimum condition according to a minute object to be moved is created by adjusting a minimum spot position and a minimum spot diameter of light to be irradiated. Patent Document 1 suggests that the larger the diameter of the light incident on the condensing optical system, the larger the incident angle of the light on the minute object and the smaller the spot diameter, which is preferable.

JP 7-31459 A

  However, in consideration of the size of various objects, the inventor does not necessarily increase the trapping force as the diameter of illumination light (hereinafter referred to as “illumination light”) incident on the entrance pupil of the objective lens increases. It was revealed. That is, this time, the present inventor has found that if the diameter of the illumination light is extremely large, the amount of light that can pass through the entrance pupil of the objective lens decreases, and conversely the trapping force becomes weak, and the diameter of the illumination light is extremely large. It was found that if it is thin, the light spot at the focal position becomes large and the trapping force becomes weak.

  Further, in Patent Document 1, it is stated that the trapping force changes when the diameter of the illumination light (beam diameter) is changed, but there is no mention of how the trapping force changes. Furthermore, when examining Patent Document 1 in more detail, “the larger the diameter of the illumination light, the stronger the trapping force” is when the entrance pupil diameter of the objective lens is sufficiently larger than the diameter of the illumination light. Just turned out to be. Therefore, in an actual optical trap device, the condition disclosed in Patent Document 1 (specifically, Formula 2) is not satisfied.

  In view of the above situation, the present inventor determines the optimal illumination light diameter based on the diameter of the trapped object (hereinafter sometimes referred to as “sample”) and the NA (numerical aperture) of the objective lens. Furthermore, the present inventors have found out the optimum range of the diameter of illumination light.

According to the present embodiment, an optical trap device that irradiates a sample with light and captures the light, and the beam diameter (hereinafter referred to as “beam diameter”) at the entrance pupil position of the objective lens of the light source and the light from the light source. Beam diameter adjustment optical system to be adjusted, objective lens that irradiates the sample with light having the adjusted beam diameter, and a beam diameter that indicates a beam diameter value that gives a light trapping force in combination of the sample size and the numerical aperture of the objective lens With reference to the storage device for storing information and the beam diameter information stored in the storage device, the beam diameter corresponding to the combination of the sample size of the sample to be captured and the numerical aperture of the objective lens used in the optical trap device And a processor that acquires a value and controls the beam diameter adjusting optical system to adjust the beam diameter of the light to the acquired beam diameter. .
Here, the sample size represents the diameter when the sample trapped by light is spherical, and the volume average diameter when the sample is a polyhedron or ellipsoid.

According to another embodiment, an optical trap device that irradiates and captures light on a sample, the light source, a beam diameter adjusting optical system that adjusts the beam diameter of light from the light source, and the beam diameter are adjusted An objective lens that irradiates the sample with light, and a light trapping force generated by irradiating the sample with light is applied to the sample in a plane perpendicular to the optical axis of the objective lens (hereinafter referred to as the “XY plane”) The ratio of the beam diameter as the diameter at which the intensity becomes 1 / e ² with respect to the maximum value and the entrance pupil diameter of the objective lens (hereinafter referred to as “pupil filling factor”) is 0.43 or more and 0.93. An optical trap device in which the beam diameter of light is adjusted by a beam diameter adjusting optical system is provided as follows.

  According to another embodiment, an optical trap device that irradiates and captures light on a sample, the light source, a beam diameter adjusting optical system that adjusts the beam diameter of light from the light source, and the beam diameter are adjusted An objective lens that irradiates the sample with light, and when the optical trapping force generated by irradiating the sample with light is applied to the sample in the optical axis direction of the objective lens (hereinafter referred to as “Z direction”), There is provided an optical trap device in which the beam diameter of light is adjusted by a beam diameter adjusting optical system so that the pupil filling factor is 0.56 or more and 0.96 or less.

  According to another embodiment, there is provided a sample separation device for separating a sample flowing in a flow path, a light source, a beam diameter adjusting optical system for adjusting a beam diameter of light from the light source, and light with adjusted beam diameter. An objective lens that irradiates the sample, a storage device that stores beam diameter information indicating a beam diameter value that gives an optical trapping force in a combination of the sample size and the numerical aperture of the objective lens, and the beam diameter for the sample is adjusted A processor that controls the irradiation of the light and sorts the sample in a desired direction in the flow path, and the processor refers to the beam diameter information stored in the storage device, and the sample size of the sample to be sorted And the beam diameter value corresponding to the combination of the numerical aperture of the objective lens used in the sample fractionator and the beam diameter adjustment optical system is controlled. Adjusting the beam diameter obtained a beam diameter of the light, the sample separation device is provided.

  According to another embodiment, a sample separation device that separates a plurality of types of samples flowing in a flow path, the light source, a beam diameter adjustment optical system that adjusts the beam diameter of light from the light source, and the beam diameter adjustment Based on the objective lens that irradiates the sample with the sampled light, the detection unit that detects the sample flowing through the flow path, and the detection result of the sample by the detection unit, it controls the irradiation of the sample with the beam diameter adjusted. A processor for separating the sample in a desired direction in the flow path, and applying a light trapping force generated by irradiating the sample with light to the sample in the XY plane, the processor has a pupil filling factor of There is provided a sample sorting device for controlling a beam diameter adjusting optical system to adjust a beam diameter of light so that it becomes 0.43 or more and 0.93 or less.

  According to another embodiment, a method of irradiating a sample with light and capturing the beam, wherein the processor indicates a beam diameter value that provides a light trapping force in a combination of sample size and objective lens numerical aperture. Referring to the information, obtain the value of the beam diameter corresponding to the combination of the sample size of the sample to be captured and the numerical aperture of the objective lens actually used, and control the beam diameter adjustment optical system to obtain the acquired beam diameter. A method is provided that includes adjusting and irradiating the sample with light having an adjusted beam diameter with an objective lens actually used.

  According to another embodiment, a method of capturing light by irradiating a sample with a sample, the optical filling force generated by irradiating the sample with light being applied to the sample in the XY plane, the pupil filling factor The beam diameter of the light from the light source is adjusted by the beam diameter adjusting optical system so that the light is adjusted to 0.43 or more and 0.93 or less, and the sample is irradiated with the light having the adjusted beam diameter by the objective lens. And a method is provided.

  According to another embodiment, a method of capturing light by irradiating a sample with a sample, wherein when the light trapping force generated by irradiating the sample with light is applied to the sample in the Z direction, the pupil filling factor is Adjusting the beam diameter of the light from the light source by the beam diameter adjusting optical system so as to be 0.56 or more and 0.96 or less, irradiating the sample with the light whose beam diameter has been adjusted by the objective lens, A method is provided.

It is a figure which shows the relationship between the sample size (for example, diameter in FIG. 1), the numerical aperture NA of an objective lens, and the pupil filling factor in the XY plane. It is a figure which shows the relationship between the sample size (for example, diameter in FIG. 2) and the numerical aperture NA of an objective lens, and a pupil filling factor in a Z direction. It is a figure which shows the range of the optimal beam diameter (optimal pupil filling factor) which can be read from FIG. It is a figure showing an example of schematic structure of optical trap device 1 by a 1st embodiment of this indication. 5 is a flowchart for explaining an operation of the optical trap device 1 according to the first embodiment of the present disclosure. It is a figure which shows the schematic structural example of the optical trap apparatus 2 by 2nd Embodiment of this indication. It is a figure which shows schematic structure of the cell sorter apparatus 3 by 3rd Embodiment of this indication. It is a figure which shows the structural example when the sorting flow path 300 used with the cell sorter apparatus 3 by 3rd Embodiment of this indication is seen from right above. It is a flowchart for demonstrating operation | movement of the cell sorter apparatus 3 by 3rd Embodiment of this indication. It is a figure which shows the schematic structural example of the optical trap system 4 by 4th Embodiment of this indication. It is a figure which shows the schematic structural example of the cell sorter apparatus 5 by the modification of 3rd Embodiment of this indication.

  Hereinafter, the present embodiment will be described with reference to the accompanying drawings. In the accompanying drawings, functionally identical elements may be denoted by the same numbers. Note that the attached drawings show an embodiment and an implementation example according to the principle of the present disclosure, but these are for understanding the present disclosure and are never used to interpret the present disclosure in a limited manner. is not. The descriptions in this specification are merely exemplary, and are not intended to limit the scope of the claims or the application in any way whatsoever.

  This embodiment has been described in sufficient detail for those skilled in the art to implement the present disclosure, but other implementations and forms are possible, without departing from the scope and spirit of the technical idea of the present disclosure. It is necessary to understand that the configuration and structure can be changed and various elements can be replaced. Therefore, the following description should not be interpreted as being limited to this.

  Further, as will be described later, the present embodiment may be implemented by software running on a general-purpose computer, or may be implemented by dedicated hardware or a combination of software and hardware.

(1) Consideration of optimum beam diameter <Presence / absence of optimum beam diameter>
The principle of optical traps has been well known in the past, but the optimum of laser light that is incident on the entrance pupil of the objective lens (the intensity of the laser light is insufficient to generate sufficient trapping force). The beam diameter has not been studied. Here, the “optimal beam diameter” means a beam diameter that maximizes the optical trapping force. As a result of various optical theoretical calculations regarding this point, the following was found.

(I) Due to the fact that the laser beam is focused by the objective lens to generate an illumination beam for the trapped object, if the beam diameter is very thin, the effective numerical aperture will be small, so it will be strongly focused at the focal position of the objective lens. Since this is not possible, the diameter of the light spot at the focal position is not reduced, and the trapping force is weakened.

(Ii) On the other hand, a laser beam generally used for an optical trap uses a laser beam whose intensity distribution in the cross section perpendicular to the propagation direction is a Gaussian distribution. The amount of light that can pass is reduced, and the trapping power is also weakened. If the spot diameter of the laser beam is the same, the trapping force is proportional to the intensity of the illumination beam. Therefore, if the light does not enter the objective lens, the trapping force becomes weak.

  Based on the above verification results, it is considered that there is an optimum value for generating a strong trapping force in the beam diameter (illumination beam diameter) when laser light is incident on the objective lens.

Ｅ（ｘ，ｙ，ｚ）をガウスビームが対物レンズ瞳に入射したことを考慮した電場振幅分布として、光トラップ力を数式２（参考文献における式（４４））により、

光が被トラップ物で屈折することによる勾配力とＦ_ｇｒａｄ（ｒ）と光が被トラップ物表面で散乱することによる散乱力Ｆ_ｓｃａｔ（ｒ）との和で計算することができる。 <Relationship between beam diameter, sample size and numerical aperture NA of objective lens>
Regarding the optical trapping force, the following can be said roughly. That is, when light enters a trapped object (for example, a sphere such as a bead), the incident light is refracted because the trapped object has a refractive index. The fact that light is refracted means that the momentum of incident light to the trapped object is different from the momentum of light emitted from the trapped object. In addition, part of the light incident on the trapped object may be scattered. Since the momentum also changes due to the scattering of light, it acts on the trapped object as a force that transmits the change in the total momentum of the light incident on the trapped object and transmitted and the scattered light. Using this fact, the optical trapping force is calculated. The magnitude of such an optical trapping force is "Optical trapping of dielectric particles in arbitrary fields", A. Rohrbach and EHK Stelzer, Opt. Soc. Am. A, Vol. 18, No. 4, (2001) 839- It can be calculated based on a calculation formula shown in 853 (hereinafter referred to as a reference). Specifically, in the following formula 1 (the formula (3) in the reference) representing the electric field amplitude of the incident beam,

Assuming that E (x, y, z) is an electric field amplitude distribution considering that the Gaussian beam is incident on the objective lens pupil, the optical trapping force is expressed by Equation 2 (Equation (44) in the reference).

Light can be calculated by the sum of the scattering power F _scat due to the gradient force and F _{grad (r)} and light due to refraction in the trap object is scattered by the trap piece surface _(r).

  The light trapping force can be generated efficiently by minimizing the loss of light to the trapped object. When the numerical aperture NA of the objective lens is large, the effect of light refracted by the trapped object is large. This is because, as the beam diameter is larger, light with an angle is incident on the trapped object, so the light trapping force is not greatly dependent on the size of the trapped object and the light incident on the trapped object is greatly bent. This is because it becomes larger. On the other hand, the effect of light scattered on the surface of the trapped object when the numerical aperture NA of the objective lens is large is that the light spot diameter decreases as the beam diameter increases. In many cases, the light is reflected and scattered in the direction opposite to the incident direction, and does not contribute to traps in the XY plane perpendicular to the optical axis. With this, it is impossible to generate the light trapping force by using the illumination light efficiently. If the object to be trapped is relatively large, the effect of scattering exceeds the above two forces. Therefore, by narrowing the beam diameter and increasing the spot diameter of the illumination light generated by the objective lens, It is better to illuminate the object with illumination light as widely as possible. From the above, it can be said that the optimum beam diameter for increasing the optical trapping force strongly depends on the size of the trapped object.

  Also, as a result of verification, for example, when the numerical aperture NA of the objective lens is large, it is found that it is better to make the beam diameter narrower and enter the objective lens (a stronger optical trapping force is generated). did. This is because light outside the light having an angle that generates the maximum trapping force does not affect the strength of the optical trapping force so much. In other words, when the numerical aperture NA of the objective lens is large, it is better to put light a little thinner than to fill the outer circumference of the objective lens so that the light is not cut much by the diaphragm. On the other hand, when the numerical aperture NA of the objective lens is small, it is better to put light into the entire outer periphery of the objective lens. As described above, the optimum beam diameter for increasing the optical trapping force also depends on the numerical aperture NA of the objective lens.

As described above, there are two optical trapping forces: the trapping force in the XY plane and the trapping force in the Z direction. Therefore, it is necessary to consider these two trapping forces separately. FIG. 1 is a diagram showing the relationship between the sample size (for example, the diameter in FIG. 1), the numerical aperture NA of the objective lens, and the pupil filling factor in the XY plane. FIG. 2 is a diagram showing the relationship between the sample size (for example, the diameter in FIG. 2), the numerical aperture NA of the objective lens, and the pupil filling factor in the Z direction. Here, the pupil filling factor is defined by the incident beam diameter / the pupil diameter of the objective lens. The incident beam diameter is defined by the diameter of 1 / e ² of the incident laser light. The curved surface of the pupil filling factor in FIGS. 1 and 2 (the relationship between the sample size and the numerical aperture NA of the objective lens and the pupil filling factor) shows an optimum relationship in which the trapping force is theoretically maximum. This theoretically maximum trapping force is called "maximum trapping force". When the sample is actually trapped, a trapping force of 90% or more of the maximum trapping force is allowed. The range of the pupil filling factor that gives a value of 90% or more of the maximum trapping force is in the range of 80% to 130% from the curved surface of the pupil filling factor of FIGS. The “storage device” described later is a beam diameter that is 90% or more of the maximum trapping force, together with beam diameter information indicating the value of the beam diameter at which the trapping force is theoretically maximum in the combination of the sample size and the numerical aperture of the objective lens. The beam diameter information indicating the value of is stored.

  The graphs shown in FIGS. 1 and 2 (the relationship between the sample size and the numerical aperture NA of the objective lens and the pupil filling factor) give a plurality of pairs of the sample size and the numerical aperture NA of the objective lens, and the maximum trap in each pair. It is obtained by calculating the force based on the above-mentioned reference and obtaining the value of the pupil filling rate at that time.

  Since light is a kind of electromagnetic wave, it has an electric field direction component and a magnetic field direction component. In FIG. 1, “the relationship between the size of the trapped object (sample) and the numerical aperture NA of the objective lens and the pupil filling factor” in the electric field direction (E field) and the “sample size and objective lens in the magnetic field direction (H field)”. The relationship between the numerical aperture NA and the pupil filling factor "is shown. As can be seen from FIG. 1, in the XY plane, there is almost no difference in the optimum pupil filling rate between the electric field direction and the magnetic field direction. Therefore, the optimum pupil filling rate does not need to be determined by separately verifying the electric field direction component and the magnetic field direction component in the laser light to be irradiated. As a result of calculation with respect to the XY plane, it has been found that there is almost no difference in the optimal pupil filling rate between the electric field direction and the magnetic field direction, so in FIG. 2 (Z direction), the characteristic of only one direction (for example, only the electric field direction) is I decided to show.

  Comparing the characteristics in the XY plane (FIG. 1) and the characteristics in the Z direction (FIG. 2), when the sample size (for example, diameter) is small, both show almost the same value, but the sample size increases. It can be seen that the gap between the two increases. That is, except when the sample size is small, in general, the pupil filling rate that gives the maximum optical trapping force in the XY plane is different from the pupil filling rate that gives the maximum optical trapping force in the Z direction. It can be said that it is necessary to control. However, even if there is a discrepancy between the two characteristics, the maximum optical trapping force is not given, but the pupil filling rate that gives a good optical trapping force both in the XY plane and in the Z direction is obtained. Is possible. It should be noted that the larger the sample size, the weaker the optical trapping force and the heavier the trapped object itself, so that the trapped object cannot be trapped. For example, the trappable sample size is preferably about 10 μm or less in diameter.

  1 and 2, fitting processing is performed on each point indicating the optimal pupil filling rate (optimal beam diameter) obtained based on the above-mentioned reference, and the optimal pupil filling rate is set as a parameter (sample size and objective lens). It shows what kind of curved surface is constituted by the change in the numerical aperture NA). Here, the amount of calculation is reduced using fitting processing, but an optimal pupil filling rate may be calculated for all combinations of the sample size and the numerical aperture NA of the objective lens.

<Conclusion about optimal beam diameter (optimal pupil filling factor)>
From the above consideration, it has been found that there is an optimum beam diameter (optimum pupil filling factor) for maximizing the optical trapping force. It was also confirmed that the optimum beam diameter value is affected (dependent) by the sample size and the numerical aperture NA of the objective lens. In particular, the sample size and the numerical aperture NA of the objective lens are highly dependent. In addition, the beam diameter (pupil filling factor) that gives the maximum optical trapping force in the XY plane and the beam diameter (pupil filling factor) that gives the maximum optical trapping force in the Z direction do not necessarily match and can be controlled independently. I also found it necessary. That is, since the behavior of the optical trapping force is different between the XY plane and the Z direction, the beam diameter (pupil filling factor) to be selected is selected between the case where the optical trap is used in the XY plane and the case where the optical trap is used in the Z direction. ) Needs to be changed. For example, when performing an operation of optically trapping beads and rolling on a substrate, it is important to select an optimum beam diameter that maximizes the optical trapping force in the XY plane. On the other hand, for example, when a bead is lifted from the substrate in the Z direction, it is important to select an optimum beam diameter that maximizes the optical trapping force in the Z direction.

  From the graphs shown in FIGS. 1 and 2, it is difficult to determine the optimum beam diameter (optimum pupil filling factor) at a glance. Therefore, FIG. 3 shows the range of the optimum beam diameter (optimum pupil filling factor) that can be read from FIGS. FIG. 3A is a table showing the range of the optimum beam diameter (optimum pupil filling factor) in the XY plane. FIG. 3B is a table showing the range of the optimum beam diameter (optimum pupil filling factor) in the Z direction. In FIG. 3, a trapped object (bead) having a size (diameter) of less than 1 μm or a trapped object (bead) having a size of 1 μm or more is used in practice. Actually, an objective lens having a relatively large numerical aperture NA such as an immersion objective lens is used (in the case of an immersion objective lens, the NA can be 1 or more), and a dry objective lens. In some cases, an objective lens having a relatively small numerical aperture NA is used (in the case of a dry objective lens, the NA cannot be 1 or more). The range of the optimum beam diameter in the dry objective lens is listed as follows. When the object to be trapped is moved in a wide range (larger than the diameter of the objective lens), the immersion objective lens can be used while maintaining the immersion state. This is because it is difficult to move the lens or the container containing the trapped object. It can be seen from FIGS. 3A and B that the optimum pupil filling factor in the XY direction is 43 to 93% (0.43 to 0.93), and the optimum pupil filling factor in the Z direction is 56 to 96% (0. 56 to 0.96). In other words, the light trapping force can be increased by making the beam diameter relatively narrow with respect to the entrance pupil of the objective lens and making the laser light enter the objective lens.

  FIG. 3C is a table showing an example of the range of the allowable beam diameter (pupil filling rate giving a value of 90% or more of the maximum trapping force) in the XY plane. FIG. 3D is a table showing an example of the range of the allowable beam diameter in the Z direction (pupil filling rate that gives a value of 90% or more of the maximum trapping force). It can be seen from FIGS. 3C and D that the acceptable pupil filling factor in the XY direction is 34.4-120.9% (0.344-1.209) and the acceptable pupil filling factor in the Z direction is 44. .8 to 124.8% (0.448 to 1.248). Here, the pupil filling rate that gives a value of 90% or more of the maximum trapping force is shown, but it is possible to make it an arbitrary ratio. For example, if a value up to 80% of the maximum trapping force is allowed, a value in the range of about 70% to 150% is used for the pupil filling rate when the trapping force is maximum (in the case of an optimal pupil filling rate). It is also possible.

  As described above, if “the relationship between the sample size and the numerical aperture NA of the objective lens and the pupil filling factor” (FIGS. 1 and 2) is obtained in advance (for example, stored in a storage device (memory) as a table). Possible), by determining the sample size (for example, diameter) and the numerical aperture NA of the objective lens, the optimum pupil filling rate (beam diameter) can be uniquely determined.

(2) First Embodiment <Configuration of Optical Trap Device>
FIG. 4 is a diagram illustrating a schematic configuration example of the optical trap device 1 according to the first embodiment of the present disclosure. The optical trap device 1 includes, for example, a light source 10, a zoom beam expander (beam diameter adjusting optical system) 20, a dichroic mirror 30, an objective lens 40, a sample mounting portion (sample mounting surface) 50, and an observation device. The optical system 60, the imaging lens 70, the imaging part 80, the control apparatus 90, and the sample mounting surface drive part 100 are provided.
The light source 10 is a light source that emits coherent light, and a laser light source can be used as an example.

  The zoom beam expander 20 changes the diameter (beam diameter) of the light emitted from the light source 10 in response to, for example, a command from the control device 90, and includes a plurality of lenses. In the zoom beam expander 20, for example, the lens 21 and the lens 22 constitute a beam expander, and the lens 23, the lens 24, and the lens 25 constitute a zoom optical system. In the zoom optical system, it is better that the incident beam diameter is sufficiently large. Therefore, the light is sufficiently expanded by the beam expander (the lens 21 and the lens 22), and the zoom optical system substantially reduces or reduces the light. As a result, a beam diameter incident on the objective lens 40 is generated. The beam diameter of the laser beam is about 1 mm, whereas the desired beam diameter at the pupil position of the objective lens 40 is about several mm to several tens of mm. Therefore, the beam expander is indispensable depending on the lens configuration. It is not a configuration.

  The dichroic mirror 30 is an optical element (mirror) having an action of reflecting light of a specific wavelength and transmitting light of other wavelengths. Thereby, the light from the light source (eg, laser light source) 10 is reflected and incident on the objective lens, while the light from the trapped object placed on the sample placement unit 50 is transmitted to the imaging unit 80. It becomes possible to guide.

  The objective lens 40 condenses the light to be irradiated on the sample (the trapped object) placed on the sample placement unit 50. When the collected light (laser light) is irradiated to the trapped object, an optical trapping force is generated according to the principle described above. When the optical trapping force acts on the trapped object, for example, a specific trapped object can be moved or captured.

The observation optical system 60 includes, for example, an illumination unit 601 that emits observation illumination light, and a condenser lens 602 for effectively condensing the observation illumination light on the sample surface. For example, it is installed to confirm the coordinate position of the trapped object or to observe how the trapped object is trapped.
The imaging lens 70 forms an image of the observation illumination light that has passed through the objective lens 40 and has not passed through the dichroic mirror 30 on the image plane.
The imaging unit 80 is configured by, for example, a CCD or a CMOS, and images a trapped object imaged on the image plane. The captured image is transmitted to the control device 90.

  The control device 90 includes, for example, a processor 91, an input device 92, an output device 93, a communication device 94, and a storage device 95, as in a normal computer.

  The storage device 95 uses, for example, a beam diameter table 951 that holds values given by “relationship between sample size and numerical aperture NA of the objective lens and pupil filling factor” in FIGS. 1 and 2, and a beam diameter table 951. And at least a control program 952 for acquiring an optimum beam diameter.

The input device 92 includes, for example, a changeover switch, a mouse, a keyboard, a touch panel type pointing device (operated with a stylus pen or a finger), a microphone, and the like, and is used to input setting information, instructions, and data. In the case of the optical trap device 1 according to the present embodiment, for example, the operator uses the input device 92 to input the size of the trapped object (sample) and the processing number NA of the objective lens 40 to the control device 90. When the size of the trapped object (sample) is unknown, the trapped object is imaged by the imaging unit 80 before being operated by irradiating the trapped object with light (laser light). The size of the sample) is specified, and an optimum beam diameter can be acquired based on the specified size and the known numerical aperture NA of the objective lens 40. Details of this processing will be described later.
The output device 93 includes, for example, a display device, a printer, a speaker, and the like, and is used for outputting data and information.

  The processor 91 is composed of, for example, a CPU, an MPU, and the like, reads a control program 952 stored in the storage device 95, refers to the beam diameter table 951, and corresponds to the pupil corresponding to the sample size and the numerical aperture NA of the objective lens 40. A filling factor is acquired, and an optimum beam diameter is calculated from the pupil filling factor. The processor 91 controls the drive unit (not shown) of the zoom beam expander 20 based on the calculated optimum beam diameter value, and adjusts the diameter (beam diameter) of the laser light emitted from the lens 25. Further, for example, when the operator instructs the movement, movement distance, and movement direction of the sample (target object) placed on the sample placement unit 50 using the input device 92, the processor 91 receives the input. In response to the instruction, the sample placement surface driving unit 100 is controlled to move the trapped object. The sample placement surface driving unit 100 is configured to be able to move the sample placement unit 50 in the XY plane and in the Z direction.

  The communication device 94 is a device that communicates with an external device or a network. In the case of this embodiment, for example, the communication device 94 receives data and information from the imaging unit 80, or drives the zoom beam expander 20 (not shown). ) Or an instruction to the sample placement surface driving unit 100.

<Operation of optical trap device>
FIG. 5 is a flowchart for explaining the operation of the optical trap device 1 according to the first embodiment of the present disclosure. The processor 91 of the control device 90 reads, for example, a control program 952 from a storage device (eg, ROM (Read Only Memory)) into an internal memory (not shown) provided in the processor 91, and executes the control program 952. . Hereinafter, the operation of the optical trap device 1 will be described with reference to FIG.

(I) Step 901
The imaging unit 80 acquires an image of the sample placement unit 50 that is illuminated with the observation illumination light from the illumination unit 601 of the observation optical system 60. For example, the image capturing unit 80 acquires each trapped object (for example, one or more trapped objects are placed) placed on the sample placing unit 50 from the acquired image of the sample placing unit 50. Calculate the coordinate position. In the present embodiment, the field of view of the objective lens 40 does not cover the entire area of the sample mounting section 50, but covers the entire area of the sample mounting section 50 by moving the sample mounting section 50 by the sample mounting surface driving section 100. Like to do. Therefore, the imaging unit 80 may not be able to calculate the coordinate positions of all the trapped objects placed on the sample placement unit 50 by one imaging. Therefore, for example, the entire area of the sample mounting unit 50 is divided into a plurality of parts in advance (for example, one divided area can be set to an arbitrary size smaller than the field of view of the objective lens 40. Then, the coordinate position in each divided region is calculated by the imaging unit 80, and these are combined to calculate the absolute coordinates of all trapped objects.
The imaging unit 80 transmits the acquired image of the sample placement unit 50 and information on the calculated coordinate position of each trapped object to the control device 90.

  The calculation of the coordinate position of the trapped object may be executed by the processor 91 of the control device 90, for example. In this case, for example, the imaging unit 80 transmits the image of the sample placement unit 50 to the control device 90 as it is.

  Note that the reference position (origin position) of the coordinates calculated by the imaging unit 80 is the reference position (origin position) of the coordinate system when the sample placement surface driving unit 100 moves the sample placement part 50 or the laser beam irradiation. It is preferable that the same coordinate system (origin position) is used. However, when these coordinate systems are different, for example, the processor 91 preferably adjusts appropriately.

(Ii) Step 902
For example, the processor 91 gives an operation order to each trapped object according to a predetermined rule. Here, as the predetermined rule, for example, the top left trapped object of the acquired image of the sample placement unit 50 is set as the first operation target, and the bottom right trapped object of the image is the last trapped object. It is possible to adopt the operation target.

  Then, the processor 91 transmits an alignment instruction to the sample placement surface driving unit 100 in order to align the irradiation position of the laser light with the position of the first trapped object. The sample placement surface driving unit 100 that has received the alignment instruction moves the sample placement unit 50 in response to the instruction, and matches the position of the trapped object to be trapped with the irradiation position of the laser beam.

(Iii) Step 903
The processor 91 determines whether an instruction to calculate the size of the trapped object (sample) is input from the input device by the operator. If an instruction to calculate the size of the trapped object (sample) is input (YES in step 903), the process proceeds to step 904. When the instruction is not input (NO in Step 903: For example, when the value of the sample size has already been input, or the size of the trapped object (sample) has already been calculated in another process) ), The process proceeds to step 905.

  If the instruction for calculating the size of the trapped object (sample) is not input and the value of the size of the trapped object (sample) is not input by the operator, the processor 91 The processing may be shifted to the step of calculating the size of the sample), or the value of the size of the trapped object (sample) may be input, or the instruction for calculating the size of the trapped object (sample) may be input An alert may be output from the output device 93.

(Iv) Step 904
The processor 91 acquires, for example, an image of the trap target to be trapped and information on the imaging magnification from the imaging unit 80, and the size (for example, sample) of the trap target (sample) from the acquired image of the trap target and the imaging magnification. The diameter is calculated when the trapped object is spherical, and the short side and the long side length are calculated when the trapped object is horizontally long.

(V) Step 905
First, the processor 91 specifies the type of the beam diameter table 951 based on an instruction input by the operator regarding whether to apply the optical trapping force in the XY plane or to apply the optical trapping force in the Z direction. When the application of the optical trapping force in the XY plane is instructed, for example, the table of FIG. 1 is selected, and when the application of the optical trapping force in the Z direction is instructed, for example, the table of FIG. Is selected.

The processor 91 refers to the selected beam diameter table 951, and the numerical aperture NA of the objective lens 40 and the pupil diameter of the objective lens 40, which are input in advance by the operator, and the trapped object (sample) that is input in advance by the operator. ) Or the size of the trapped object (sample) calculated in step 904, the irradiation beam diameter of the laser light is calculated. That is, as described above, the beam diameter table 951 stores the relationship between the sample size and the numerical aperture NA of the objective lens and the pupil filling factor (for example, information in FIGS. 1 and 2). If the numerical aperture NA of the objective lens is known, the pupil filling factor (= (diameter of 1 / e ² of the irradiation laser beam) / (pupil diameter of the objective lens)) can be known, and the beam diameter of the irradiation laser beam (1 of the irradiation laser beam). / E ² diameter).

(Vi) Step 906
As an example, the processor 91 moves the lens 23 and the lens 24 of the zoom beam expander 20 by a drive unit (not shown) of the zoom beam expander 20 based on the value of the beam diameter calculated in step 905, and Adjust the beam diameter to the calculated value.

(Vii) Step 907
The processor 91 instructs the light source (laser light source) 10 to start irradiating the trapped object (trap target) with laser light. When the object to be trapped is irradiated with the laser beam, the maximum trapping force is generated under the condition of the given sample size and the numerical aperture NA of the objective lens 40, and the object to be trapped can be trapped. It becomes like this.

(Viii) Step 908
The processor 91 acquires information on the moving direction and moving distance input by the operator using the input device, and moves the sample mounting unit 50 in the input moving direction by the moving distance. 100 is controlled. Then, the position of the trapped object held by the irradiation laser beam by the optical trapping force relatively changes in the sample placement unit 50. The optical trap device 1 illustrated in FIG. 4 is configured to relatively change the position of the trapped object by moving the sample mounting unit 50 because the position of the laser light irradiation optical system is fixed. However, for example, when the optical trap device 1 has a shape that can be operated by a micromanipulator (for example, a configuration that can be attached to a device such as a pen-type optical trap device or a micropipette manipulator) If the irradiation position of the laser beam is changed while holding the trapped object by the optical trap device 1, the position of the trapped object can be moved. The pen-type optical trap device will be further described in the fourth embodiment.

(Ix) Step 909
For example, the processor 91 controls the imaging unit 80, captures an image of how the object to be trapped is held and moved by the optical trapping force, and displays the captured image on a display screen of an output device (for example, a display). The captured video is stored in the storage device 95.

(X) Step 910
For example, the processor 910 refers to the processing order information given in step 902 and determines whether there is an unprocessed trapped object. If there is an unprocessed trapped object (YES in step 910), the process returns to step 907. If all trapped objects to be trapped have been operated (NO in step 910), the process proceeds to step 911.

(Xi) Step 911
The processor 911 stops the irradiation of laser light from the light source (for example, laser light source) 10.

<Summary of First Embodiment>
In the optical trap device 1 according to the first embodiment, as an example, referring to the optimum beam diameter information (corresponding to the beam diameter table 951), the numerical aperture of the objective lens 40 used in the optical trap device 1 and the target coverage Information on the beam diameter corresponding to the combination with the size (for example, diameter) of the trapped object (sample) is acquired. When the trapped object is irradiated with light (for example, laser light) with the acquired beam diameter, the trapped object can be captured with an appropriate trapping force (for example, maximum value). Since the beam diameter is determined using the optimum beam diameter information prepared in advance as described above, the optimum beam diameter corresponding to the combination of the numerical aperture of various objective lenses 40 and the size of the trapped object (sample). Will be able to determine.

  For example, if the sample size is known, the operator inputs information about the sample size and the numerical aperture of the objective lens 40 from the input device 92 to the optical trap device 1, and sets the beam diameter corresponding to the combination to the beam diameter table 951. It may be determined in advance based on the above. If the sample size is almost uniform, the optimum beam diameter has only to be obtained once, so that the operator can efficiently operate the trapped object with an appropriate trapping force. On the other hand, for example, when the sample size is unknown or when trapped objects of a plurality of sizes are mixed, the size is specified from the image of the trapped object captured by the imaging unit 80 each time the operation is performed, and the beam Based on the diameter table, an optimum beam diameter corresponding to the specified sample size and the numerical aperture of the objective lens 40 may be obtained. As a result, even when various types of trapped objects are mixed, the optimum beam diameter can be automatically acquired.

  In the first embodiment, for example, the position of the object to be trapped on the sample placement surface is changed by moving the sample placement portion (sample placement surface) 50. In this case, the size of the condenser lens 602 of the observation optical system 60 can be reduced. As the objective lens 40, both an immersion objective lens and a dry objective lens can be used. For example, the sample placement portion (sample placement surface) 50 can be used in a wide range (for example, the tip lens of the objective lens). When moving it over a wider range than the range of the diameter, it is preferable to use a dry objective because there is a risk that the immersion liquid may run out.

(3) Second Embodiment In the first embodiment, the object to be trapped is moved in the sample mounting unit 50 by moving the sample mounting unit 50 by the sample mounting surface driving unit 100. In the second embodiment, the sample placement unit 50 is fixed, and the laser beam irradiation optical system 200 is moved by the stage 210 to move the trapped object in the sample placement unit 50.

  FIG. 6 is a diagram illustrating a schematic configuration example of the optical trap device 2 according to the second embodiment of the present disclosure. FIG. 6A shows an example of the overall configuration of the optical trap device 2. FIG. 6B shows a configuration example of the laser light irradiation optical system 200. In FIG. 6, the same components as those of the optical trap device 1 (see FIG. 4) according to the first embodiment are denoted by the same reference numerals.

  6A, the optical trap device 2 includes, for example, an irradiation optical system 200, a stage 210 for moving the irradiation optical system 200 in the XY plane and in the Z direction, an observation optical system 60, and a control device 90. And.

  As in the first embodiment, the observation optical system 60 includes, for example, an illumination unit 601 that emits observation illumination light, and a condenser lens 602 that effectively collects the observation illumination light on the sample surface. Prepare. The observation optical system 60 may be configured to move in synchronization with the movement operation of the irradiation optical system 200 by the stage 210, but here, for example, the observation illumination light is applied to the entire region of the sample placement unit 50. The observation optical system 60 is configured so that it can be illuminated. For this reason, for example, a condenser lens 602 having a larger diameter than that in the first embodiment is used. By doing so, the observation optical system 60 can be fixed, and it is not necessary to provide a drive system for moving the observation optical system 60, so that a cost advantage can be obtained.

  The configuration and operation of the control device 90 in the second embodiment are substantially the same as in the case of the first embodiment, but in the second embodiment, the sample placement surface driving unit 100 of the first embodiment is configured. Instead, the operation of the stage 210 is controlled. Since other operations are the same as those of the control device 90 in the first embodiment, a description thereof will be omitted. In the second embodiment, the “sample placement surface drive unit” (corresponding to the sample placement surface drive unit 100) in Step 908 of FIG. 5 is read as a stage (corresponding to the stage 210).

<Summary of Second Embodiment>
Similar to the first embodiment, since the beam diameter is determined using the optimum beam diameter information prepared in advance, the optimum beam diameter corresponding to various combinations of numerical apertures and sample sizes of the objective lens 40 is used. Will be able to determine.
The determination of the optimum beam diameter when the sample size is known, when the sample size is unknown, or when trapped objects of a plurality of sizes are mixed is the same as in the first embodiment.

  On the other hand, in the second embodiment, unlike the first embodiment, for example, the position of the trapped object on the sample mounting surface is changed by moving the laser light irradiation optical system 200. In this case, it is preferable to increase the size of the condenser lens 602 of the observation optical system 60. For example, by linking the observation optical system 60 with the irradiation optical system 200, the condenser lens 602 is made to be the same as that of the first embodiment. Can be the same size as the case. As in the first embodiment, as the objective lens 40, both an immersion objective lens and a dry objective lens can be employed. For example, when the laser light irradiation optical system 200 is moved over a wide range (for example, over a wide range than the range determined by the pupil diameter of the objective lens), it is preferable to use a dry objective lens.

(4) Third Embodiment The third embodiment of the present disclosure is a cell sorter device (sample sorting device) using the optical trap device 1 according to the first embodiment or the optical trap device 2 according to the second embodiment. 3 is disclosed.

<Configuration of cell sorter>
FIG. 7 is a diagram showing a schematic configuration of the cell sorter device 3 according to the third embodiment. The cell sorter device 3 includes, for example, a light source 10, a zoom beam expander 20, a position adjusting mirror pair 311, a relay lens 313, a dichroic mirror 30, an objective lens 40, a sorting channel 300, and an observation optical system. 60, an imaging lens 70, a mirror driving unit 310, an imaging unit 320, and a control device 330.

  The position adjustment mirror pair 311 is provided to change the irradiation position of the laser beam, and is configured by an X direction position adjustment mirror 3111 and a Y direction position adjustment mirror 3112 as an example. Yes. However, the mirror 3112 may be used for X direction adjustment, and the mirror 3111 may be used for Y direction adjustment.

  The relay lens 313 is installed to place the pupil conjugate plane 312 of the objective lens 40 at a substantially central position between the X-direction position adjustment mirror 3111 and the Y-direction position adjustment mirror 3112.

  In the case of the configuration shown in FIG. 7, the center position of the beam (irradiated laser light) moves in the XY direction on the pupil plane of the objective lens 40, but the X-direction position adjustment mirror 3111 and the Y-direction position adjustment By making the mirror 3112 as close as possible, the shift of the center position of the beam can be suppressed to a small level, which can be suppressed to a level that is practically no problem.

  If the focal lengths of the two lenses constituting the relay lens system 313 are f1 and f2, respectively, from the objective lens 40 side, the lenses are respectively installed at a distance of f1 and a position of 2f1 + f2 from the pupil plane of the objective lens 40. In this case, a pupil conjugate plane 312 is generated at a position 2f1 + 2f2 measured from the pupil plane of the objective lens 40. The simplest case is when f1 = f2, but when f1 ≠ f2, the diameter of the beam incident on the pupil of the objective lens 40 is f1 / f2 times. For this reason, it is necessary to generate a beam diameter in consideration of this with the zoom beam expander 20.

  Since the configurations and functions of the light source 10, the zoom beam expander 20, the dichroic mirror 30, the objective lens 40, the observation optical system 60, and the imaging lens 70 are the same as those in the first embodiment, detailed description thereof is omitted. To do.

  In the cell sorter device 3, a sorting flow path 300 is provided instead of the sample placement unit 50 in the first embodiment. The sorting flow path 300 is a device for selecting a plurality of types of cells (for example, red blood cells, white blood cells, and platelets in blood) and collecting the same type of cells. Examples of selection and separation targets include cells (normal and abnormal cells), sperm, viruses, bacteria, organelles or small parts, spherical structures, colloidal suspensions, lipids and fat globules, gels, immiscible particles , Blastomeres, cell aggregates, microorganisms and other biological materials.

  In the cell sorter device 3, each mirror of the position adjustment mirror pair 311 is configured such that the angle can be adjusted by, for example, a mirror driving unit 310 driven by an instruction from the control device 330. The angle of each mirror of the position adjustment mirror pair 311 can be adjusted independently. When the angle of each mirror changes, the position of the laser beam irradiated to the sorting flow path 300 changes. As a result, an object to be trapped (sample (cells, etc.)) that has reached the flow path branching portion 307 of the sorting flow path 300 can be trapped by the optical trapping force of the laser light and sorted into a desired sorting destination flow path. It becomes. The configuration of the sorting channel 300 will be described later (see FIG. 8).

  For example, in addition to the operation of the imaging unit 80 according to the first embodiment, the imaging unit 320 images a sample flowing through the sorting channel 300, and based on the captured image, the sample size (including shape), and the sample The position in the flow path is calculated, and the calculated information is transmitted to the control device 330. The calculation of the sample size (including the shape) and the calculation of the position of the sample may be executed by the processor 91. In this case, the imaging unit 320 only transmits the captured sample image to the control device 330.

  The control device 330 has substantially the same configuration as that of the control device 90 in the first embodiment. For example, the control device 330 is further provided with a sorting program 3301 instead of the control program 952 in the storage device 95. It is different from 90. The sorting program 3301 is executed by the processor 91. For example, based on the sample size (including shape) and position information received from the imaging unit 320, the sorting program 3301 is provided at the end of the sorting channel 300 from the channel branching unit 307. It is judged (selection judgment result) which sample channel should flow through. Then, the sorting program 3301 determines the position where the laser beam is irradiated based on the sorting determination result, and controls the mirror driving unit 310 so as to guide the trapped sample in a desired direction, thereby adjusting the position. The angle of each mirror of the mirror pair 311 is adjusted.

<Configuration of sorting flow path>
FIG. 8 is a diagram illustrating a configuration example when the sorting flow path 300 used in the cell sorter device 3 according to the third embodiment is viewed from directly above.
The sorting channel 300 includes, for example, a liquid reservoir 301 that stores a mixed solution (for example, a liquid containing a plurality of types of cells) before sorting, a main channel 302, and a first branch channel 303 branched from the main channel 302. And the second branch flow channel 304, the first output unit 305 that is a part for collecting and taking out the sorted first type sample (for example, first type cell) 308, and the sorted second type sample. A second output unit 306 which is a part for storing and taking out (for example, second type cells) 309. FIG. 8 shows an example in which the mixed liquid is separated into two and taken out. However, the number of separations (that is, the number of branch channels) is not limited to two, depending on the application. It is possible to set more than that.

  The flow path branching portion 307 in the sorting flow path 300 is, for example, a portion that is divided into the branch flow paths 303 and 304 from the main flow path 302, and at the same time, the laser light emitted from the light source 10 is a sample (for example, a first type cell). And a second type cell), and the light trapping force is provided to the sample.

  The movement of the sample (the first type cell and the second type cell) from the liquid reservoir 301 to the output units 305 and 306 via the main flow path 302 is performed by, for example, flowing sheath liquid from the liquid reservoir 301 to the main flow path 302. Or by charging each sample and applying an electric field to the liquid reservoir 301 to output each sample from the liquid reservoir 301 to the main flow path 302. For example, it is also possible to introduce a sample using an electroosmotic flow. In this case, it is preferable to use a hydrophilic material such as glass as the material of the sorting channel 300. Since the glass is negatively charged, positive ions in the sample are pulled by the negative charge of the glass, and an electric double layer is formed. When a voltage is applied here, the charged portion in the sample moves, and the entire sample solution flows from the liquid reservoir 301 to the main flow path 302 by being pulled by this. This is the principle of sample introduction using electroosmotic flow.

<Operation of cell sorter>
FIG. 9 is a flowchart for explaining the operation of the cell sorter device 3 according to the third embodiment of the present disclosure. For example, the processor 91 of the control device 330 reads a sorting program 3301 from a storage device (for example, ROM (Read Only Memory)) 95 into an internal memory (not shown) provided in the processor 91 and executes the sorting program 3301. To do. Hereinafter, the operation of the cell sorter apparatus 3 will be described with reference to FIG.

(I) Step 1301
The processor 91 inputs the size of various samples (for example, a plurality of types of cells contained in the mixed solution) input by an operator (for example, the diameter is a sample when the sample is spherical, and the size is short when the sample is a horizontally long shape). Information on the numerical aperture NA of the objective lens 40 used in the cell sorter device 3 is read from, for example, the storage device 95, and the optimum beam diameter corresponding to various samples is calculated. . The optimum beam diameter varies depending on the sample size. For example, when the difference in the size of various samples is less than or equal to a predetermined value, only one beam diameter value is prepared and used. Anyway. Furthermore, for example, the difference in size of k (2 ≦ k <n) integer samples among n (n ≧ 3) types of samples is less than a predetermined value, and the difference in size of the remaining types of samples. Is larger than a predetermined value, a common beam diameter may be applied only to k types of samples whose size difference is equal to or smaller than the predetermined value.

In the case of the cell sorter apparatus 3, since the optical trapping force is applied in the XY plane, for example, the table of FIG. 1 is selected as the beam diameter table 951. The processor 91 refers to the selected beam diameter table 951, and sets various samples based on the numerical aperture NA of the objective lens 40, the pupil diameter of the objective lens 40, and the sample sizes of the various samples, which are input in advance by the operator. The optimum irradiation beam diameter of the corresponding laser beam is calculated. That is, as described above, since the beam diameter table 951 stores the relationship between the sample size and the numerical aperture NA of the objective lens and the pupil filling factor (see FIG. 1), the sample size and the numerical aperture NA of the objective lens are stored. Is known, the pupil filling factor (= (diameter of 1 / e ² of the irradiation laser light) / (pupil diameter of the objective lens)) is known, and the beam diameter of the irradiation laser light (1 / e ² of the irradiation laser light). Can be requested.

(Ii) Step 1302
The processor 91 instructs the imaging unit 320 to image the sample passing through the flow path branching unit 307 of the sorting flow channel 300, and acquires an image of the sample passing through the flow path branching unit 307 from the imaging unit 320. .

  For example, the imaging unit 320 may calculate the size of the sample from the acquired sample image. In this case, the processor 91 also acquires sample size information from the imaging unit 320. For example, the size of the sample may be calculated from the image acquired by the processor 91 from the imaging unit 320 and the imaging magnification.

(Iii) Step 1303
Based on the sample image acquired from the imaging unit 320, the processor 91 identifies the type of sample that is passing through the flow path branching unit 307. For example, the processor 91 extracts feature points from the acquired sample image, and compares and collates the extracted feature point information with the feature point information of various samples stored in the storage device 95 in advance. The type of sample can be specified. At that time, information on the size of the sample may be further used.

(Iv) Step 1304
Based on the type of sample specified in step 1303, the processor 91 obtains a beam diameter value (calculated in step 1301) used when sorting the sample, and drives the zoom beam expander 20 (not shown). To adjust the beam diameter of the laser beam to an optimum beam diameter.

(V) Step 1305
In the flow path branching unit 307, the processor 91 irradiates the sample to be sorted with the laser beam having the beam diameter adjusted in step 1304, generates an optical trapping force, and traps the sample. For example, since the processor 91 recognizes the type of the trapped sample at this point, the sample trapped in which branch flow path among the plurality of branch flow paths extending from the flow path branching unit 307 can be guided. You can judge whether it is okay.

(Vi) Step 1306
The processor 91 controls the mirror driving unit 310 to change the angle of at least one of the position adjusting mirror pair 311 placed at a position conjugate to the pupil plane of the objective lens 40, and the sample trapped in step 1305 is changed. It guides to a desired branch channel (a branch channel connected to an output unit for collecting a sample to be sorted). For example, the induced sample passes through the desired branch channel (the first branch channel 303 or the second branch channel 304) and arrives at the first output unit 305 or the second output unit 306. And collected.

(Vii) Step 1307
The processor 91 acquires an image (moving image or still image) obtained by imaging the sample sorting state from the imaging unit 320, outputs the image to the output device (for example, displays on the screen of the display device), and stores the image in the storage device 95. To do.
If there is no need, the processing corresponding to step 1307 may not be executed.

(Viii) Step 1308
The processor 91 determines whether or not the sorting process has been completed for all the samples held in the liquid reservoir 301. If the sorting process has been completed for all samples (YES in step 1308), the process proceeds to step 1309. If a sample that has not been sorted remains (NO in step 1308), the process proceeds to step 1302 (returns).

(Ix) Step 1309
The processor 91 controls the light source (for example, laser light source) 10 and stops the irradiation of the laser light.

<Summary of Third Embodiment>
The principle of the optical trap apparatus described in the first embodiment and the second embodiment can be applied to a cell sorter apparatus (sample sorting apparatus) 3. In the cell sorter device 3, for example, a sorting flow path 300 in which a plurality of branch flow paths 303 to 304 are provided on the output side of the main flow path 302 is installed, and any of the branch flow paths 303 to 304 extends from the main flow path 302. The optical trapping force is used when sorting into two. Also in this case, an optimum beam diameter is determined in order to generate an appropriate trapping force (maximum trapping force). The optimum beam diameter is determined by, for example, referring to the beam diameter table 951 and irradiating the cell with the size of the cell (sample) to be sorted and the laser light, as in the first and second embodiments. It can be uniquely determined from the combination with the numerical aperture of the objective lens 40 used for classification.

  In the third embodiment, for example, the size of each sample is specified from each image obtained by imaging a plurality of types of samples with the imaging unit 320, and based on this, the sample passing through the flow path branching unit 307 is determined. The optimum beam diameter of the laser beam to be applied is determined. For example, if the sample passing through the flow path branching portion 307 changes (assuming that the size is different for each sample), the optimum beam diameter also changes, so that laser beam irradiation is performed while switching the beam diameter. . By doing so, it becomes possible to divide the sample by irradiating laser beams with an optimum beam diameter to various samples and applying an optical trapping force.

  On the other hand, as will be described later in the modification shown in FIG. 11, for example, before labeling (fluorescent labeling) various samples and sorting the samples, laser light having a specific wavelength is emitted from at least one detection light source 1501 to 1502. Then, each sample flowing through the flow path may be irradiated to detect scattered light and fluorescence from each sample, and various samples may be specified based on the detection result. The optimum beam diameter is determined in accordance with the combination of the specified various sample sizes and the numerical aperture of the objective lens 40 of the irradiation optical system 200, as described above.

(5) Fourth Embodiment FIG. 10 is a diagram illustrating a schematic configuration example of an optical trap system 4 according to a fourth embodiment of the present disclosure. FIG. 10A shows the overall configuration of the optical trap system 4. FIG. 10B shows a configuration example of the laser light irradiation optical system 410 of the optical trap system 4.

  Referring to FIG. 10A, an optical trap system 4 includes, for example, a pen-shaped laser light irradiation device 400, a sample placement unit 50 on which an object to be trapped is placed, an observation optical system 60, and an observation objective. A lens 420, an imaging lens 70, an imaging unit 80, a control device 90, and a micropipette manipulator (not shown) are provided. Except for the laser light irradiation device 400 and the observation objective lens 420, they are components having the same configuration and function as the optical trap device 1 according to the first embodiment.

The laser light irradiation device 400 having a pen shape accommodates a laser light irradiation optical system 410 inside and is held by a micropipette manipulator. The micropipette manipulator is configured to operate in accordance with instructions input by an operator via a computer. The laser light irradiation optical system 410 has the same configuration as the laser light irradiation optical system of the first optical trap device 1 except that it does not have the dichroic mirror 30, and has a light source (for example, a laser light source) 10. The zoom beam expander 20 and the objective lens 40 are provided.
An image of the trapped object is formed on the image plane of the imaging unit 80 by the observation objective lens 420 and the imaging lens 70.

  The illumination unit 601 and the condenser lens 602 constituting the observation optical system 60 are configured to have a size that can irradiate observation illumination light over the entire area of the sample placement unit 50, for example. In addition, the observation objective lens 420 and the imaging lens 70 are also sized to capture an image of the entire area of the sample placement unit 50. As described above, by increasing the size of the observation optical system 60, the observation objective lens 420, and the imaging lens 70 as compared with the optical trap device 1 according to the first embodiment, the optical trapping force is applied and the trapped object is removed. There is an advantage that it is not necessary to move the sample mounting unit 50, the observation optical system 60, or the imaging unit 80 in order to capture an image of the position to be operated.

  The operation of the optical trap system 4 is the same except that step 908 in the flowchart (FIG. 5) for explaining the operation of the optical trap device 1 according to the first embodiment is not necessary, and the description thereof is omitted. To do.

<Summary of Fourth Embodiment>
According to the optical trap system 4 according to the fourth embodiment, the laser beam irradiation device 400 having a laser beam pen shape is adopted, and the operator operates the micropipette manipulator. It becomes possible to handle.

(6) Application of optical trap device (i) The optical trap device according to each embodiment is evaluated to be optimal using a predetermined evaluation method in a state where a large number of samples (for example, beads) are arranged, for example. This can be used when removing a sample. In this case, since the sample is spread on the sample mounting surface, the laser beam irradiation position must be moved over a wide range (the sample mounting surface drive unit 100 and the stage 210 are moved over a wide range) when taking out a specific sample. I must. For this reason, when an immersion objective lens (a water immersion objective lens or an immersion objective lens) is used, there is a possibility that the liquid will run out. Therefore, in such an application, it is preferable to configure the optical trap device using a dry objective lens.

  However, in general, it is known that the optical trapping force itself is larger when the immersion objective lens is used than when the dry objective lens is used. Therefore, when using the dry objective lens, it is as efficient as possible. It is desirable to construct an optical system. If an efficient optical system is configured, there is no room for design on the light source side. Therefore, in order to effectively use the light amount of the light source, in this disclosure, the beam diameter of the irradiated laser beam is set to an optimal size and the maximum trap I try to produce power.

(Ii) The optical trap device according to each embodiment can be used, for example, for measuring an interaction between proteins (use for biological research). In this case, an immersion objective lens can also be used.

  For example, various proteins appear on the surface of cells, but what kind of protein it is, or how much affinity it binds to substances that have short peptides (amino acids). In order to do this, a previously identified peptide is attached to the sphere. When a cell is brought close to this sphere, the cell sticks to the sphere. And when these are separated using optical trapping force, the interaction between proteins can be determined by measuring how much force (which can be converted from the amount of irradiation light when generating optical trapping force) can be separated. Can be measured. If they can be separated easily (with a weak force), they will simply adsorb, and if they cannot be separated easily, they will interact, and what kind of protein will appear on the cell surface. You will be able to know if you are.

(7) Modification (i) In the first and second embodiments, the irradiation position of light (for example, laser light) is changed by moving the sample surface driving unit 100 and the stage 210. For example, As in the third embodiment, a position adjustment mirror pair may be provided, and the light irradiation position may be changed by changing the angle. However, in this case, since the range of the light irradiation position is smaller than in the form in which the stage 210 or the like is moved, the application may be limited as compared with the case where the stage 210 or the like is moved.

(Ii) In the first to fourth embodiments, the processor 91 determines the optimum beam diameter value by referring to the beam diameter table 951. If the sample size is known, the processor 91 sets the sample size in advance. Correspondingly, an optimum beam diameter value may be determined. For example, as shown in FIG. 3, when applying the optical trapping force in the XY plane, applying the optical trapping force in the Z direction within a range where the pupil filling factor is 0.43 or more and 0.93 or less. The operator may adjust the zoom beam expander 20 using the control device 90 and set the beam diameter of the laser beam in advance so that the pupil filling factor is in the range of 0.56 to 0.96. it can.

(Iii) In the cell sorter device 3 according to the third embodiment, the angle of the position adjusting mirror pair 311 is changed, and the captured sample is moved and guided to a desired branch flow path. The sample may be guided to a desired branch flow path by changing the amount of light irradiation and changing the generated optical trapping force (by changing the intensity according to the type of sample) and sorting. Further, for example, the sample may be sorted by controlling ON / OFF of laser light irradiation.

(Iv) In the cell sorter device 3 according to the third embodiment, the sample size is specified based on the image of the sample passing through the flow path branching unit 307 and the beam diameter of the laser light is determined. May be used to determine the beam diameter by specifying the type of sample. For example, scattering obtained by labeling various samples in advance (for example, fluorescent labeling) and irradiating the sample flowing through the sorting flow path 300 with laser light of a specific wavelength (different from laser light for optical trapping). Light or fluorescence is detected by the detection unit. Since the scattered light and fluorescence detected by the detection unit exhibit different characteristics for each sample, the type of sample can be specified by analyzing the scattered light and fluorescence. Hereinafter, the cell sorter device 5 for realizing such cell sorting will be briefly described. It should be noted that the same reference numerals are assigned to the same components as those already described.

  FIG. 11 is a diagram illustrating a schematic configuration example of a cell sorter device 5 according to a modification of the third embodiment. The cell sorter device 5 includes, for example, an irradiation optical system 1500, an observation optical system and an imaging unit (not shown in FIG. 11) (corresponding to the observation optical system 60 and the imaging unit 80 described above), a control device 90, and a plurality of detections. Light sources 1501 to 1502 and a plurality of detectors 1503 to 1504, for example, a removable sorting channel 300 is installed, and a light trapping force is applied to the sample to sort the sample (for example, cells). Is.

  The irradiation optical system 1500 has the same configuration as that of the irradiation optical system 200 described in the second embodiment. For example, as in the third embodiment, by changing the angle of the position adjustment mirror pair 311, the sample trapped (captured) by laser light irradiation can be obtained in the desired first branch flow path 303 or the second branch. The trapped sample may be guided to the desired first branch channel 303 or the second branch channel 304 by moving the stage 210 as in the second embodiment. You may do it.

  As in the first embodiment, the observation optical system 60 includes, for example, an illumination unit 601 that emits observation illumination light, and a condenser lens 602 that effectively collects the observation illumination light on the sample surface. Prepare. As in the second embodiment, the observation optical system 60 can be configured so that, for example, the observation illumination light can illuminate the entire region of the sample placement unit 50.

  In the sorting channel 300, the liquid reservoir 301 stores a sample liquid in which a plurality of types of samples (cells and the like) are mixed. For example, each sample is fluorescently labeled and scatters or emits fluorescence when irradiated with light of a specific wavelength. As an example, multiple staining using a plurality of fluorescent dyes is also possible.

  In the cell sorter device 5, a plurality of detection light sources 1501 to 1502 are provided as an example. This is to cope with multiple staining. Each detection light source emits light having a different wavelength (for example, laser light). Since a plurality of detection light sources are provided, a plurality of detectors are also provided here.

  Detectors 1503 to 1504 detect scattered light and fluorescence generated when each sample is irradiated with light from each of the detection light sources 1501 to 1502. The detected scattered light and fluorescence are converted into an electrical signal proportional to the optical signal, for example, and transmitted to the control device 90.

  For example, the control device 90 calculates the size of each sample based on the electrical signal corresponding to the scattered light or fluorescence detected for each sample, and identifies the type of each sample based on the information on the size. Thereby, for example, since the control device 90 can recognize what kind of sample and what kind of sample, when the sample enters the flow path branching unit 307, it is determined which branch flow path should be sorted. be able to. The control device 90 traps the target sample by controlling the irradiation of the laser light from the laser light source 10 of the irradiation optical system 200. Then, the control device 90 controls the mirror driving unit 310 to change the angle of the dichroic mirror 30 or to move the stage 210 so that the sample trapped by the optical trapping force is a desired branch flow. Guide to the path (branch flow path corresponding to the type). Other operations and processes of the control device 90 are the same as the operations and processes of the control device 90 in the first embodiment. That is, the optimum beam diameter is determined in the same way in the modification. In addition, when the size of each sample is known, for example, the beam diameter is calculated in advance (the pupil filling rate is calculated within the range of 0.43 to 0.93), and which kind of sample flows through the flow path. Depending on the situation, the zoom beam expander 20 may be controlled so as to simply switch the beam diameter.

(8) Other Embodiments The functions of the embodiments can also be realized by software program codes. In this case, a storage medium in which the program code is recorded is provided to the system or apparatus, and the computer (or CPU or MPU) of the system or apparatus reads the program code stored in the storage medium. In this case, the program code itself read from the storage medium realizes the functions of the above-described embodiment, and the program code itself and the storage medium storing it are included in this embodiment. As a storage medium for supplying such program code, for example, a flexible disk, CD-ROM, DVD-ROM, hard disk, optical disk, magneto-optical disk, CD-R, magnetic tape, nonvolatile memory card, ROM Etc. are used.

  Also, based on the instruction of the program code, an OS (operating system) running on the computer performs part or all of the actual processing, and the functions of the above-described embodiments are realized by the processing. May be. Further, after the program code read from the storage medium is written in the memory on the computer, the computer CPU or the like performs part or all of the actual processing based on the instruction of the program code. Thus, the functions of the above-described embodiments may be realized.

  Furthermore, by distributing the program code of the software that realizes the functions of the embodiments via a network, it is stored in a storage means such as a hard disk or memory of a system or apparatus or a storage medium such as a CD-RW or CD-R. The program code stored in the storage means or the storage medium may be read out and executed by the computer (or CPU or MPU) of the system or apparatus during storage.

  The processes and techniques described herein are not inherently related to any particular apparatus and can be implemented by any suitable combination of components. In addition, various types of devices for general purpose can be used in accordance with the methods described herein. It may be beneficial to build a dedicated device to perform the method steps described herein. Various inventions can be formed by appropriately combining a plurality of constituent elements disclosed in the embodiments. For example, some components may be deleted from all the components shown in the embodiment. Furthermore, constituent elements over different embodiments may be appropriately combined.

  Other implementations of the present disclosure will become apparent to those having ordinary skill in the art from consideration of the specification and embodiments of the present disclosure disclosed herein. Various aspects and / or components of the described embodiments can be used singly or in any combination.

DESCRIPTION OF SYMBOLS 1, 2 Optical trap apparatus 3, 5 Cell sorter apparatus 4 Optical trap system 10 Light source 20 Zoom beam expander 30 Dichroic mirror 40 Objective lens 50 Sample mounting part 60 Observation optical system 70 Imaging lens 80, 320 Imaging part 90, 330 Control Apparatus 100 Sample placement surface drive unit 200, 1500 Irradiation optical system 210 Stage 300 Sorting channel 301 Liquid reservoir 302 Main channel 303 First branch channel 304 Second branch channel 305 First output unit 306 Second Output section 307 Flow path branching section 308 First kind sample 309 Second kind sample 310 Mirror driving section 311 Position adjustment mirror pair 313 Relay lens 400 Laser light irradiation device 410 Laser light irradiation optical system 420 Observation objective lenses 1501 and 1502 Detection light source 1503, 1504 Part

Claims (26)

An optical trap device for irradiating and capturing light on a sample,
A light source;
A beam diameter adjusting optical system for adjusting a beam diameter of light from the light source;
An objective lens for irradiating the sample with light having an adjusted beam diameter;
A storage device for storing beam diameter information indicating a value of a beam diameter that gives an optical trapping force in a combination of a sample size and a numerical aperture of an objective lens;
By referring to the beam diameter information stored in the storage device, a beam diameter value corresponding to a combination of the sample size of the sample to be captured and the numerical aperture of the objective lens used in the optical trap device is obtained. A processor that controls the beam diameter adjusting optical system to adjust the beam diameter of the light to the acquired beam diameter;
An optical trap device. The claim 1, further comprising:
An observation optical system for illuminating the sample with illumination light for observation;
An imaging unit that images the sample illuminated by the observation illumination light;
An optical trap device. In claim 2,
The processor applies the sample size of the sample to be captured and the numerical aperture of the objective lens calculated based on the image of the sample captured by the imaging unit to the beam diameter information, and the sample to be captured An optical trap device that obtains the value of the beam diameter corresponding to the sample size and the numerical aperture of the objective lens. In any one of Claims 1-3,
Furthermore, the first drive unit for moving the sample mounting unit for mounting the sample,
In response to the input instruction, the processor controls the first driving unit to move the position of the sample captured by the light irradiation on the sample mounting unit. In any one of Claims 1-3,
In addition, a light irradiation optical system including the light source, the beam diameter adjustment optical system, and the objective lens is mounted, and includes a moving stage that moves a light irradiation position by the light irradiation optical system,
In response to an input instruction, the processor controls the moving stage to move the position of the sample captured by the light irradiation. In any one of Claims 1-3,
Further, a light irradiation position adjusting mirror that rotates around a rotation axis and changes a traveling direction of the light emitted from the light source;
A second drive unit that changes the angle of the light irradiation position adjusting mirror,
In response to the input instruction, the processor controls the second drive unit to change the angle of the light irradiation position adjusting mirror to change the light irradiation position, and is captured by the light irradiation. An optical trap device for moving the position of the sample. In claim 6,
The light irradiation position adjusting mirror is an optical trap device arranged at a position conjugate with the pupil plane of the objective lens. In any one of Claims 1-7,
The objective lens is an optical trap device, which is a dry lens having a numerical aperture of less than one. In any one of Claim 1 to 8,
The beam diameter adjusting optical system is an optical trap device including a zoom beam expander. In any one of Claim 1 to 9,
The storage device includes, as the beam diameter information, first beam diameter information used when the sample is captured and moved in the XY plane, and a second beam used when the sample is captured and moved in the Z direction. An optical trap device for storing diameter information. An optical trap device for irradiating and capturing light on a sample,
A light source;
A beam diameter adjusting optical system for adjusting a beam diameter of light from the light source;
An objective lens that irradiates the sample with light having an adjusted beam diameter,
When the light trapping force generated by irradiating the sample with the light is applied to the sample in the XY plane, the beam diameter adjustment is performed so that the pupil filling factor is 0.43 or more and 0.93 or less. An optical trap device in which a beam diameter of the light is adjusted by an optical system. An optical trap device for irradiating and capturing light on a sample,
A light source;
A beam diameter adjusting optical system for adjusting a beam diameter of light from the light source;
An objective lens that irradiates the sample with light having an adjusted beam diameter,
When the optical trapping force generated by irradiating the sample with the light is applied to the sample in the Z direction, the beam diameter adjusting optics is set so that the pupil filling factor is 0.56 or more and 0.96 or less. An optical trap device in which a beam diameter of the light is adjusted by a system. A sample separation device for separating a plurality of types of samples flowing in a flow path,
A light source;
A beam diameter adjusting optical system for adjusting a beam diameter of light from the light source;
An objective lens for irradiating the sample with light having an adjusted beam diameter;
A storage device for storing beam diameter information indicating a value of a beam diameter that gives an optical trapping force in a combination of a sample size and a numerical aperture of an objective lens;
A processor for controlling the irradiation of light with the beam diameter adjusted to the sample, and sorting the sample in a desired direction in the flow path,
The processor refers to the beam diameter information stored in the storage device, and the beam diameter corresponding to the combination of the sample size of the sample to be sorted and the numerical aperture of the objective lens used in the sample sorting device And a beam size adjusting optical system to adjust the beam diameter of the light to the acquired beam diameter. In claim 13,
Furthermore, a detection unit for detecting the characteristics of the sample,
The processor identifies the type of the sample based on the characteristics of the sample detected by the detection unit, and controls the irradiation of light having the beam diameter adjusted according to the identified sample type. Sample sorting device. The claim 13, further comprising:
An observation optical system for illuminating the sample with illumination light for observation;
An imaging unit that images the sample illuminated by the observation illumination light; and
The processor specifies the type of the sample based on the image of the sample imaged by the imaging unit, and controls the irradiation of the light whose beam diameter is adjusted according to the type of the specified sample. Sample sorting device. In claim 15,
The sample sorting device, wherein the processor identifies the type of the sample from information on a sample size of the sample calculated based on an image of the sample. In claim 16,
The processor refers to the beam diameter information, acquires the value of the beam diameter according to the sample size of the sample, and controls the beam diameter adjustment optical system to adjust to the acquired beam diameter. apparatus. In any one of Claims 13 to 17,
The processor controls the amount of light with the beam diameter adjusted for the sample based on the sample type information, and guides the sample in a desired direction in the flow path. Sorting, sample sorting device. In claim 18,
The processor separates the sample by controlling ON / OFF the irradiation of the light with the beam diameter adjusted to the sample based on the information on the type of the sample. In any one of Claims 14 to 16,
Furthermore, a light irradiation position adjusting mirror that rotates around the rotation axis and changes the traveling direction of the light whose beam diameter is adjusted,
A mirror driving unit that changes an angle of the light irradiation position adjusting mirror, and
The processor is captured by the light irradiation by controlling the mirror driving unit according to the type of the sample to change the light irradiation position by changing the angle of the light irradiation position adjusting mirror. A sample separation device for separating the sample by guiding the sample in the desired direction. In claim 20,
The light irradiation position adjusting mirror is a sample sorting device arranged at a position conjugate with the pupil plane of the objective lens. A sample separation device for separating a plurality of types of samples flowing in a flow path,
A light source;
A beam diameter adjusting optical system for adjusting a beam diameter of light from the light source;
An objective lens for irradiating the sample with light having an adjusted beam diameter;
A detection unit for detecting the sample flowing through the flow path;
A processor that controls irradiation of the light with the beam diameter adjusted to the sample based on a detection result of the sample by the detection unit, and sorts the sample in a desired direction in the flow path. ,
When an optical trapping force generated by irradiating the sample with the light is applied to the sample in the XY plane, the processor is configured so that the pupil filling factor is 0.43 or more and 0.93 or less. A sample sorting device for controlling a diameter adjusting optical system to adjust a beam diameter of the light. In claim 22,
The detection unit detects scattered light or fluorescence generated when the sample is irradiated with detection light as a feature of the sample,
The processor identifies the type of the sample based on the characteristics of the sample, controls the beam diameter adjustment optical system based on the identified sample type, adjusts the beam diameter, and the classification target Sample sorting device that irradiates the sample. A method of irradiating and capturing light on a sample,
The processor refers to the beam diameter information indicating the value of the beam diameter that gives the optical trapping force in the combination of the sample size and the numerical aperture of the objective lens, and refers to the sample size of the sample to be captured and the actual aperture of the objective lens used. Obtaining a beam diameter value corresponding to the combination with the number, controlling the beam diameter adjusting optical system to adjust the beam diameter of the light from the light source to the acquired beam diameter;
Irradiating the sample with light having the beam diameter adjusted with the objective lens actually used;
Including a method. A method of irradiating and capturing light on a sample,
When the optical trapping force generated by irradiating the sample with light is applied to the sample in the XY plane, the light source is adjusted by the beam diameter adjusting optical system so that the pupil filling factor is 0.43 or more and 0.93 or less. Adjusting the beam diameter of the light from
Irradiating the sample with light having the beam diameter adjusted by an objective lens;
Including a method. A method of irradiating and capturing light on a sample,
When the light trapping force generated by irradiating the sample with light is applied to the sample in the Z direction, the beam diameter adjustment optical system is used to adjust the pupil filling rate from 0.56 to 0.96 from the light source. Adjusting the beam diameter of
Irradiating the sample with light having the beam diameter adjusted by an objective lens;
Including a method.

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