JP5306835B2 - Optical measurement method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical measuring method, capable of preventing the contamination of a minute particle fractionating apparatus by the minute particle-containing solution built in a substrate and being miniaturized, by reducing the number of optical members to be arranged. <P>SOLUTION: The substrate 110, a substrate-fixing part 120, a light route forming part 130, a light connector 134 and first-third optical fibers 135a, 135b and 135c are housed inside a clean bench 101 forming a closed space. A measurement control part 140 is arranged outside the clean bench 101 and is connected to the first-third optical fibers 135a, 135b and 135c via a connecting optical fiber 141. According to this constitution, the measurement and fractionation of the minute particles contained in the minute particle-containing solution 11 are performed, while being controlled from the outside of the clean bench 101 and the contamination possibility of a peripheral environment, or the like, by the minute particles during fractionation work is eliminated. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to an optical measurement method for measuring and sorting fine particles contained in a solution.

  In recent years, a solution in which fine particles to be measured are dispersed in a predetermined liquid is flowed through a very thin channel, and light is irradiated onto this to acquire characteristic information such as the shape of the fine particles. Attention has been focused on a particle measurement and separation method and a particle separation apparatus for identification and separation.

  An example of a conventional fine particle sorting apparatus is shown in FIG. In the fine particle sorting apparatus 900 shown in the figure, a fine particle-containing solution containing fine particles to be measured is caused to flow through a flow path 902 formed in a glass substrate 901, and laser light is supplied from a laser light source 903 via a lens 904a. Is irradiated. The light scattered by the fine particles is arranged in a direction substantially perpendicular to the irradiation optical axis from the glass substrate 901 and the first light receiving unit 905 arranged on the opposite side to the laser light source 903 and the lens 904a on the irradiation optical axis. The received light is received by the second light receiving unit 906. The light receiving unit 905 receives forward scattered light excluding irradiation light through the lens 904b, and the light receiving unit 906 receives side scattered light and fluorescence through the lens 904c.

  Conventionally, fine particles are identified by analyzing information such as forward scattered light, side scattered light, and fluorescence received by the light receiving units 905 and 906, and the fine particles are charged by droplet charging or mechanical methods based on the results. Sorting was done.

  Further, in the fine particle sorting apparatus 910 shown in FIG. 7 described in Patent Document 1, as another method of sorting the measured fine particles, a stimulus applying means for applying a stimulus to the sample solution flowing through the flow path 912 of the substrate 911 is provided. ing. Here, a microheater 913, a semiconductor laser, or the like that locally heats the solution is used as the stimulus applying means. When the solution is locally heated, the heated portion is changed from the sol state to the gel state, thereby controlling the flow of the solution to separate the fine particles.

JP 2004-152218 A

  However, the optical measurement method using the conventional fine particle sorting apparatus has the following problems. A substrate through which a fine particle-containing solution to be measured flows is fixed to a fine particle sorting apparatus as part of the apparatus. Therefore, in order to measure and separate another fine particle-containing solution, it is necessary to clean the flow path of the substrate. However, if the cleaning is not sufficient, the fine particle-containing solution is contaminated with another fine particle-containing solution. There was a problem. In particular, when the fine particles contained in the solution are infectious cells, the surrounding environment may be contaminated during washing, or another fine particle-containing solution may be contaminated to cause secondary infection.

  Further, in the conventional optical measurement method using the fine particle sorting apparatus, when the fine particle-containing solution is to be measured at a plurality of measurement points, it is necessary to arrange the lens and the light receiving unit as a set toward each measurement point. . Similarly, in order to irradiate a solution containing fine particles with a plurality of irradiation lights, it is necessary to arrange each light source and lens as a set. Even when laser light is used as the stimulus applying means for controlling the flow of the fine particle-containing solution, it is necessary to separately provide an optical system member such as a lens for irradiating the laser light. As described above, in the optical measurement method using the conventional fine particle sorting apparatus, it is necessary to arrange optical system members such as a plurality of lenses around the substrate. When the number of components increases and the apparatus becomes large, it becomes difficult to use in a clean environment.

  Accordingly, the present invention has been made in view of the above-described problems of the prior art, and an object thereof is to provide an optical measurement method capable of realizing downsizing by reducing the number of optical system members to be arranged.

  In a first aspect of the optical measurement method according to the present invention, functional light acting on an object to be irradiated is incident on an optical path forming unit that forms two or more optical paths so as to have functional points at two or more positions. The function light is emitted to any one of the functional points via any one of the light paths, and the measurement light is emitted from the measurement point by irradiating the predetermined light receiving unit from any one of the light paths. The irradiated object is measured by guiding light, and the irradiated object is irradiated with functional light different from the functional light via any one of the optical paths.

  Another aspect of the optical measurement method according to the present invention is characterized in that the predetermined inclusions included in the irradiated object are separated by irradiating the irradiated object with the different functional light.

  Another aspect of the optical measurement method according to the present invention is characterized in that two or more measurement lights including a fluorescence wavelength are guided to at least one of the optical paths.

  Another aspect of the optical measurement method according to the present invention is characterized in that the functional light is emitted and the measurement light is measured at a position separated from the irradiated object and the optical path forming unit.

  Another aspect of the optical measurement method according to the present invention is characterized in that the optical path forming unit forms the two or more optical paths with a single lens system.

  Another aspect of the optical measurement method according to the present invention is characterized in that the optical path forming unit forms one or more optical paths by arranging one or more fibers without using a lens.

  In another aspect of the optical measurement method according to the present invention, a light is incident on a position conjugate to a position where the functional light is irradiated to the irradiated object on a side opposite to the irradiated object of the optical path through a lens. An emission point is arranged.

  In another aspect of the optical measurement method according to the present invention, as the measurement light, the functional light is scattered light scattered by the irradiated object or fluorescence emitted by being excited by the functional light is measured. And

  Another aspect of the optical measurement method according to the present invention is characterized in that the scattered light is backscattered light that is scattered coaxially with the functional light.

  Another aspect of the optical measurement method according to the present invention is characterized in that the scattered light is side scattered light scattered in a direction substantially perpendicular to the irradiation direction of the functional light.

  Another aspect of the optical measurement method according to the present invention is characterized in that when measuring the fluorescence having different wavelengths, the fluorescence on the long wavelength side is measured first.

  In another aspect of the optical measurement method according to the present invention, the irradiated object is an irradiated solution in which a solution containing fine particles containing fine particles is sheathed with a sheath solution, and the irradiated solution is provided in a predetermined substrate. The fine particles are identified and separated by flowing in a flow path.

  In another aspect of the optical measurement method according to the present invention, a stimulus-sensitive substance is added to at least one of the fine particle-containing solution or the sheath solution, and a predetermined stimulus is applied to the stimulus-sensitive substance with the other functional light. To control the flow of the irradiated solution.

  Another aspect of the optical measurement method according to the present invention is characterized in that a disposable substrate is used as the substrate.

  Another aspect of the optical measurement method according to the present invention is characterized in that the substrate and the optical path forming unit are housed in a clean bench.

  Another aspect of the optical measurement method according to the present invention is characterized in that an optical fiber is connected to the light incident / exit point and the measurement light is incident and the functional light is emitted.

  Another aspect of the optical measurement method according to the present invention is characterized in that two or more optical fibers are arranged in parallel at the light incident / exit point.

  In another aspect of the optical measurement method according to the present invention, the measurement light emitted from the functional point by irradiation of the functional light is guided to the same optical fiber as that guided by the functional light. It is characterized by.

  Another aspect of the optical measurement method according to the present invention is characterized in that the two or more optical fibers are connected to the optical path forming unit using an optical connector.

  In another aspect of the optical measurement method according to the present invention, the two or more functional points are provided in the flow direction of the irradiated solution, and the flow rate of the irradiated solution is determined from the two or more measurement lights incident from the functional points. It is characterized by estimating.

  Another aspect of the optical measurement method according to the present invention is characterized in that the backscattered light is received at the two or more functional points to estimate the flow rate of the irradiated solution.

  Another aspect of the optical measurement method according to the present invention is characterized in that the side scattered light is received at the two or more functional points to determine the flow rate of the irradiated solution.

  Another aspect of the optical measurement method according to the present invention is characterized in that the flow rate of the fine particles contained in the fine particle-containing solution is individually obtained, and the fine particles are classified by calculating the timing from the flow rate each time. To do.

  In another aspect of the optical measurement method according to the present invention, the fine particle-containing solution or the sheath solution to which the stimulus-sensitive substance is added is irradiated with a predetermined functional light to be heated, thereby changing the flow velocity resistance and The distance between the fine particles is adjusted, or the fine particles are selectively flowed to another flow path branched from the flow path.

  Another aspect of the optical measurement method according to the present invention is characterized in that infrared light is used as the functional light.

  Another aspect of the optical measurement method according to the present invention is characterized in that a position apart from a straight line connecting the two or more measurement points is heated with the infrared light.

  Another aspect of the optical measurement method according to the present invention is characterized in that a position on a straight line connecting the two or more measurement points is heated with the infrared light.

  Another aspect of the optical measurement method according to the present invention is characterized in that the downstream side of the measurement point is heated with the infrared light.

  Another aspect of the optical measurement method according to the present invention is characterized in that the fine particles are cells.

  According to the present invention, by using an optical path forming unit that forms two or more optical paths so as to have functional points at two or more different positions of the irradiated object, the number of optical system members to be arranged can be reduced and miniaturization can be achieved. An optical measurement method that can be realized can be provided.

It is a schematic block diagram which shows one Example of the fine particle sorter used with the optical measurement method of 1st Embodiment. It is a figure which shows the flow path of a fine particle sorter in detail. It is a schematic block diagram which shows one Example of the fine particle sorter used with the optical measurement method of 2nd Embodiment. It is a schematic block diagram which shows the conventional fine particle sorting apparatus. It is a schematic block diagram which shows another conventional fine particle sorting apparatus. It is a schematic block diagram which shows one Example of the fine particle sorter used with the optical measurement method of 3rd Embodiment. It is a schematic block diagram which shows one Example of the fine particle sorter used with the optical measurement method of 4th Embodiment.

  An optical measurement method according to a preferred embodiment of the present invention will be described with reference to the drawings. The fine particle sorting apparatus used in the first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a schematic configuration diagram showing a fine particle sorting apparatus used in the present embodiment. The fine particle sorting apparatus 100 used in the present embodiment includes a substrate 110, a substrate fixing unit 120 for fixing the substrate 110, an optical path forming unit 130, a measurement control unit 140, and a pneumatic control unit 150.

  The substrate 110 contains the fine particle-containing solution 11 and the sheath solution 12, and has a structure in which the irradiation solution 13 in which the fine particle-containing solution 11 is sheathed with the sheath solution 12 flows through the flow path 111 at a predetermined flow rate. . The substrate 110 is positioned and fixed to the substrate fixing unit 120 with high accuracy. In order to control the flow rate of the solution to be irradiated 13, the air pressure controller 150 is connected to the substrate 110 through the hollow tube 151. The substrate 110 can be formed using a silicone resin (for example, PDMS) and glass. The channel 111 is formed in the silicone resin 117, and the silicone resin 117 on the surface where the channel 111 is formed is covered with the cover glass 116. It can be formed by covering. As the cover glass 116 that covers the silicone resin 117, a glass that can transmit the functional light emitted from the measurement / separation control unit 140 is used.

  Details of the flow path 111 are shown in FIG. In the channel 11, two measurement points 111a and 111b and one heating point 111c are set at different positions as functional points. The downstream side of the flow path 111 is connected to the sheath solution discharge part 115, but another flow path branched from the flow path 111 is provided on the upstream side of the heating point 111c. It is connected to the collection unit 114. In this embodiment, two measurement points are set in the flow path 111. However, the present invention is not limited to this, and two or more measurement points may be provided.

  The optical path forming unit 130 uses the lens 131 to form three optical paths 132a and 132b so as to form a focal point at two measurement points 111a and 111b set at different positions in the flow path 111 and one heating point 111c. , 132c. In this embodiment, there are two measurement points. However, the present invention is not limited to this, and even when more measurement points are provided, the optical path forming unit 130 forms an optical path corresponding to each measurement point. Can do. In addition, instead of the lens 131, the optical paths 132a, 132b, and 132c can be formed using optical fibers.

  Light input / output points 133a, 133b, and 133c are provided on the surface of the optical path forming unit 130 opposite to the substrate 110, and light incident on the light input / output points is respectively optical paths 132a, 132b, and 132c. The focal points are formed at the measurement points 111a and 111b and the heating point 111c via the. Light emitted from the measurement points 111a and 111b forms a focal point at the light input / output points 133a and 133b via the optical paths 132a and 132b, respectively.

  First to third optical fibers 135a, 135b, and 135c are connected to the optical input / output points 133a, 133b, and 133c using an optical connector 134, respectively. Furthermore, the measurement control unit 140 is connected via a connection optical fiber 141 connected to the first to third optical fibers 135a, 135b, and 135c. The connection optical fiber 141 can be formed by bundling the first to third optical fibers 135a, 135b, and 135c.

  As described above, the optical path forming unit 130 of the present embodiment shares the internal lens 131 by arranging a plurality of optical fibers on the surface opposite to the substrate 110 to form a plurality of light input / output points. Thus, a plurality of optical paths are formed. By adopting such a configuration, even when measurement is performed at a plurality of measurement points, it is not necessary to separately arrange an optical path forming unit (lens, optical fiber, etc.) corresponding to each measurement point, and the particulate separation device 100 Can be miniaturized.

  The measurement control unit 140 includes a light source 142, a light receiving unit 143, a measuring unit 144, and a stimulus applying unit 145. The irradiation light emitted from the light source 142 is applied to the fine particle-containing solution in the substrate 110 via the connection optical fiber 141 and the optical path forming unit 130, while the scattered light from the fine particles (measurement light) is in the reverse path. The light is incident on the light receiving unit 143, and the measurement unit 144 analyzes the fine particles based on the signal from the light receiving unit 143. Further, based on the analysis result by the measuring means 144, the stimulus applying means 145 gives a predetermined stimulus to the fine particle-containing solution to sort the fine particles.

  The pneumatic control unit 150 is connected to the substrate 110 through the hollow tube 151. In order to control the flow rate or the like of the solution to be irradiated that flows through the channel 111 in the substrate 110, the hollow tube 151 is connected to the upstream side and the downstream side of the channel 111, and the pressure applied to each is adjusted.

  In the present embodiment, the substrate 110, the substrate fixing unit 120, the optical path forming unit 130, the optical connector 134, and the first to third optical fibers 135a, 135b, and 135c are accommodated in the clean bench 101 that forms a closed space. Yes. The measurement control unit 140 and the pneumatic control unit 150 are arranged outside the clean bench 101, and the measurement control unit 140 is connected to the first to third optical fibers 135a, 135b, and 135c via the connection optical fiber 141, The control unit 150 is connected to the substrate 110 through the hollow tube 151. Further, the substrate 110 of the present embodiment is detachably formed on the substrate fixing unit 120 and has a disposable structure that does not leak the built-in fine particles to the outside.

  By configuring the fine particle separation device 100 used in the present embodiment as described above, the measurement and separation of the fine particles contained in the fine particle-containing solution 11 can be controlled from outside the clean bench 101, and There is no risk of contamination of the surrounding environment by fine particles during sorting. In addition, when the substrate 110 that has been measured and sorted is replaced with another substrate, it is possible to replace the sealed substrate 110 together with the fine particles, so that there is no possibility of being contaminated with the fine particles when the substrate 110 is replaced. According to the present embodiment, it is possible to provide a contamination-free optical measurement method that prevents contamination by fine particles.

  The structure of the substrate 110 will be described in more detail with reference to FIGS. A fine particle solution injection unit 112 and a sheath solution injection unit 113 are provided on the upstream side of the flow channel 111, and a fine particle recovery unit 114 and an irradiated solution discharge unit 115 are provided on the downstream side of the flow channel 111. The irradiated solution 13 in a sheath flow state in which the fine particle-containing solution 11 and the sheath solution 12 are injected into the flow path 111 from the fine particle-containing solution injection portion 112 and the sheath solution injection portion 113, respectively, The channel 111 is formed and flows downstream.

  The channel 111 is covered with a cover glass 116, and measurement points 111 a and 111 b and a heating point 111 c are provided at predetermined positions of the channel 111. Irradiated light that has passed through a predetermined light path from the measurement control unit 140 passes through the cover glass 116 and is irradiated to the irradiated solution 13 that passes through the measurement points 111a and 111b. In addition, measurement light emitted from the measurement points 111a and 111b is transmitted to the measurement control unit 140 via a predetermined optical path, and here, classification control is performed based on the measurement result of the fine particles 10. The separated fine particles are collected in the fine particle collecting unit 114, and the other irradiated solution 13 is discharged to the irradiated solution discharging unit 115.

  In this embodiment, a solution that undergoes sol-gel transition at a predetermined temperature is used as the fine particle-containing solution 11 or the sheath solution 12. When such a solution is used, when the heating point 111c is irradiated with infrared light as the stimulus imparting means 145, the heating point 111c is heated and transitions from the sol state to the gel state. When transitioning to the gel state, the viscosity of the solution increases, and the flow rate of the heating point 111c decreases or stops. On the other hand, when the infrared light irradiation is stopped, the temperature is lowered to return to the sol state again, and the irradiated solution 13 flows at the original flow rate through the heating point 111c.

  In the present embodiment, the solution having the characteristics as described above is used for the fine particle-containing solution 11 or the sheath solution 12, thereby controlling the irradiation of infrared light by the stimulus applying means 145 and adjusting the flow rate of the irradiated solution 13. Thus, only predetermined fine particles can be collected in the fine particle collecting unit 114, and the other irradiated solution 13 can be discharged to the sheath solution discharging unit 115 for separation.

  The substrate 110 has a structure that can be attached to and detached from the substrate fixing unit 120, but the measurement points 111 a and 111 b and the heating point 111 c on the substrate 110 are positioned with high accuracy with respect to the optical path forming unit 130, so It is supposed to be fixed. In the embodiment shown in FIG. 1, positioning pins 121 are provided in the substrate fixing portion 120 as positioning means, and an insertion hole for inserting the positioning pins 121 is formed in the substrate 110. By inserting the insertion hole into the positioning pin 121, the substrate 110 is positioned with high accuracy on the substrate fixing portion 120. The positional relationship between the substrate fixing unit 120 and the optical path forming unit 130 is fixed, and when the substrate 110 is positioned with high accuracy on the substrate fixing unit 120, the optical path forming unit 130 is also positioned with high accuracy. The

  In order to control the flow rate of the irradiated solution 13 flowing through the flow path 111 and the collection of the fine particles, one end of the hollow tube 151 is provided in each of the fine particle solution injection unit 112, the sheath solution injection unit 113, and the irradiated solution discharge unit 115. Are connected, and the other end is connected to the pneumatic controller 150. The flow rate of the irradiation target solution 13 is adjusted by the difference between the air pressure applied to the fine particle solution containing injection unit 112 side and the air pressure applied to the irradiation solution discharge unit 113 side. The air pressure control unit 150 controls the air pressure applied to the fine particle solution injection unit 112 side and the irradiated solution discharge unit 113 side. The air pressure control unit 150 is installed outside the clean bench 101, and there is no possibility of being contaminated with the fine particles 10 while controlling the flow rate of the irradiated solution 13.

  Next, the structure of the optical path forming unit 130 will be described in more detail. The light path forming unit 130 uses the lens 131 provided therein, and light incident from the light input / output points 133a, 133b, and 133c passes through the light paths 132a, 132b, and 132c, and the measurement points 111a and 111b on the flow path 111, respectively. And it is comprised so that a focus may be formed in the heating point 111c. The light input / output points 133a, 133b, and 133c, the measurement points 111a and 111b, and the heating point 111c are configured to have a conjugate relationship with each other via the common lens 131.

  The optical path forming unit 130 and the measurement control unit 140 are connected using an optical fiber. The optical input / output points 133a, 133b, and 133c of the optical path forming unit 130 are arranged in parallel with the first to third optical fibers 135a, 135b, and 135c using an optical connector 134, respectively. Each optical fiber 135a, 135b, 135c transmits both irradiation light emitted from the light source 142 and measurement light emitted from the measurement points 111a, 111b. Each of the optical fibers 135a, 135b, and 135c is connected to the connection optical fiber 141 and configured to perform optical transmission with the measurement control unit 140.

  Next, the detailed structure of the measurement control unit 140 will be described below. The measurement control unit 140 performs predetermined measurement based on a light source 142 that emits predetermined irradiation light, a light receiving unit 143 that receives measurement light emitted from the measurement points 111a and 111b, and a signal from the light receiving unit 143. It has the measurement means 144 to perform, and the stimulus imparting means 145 which gives a predetermined stimulus to the irradiated solution 13. The measuring means 144 controls to irradiate the irradiated solution 13 located at the measurement point 111a or 111b by outputting predetermined irradiation light from the light source 142 at predetermined timing. Thereby, measurement light is emitted from the irradiated solution 13 at the measurement point 111a or 111b, and this light is received by the light receiving unit 142 and subjected to predetermined signal processing.

  As measurement light emitted from the irradiated solution 13 located at the measurement points 111a and 111b, for example, there is backscattered light in which the irradiation light is scattered by the fine particles contained in the fine particle-containing solution 11. Moreover, the predetermined fluorescence which is excited by the irradiation light and emits light can be used as the measurement light. When fluorescence having different wavelengths is used as measurement light, it is preferable to measure fluorescence on the long wavelength side first. Thereby, the fluorescence deterioration of microparticles | fine-particles can be prevented.

  In the embodiment shown in FIG. 1, the backscattered light is measured at the measurement points 111a and 111b, whereby characteristic information such as the size, shape, and internal structure of the fine particles 10 can be obtained. Further, fluorescence is also measured at the measurement point 111b, and the identification of the fine particles can be performed by combining the information of the backscattered light and the fluorescence. The measuring means 144 controls the stimulus applying means 145 so as to classify the fine particles according to whether or not the identified fine particles are of interest.

  In the present embodiment, two or more measurement points are provided in the flow direction of the solution to be irradiated 13, and the flow rate of the solution to be irradiated can be measured using measurement light emitted from each. In FIG. 1, two measurement points 111 a and 111 b are provided, and the flow rate of the irradiated solution 13 is calculated by the measuring means 144 based on the backscattered light emitted from each. The flow rate calculated by the measuring unit 144 can be used to control the flow rate of the irradiation target solution 13 by the air pressure control unit 150.

  The flow rate of the irradiated solution 13 calculated by the measuring means 144 can also be used to determine the timing for separating the fine particles. That is, the measuring means 144 determines the timing for separating the fine particles 10 from the flow rate of the irradiated solution 13, and controls the stimulus applying means 145 or the air pressure control unit 150 based on this to cause the fine particles 10 to be separated. Can do.

  The stimulus imparting means 145 controls the flow velocity resistance or the flow direction of the solution to be irradiated 13 based on the result of processing the measurement light by the measuring means 144. In this embodiment, when the predetermined fine particles 10 are detected by the measuring unit 144, the stimulus applying unit 145 emits infrared light from the light source 142 to irradiate the heating unit 111c. As a result, the solution to be irradiated 13 is heated and the flow toward the sheath solution discharge part 115 is decelerated or stopped, and the predetermined fine particles 10 are flowed toward the fine particle recovery part 114 side and separated.

  As the fine particle-containing solution 11, a solution that increases in viscosity when heated by infrared light can be used, and the flow direction and flow velocity resistance of the solution can be changed by changing the viscosity. Alternatively, such a solution can be used for the sheath solution 12. As such a solution, a solution that undergoes sol-gel transition at a predetermined temperature can be used.

  The set position of the heating point 111c can be on a straight line connecting the measurement points 111a and 111b. In this case, when the heating point 111c is irradiated with predetermined irradiation light and heated, the viscosity of the solution to be irradiated 13 is increased and the flow rate is lowered or stopped. Thereby, the droplet containing the target fine particles can be separated, and can be separated and collected by a predetermined sorting method.

  Alternatively, the setting position of the heating point 111c can be provided at a position away from the line connecting the measurement points 111a and 111b. In this case, the flow direction of the irradiation target solution 13 can be changed two-dimensionally. In this embodiment, another flow path that branches the flow path 111 is provided before the heating point 111c. When the heating point 111c is heated to increase the viscosity, the irradiated solution 13 is transferred to the other flow path. And is collected by the particulate collection unit 114.

  The irradiation target solution 13 is controlled by the stimulus applying unit 145 based on the result of the measuring unit 144. That is, the measurement light received by the light receiving unit 143 is processed by the measuring unit 144, and the stimulus applying unit 145 controls the flow of the irradiated solution 13 based on the determination result. Thus, in order to control the flow of the irradiated solution 13 based on the measurement result, in this embodiment, the arrangement of the heating points 111c is set downstream of the measurement points 111a and 111b.

  As described above, in the fine particle sorting apparatus 100 used in this embodiment, the substrate 110, the optical path forming unit 130, and the like are housed in the clean bench 101 in the closed space, and the measurement control unit 140 provided outside thereof. In addition, the fine particle solution 11 in the substrate 110 is measured and controlled using the air pressure control unit 150. In addition, the substrate 110 is configured to be detachable from the substrate fixing portion 120, and the built-in fine particles 10 are separated in the substrate 110, thereby having a disposable structure that does not leak outside. By adopting such a configuration of the fine particle sorting apparatus 100 used in the present embodiment, it is possible to provide a contamination-free optical measurement method that prevents contamination by the fine particles 10.

  The fine particles 10 can be, for example, predetermined cells. In the present embodiment, since the solution containing cells as the fine particle-containing solution 11 is built in the substrate 110 and fixed in the clean bench 101, and measurement / sorting can be performed, the cells are stored in the clean bench 101. There is no risk of leakage to the outside, and there is no risk of infection by cells. In addition, since the entire substrate 110 can be disposed after the measurement / sorting is completed, there is no risk of infection by cells.

  An optical measurement method according to the second embodiment of the present invention will be described with reference to FIG. The fine particle sorting apparatus 200 used in the present embodiment includes a second optical path forming unit 230 in addition to the optical path forming unit 130 of the first embodiment. Similar to the first embodiment, the optical path forming unit 130 is arranged in a direction perpendicular to the flow direction of the flow path 111, and is configured to receive measurement light and transmit the measurement light to the measurement control unit 240.

  The second optical path forming unit 230 added in the present embodiment is arranged on one side (upstream side in FIG. 3) in the flow direction of the flow path 111. Irradiated light emitted from the light source 142 in the measurement control unit 240 is incident on the second optical path forming unit 230 via the connection optical fiber 241 and the fourth optical fiber 235, and is irradiated using the lens 231 here. An optical path 232 is formed so as to form a focal point at a predetermined position of the solution 13.

  In the fine particle sorting apparatus 200 used in the present embodiment, side scattered light can be measured in addition to the backscattered light and fluorescence that can be measured by the fine particle sorting apparatus 100 used in the first embodiment. . As an example, when the backscattered light irradiation light is incident on the irradiated solution 13 from the first light path forming unit 130 side, the backscattered light is incident on the light path forming unit 130 from a predetermined measurement point. Further, when the side scattered light irradiation light enters the irradiated solution 13 from the second light path forming unit 230 side, the side scattered light enters the light path forming unit 130 from a predetermined measurement point. Further, when the irradiation light for fluorescence enters the irradiated solution 13 from the first light path forming unit 130 side, the fluorescence enters the light path forming unit 130 from a predetermined measurement point. In the present embodiment, it is possible to further measure the side scattered light, thereby realizing highly sensitive measurement.

  The heating infrared light emitted from the measurement control device 240 for controlling the flow of the irradiated solution 13 is a predetermined heating point of the irradiated solution 13 from the optical path forming unit 130 as in the first embodiment. Is irradiated. In the present embodiment, by measuring side scattered light in addition to back scattered light and fluorescence, it becomes possible to identify fine particles with higher accuracy, and using the given predetermined fine particles using the stimulus applying means 145. Can be separated.

  An optical measurement method according to the third embodiment of the present invention will be described with reference to FIG. The particle sorting apparatus 300 used in this embodiment forms an optical path using only the optical fiber 135 (135a, 135b, 135c) without using a lens, instead of the optical path forming unit 130 used in the first embodiment. An optical path forming unit 330 is provided.

  In the fine particle sorting apparatus 300 used in the present embodiment, an optical path for irradiating the irradiation target solution 13 or making measurement light incident is formed by using only an optical fiber without using a lens, so that it is stored in a clean bench. It is possible to further reduce the size of the device.

  An optical measurement method according to the fourth embodiment of the present invention will be described with reference to FIG. The fine particle sorting apparatus 400 used in the present embodiment replaces the optical path forming unit 130 used in the second embodiment, and uses an optical fiber 135 (135a, 135b, 135c) includes an optical path forming unit 330 that forms an optical path only. Further, in place of the second optical path forming unit 230 used in the second embodiment, an optical path is formed only by the fourth optical fiber 235 without using a lens.

  Even in the fine particle sorting apparatus 400 used in the present embodiment, the optical path for irradiating the irradiation target solution 13 or entering the measurement light is formed by using only an optical fiber without using a lens, so that it is stored in the clean bench. It is possible to further reduce the size of the device.

The optical measurement method of the present invention is not limited to the above-described embodiment, and for example, the following is also possible.
A substrate containing an object to be irradiated can be configured to be detachable from the substrate fixing portion, and positioning means capable of positioning with high accuracy by bonding the substrate and the substrate fixing portion to each other may be provided. A disposable substrate can be used. The substrate and the optical path forming unit may be housed in a clean bench. Furthermore, the measurement result may be acquired from the measurement light at a position separated from the substrate and the optical path forming unit, or the functional light may be emitted.

  Further, the flow of the irradiated solution may be controlled by adding a stimulus-sensitive substance to at least one of the fine particle-containing solution or the sheath solution and applying a predetermined stimulus to the stimulus-sensitive substance with another functional light. Good. By irradiating a predetermined functional light to a solution containing fine particles or a sheath solution to which a stimulus-sensitive substance is added and heating it, the flow resistance is changed to adjust the interval between the fine particles, or another branched from the flow path The fine particles can be selectively passed through the flow path. Infrared light can be used as the functional light. Furthermore, the position away from the straight line connecting two or more measurement points is heated with infrared light, or the position on the straight line connecting two or more measurement points is heated with infrared light, or the downstream side from the measurement point A method such as heating with infrared light may be used.

  In addition, the description in this Embodiment shows an example of the optical measurement method based on this invention, and is not limited to this. The detailed configuration and detailed operation of the optical measurement method in the present embodiment can be changed as appropriate without departing from the spirit of the present invention.

11 Fine particle solution 12 Sheath solution 13 Irradiated solution 100, 200, 900, 910 Fine particle separator 101 Clean bench 110, 901, 911 Substrate 111, 902, 912 Channel 111a, 111b Measurement point 111c Heating point 112 Injection of fine particle solution Unit 113 sheath solution injection unit 114 particulate collection unit 115 sheath solution discharge unit 116 cover glass 117 silicone resin 120 substrate fixing unit 121 positioning pin 130, 230 optical path forming unit 131, 231 lens 132a, 132b, 132c, 232 optical path 133a, 133b, 133c Light incident / exit point 134 Optical connectors 135a, 135b, 135c, 235 First to fourth optical fibers 140, 240 Measurement control units 141, 241 Connection optical fiber 142 Light source 143 Light receiving unit 144 Measuring means 145 Stimulation applying means 150 Pneumatic pressure control unit 151 Hollow tube

Claims (31)

Functional light acting on the object to be irradiated is incident on an optical path forming unit that forms two or more optical paths so as to have functional points at two or more positions.
Emitting the functional light to any of the functional points via any of the light paths;
Measuring the irradiated object by guiding measurement light emitted from a measurement point by irradiation of the functional light to a predetermined light receiving unit from any of the optical paths,
An optical measurement method of irradiating the irradiated object with functional light different from the functional light via any of the optical paths ,
The irradiated object is an irradiated solution in which a fine particle-containing solution containing fine particles is sheathed with a sheath solution, and the irradiated solution is passed through a channel provided in a substrate to identify and separate the fine particles. ,
The substrate is disposable, and is placed on a substrate fixing portion. The channel is formed on the surface of the substrate, the channel is covered with a cover glass, and the cover glass is fixed to the substrate. An optical measurement method characterized by being placed so as to face the upper surface of the part . The optical measurement method according to claim 1 , wherein the substrate and the optical path forming unit are housed in a clean bench. The optical measurement method according to claim 1, wherein the substrate is detachably positioned and fixed to the substrate fixing portion. The substrate fixing portion has a positioning pin, the substrate has an insertion hole for inserting the positioning pin, and the positioning pin is inserted into the insertion hole to be positioned and fixed to the substrate fixing portion. The optical measurement method according to claim 3. The optical measurement method according to claim 1, wherein the functional light is emitted toward the cover glass from below the substrate . The optical measurement according to any one of claims 1 to 4 , wherein a predetermined inclusion contained in the irradiated object is separated by irradiating the irradiated object with the other functional light. Method. At least one optical measuring method according to any one of claims 1 to 6 2 or more measuring light including the fluorescence wavelength and wherein the guided of the light path. The optical measurement method according to any one of claims 1 to 7, characterized in that the emission and measurement of the measuring light of the functional light at a position separated from the irradiated object and the light path forming unit . In the light path forming unit, optical measurement method according to any one of claims 1 to 8, characterized by forming the two or more light paths in one lens system. In the light path forming unit, optical measurement method according to any one of claims 1 to 8 to place one or more fiber without passing through the lens and forming the two or more light paths . Opposite the the irradiated object is via the lens of the optical path, claim 9, wherein the disposing the light incident and exit points to the position conjugate with the position of irradiating the functional light irradiated object The optical measurement method described in 1. As the measuring light, in any one of claims 1 to 11, characterized in that measuring fluorescence, wherein the functional light is emitted said excited by the scattered light or the functional light scattered by an object to be irradiated The optical measurement method described. The optical measurement method according to claim 12 , wherein the scattered light is backscattered light scattered in a coaxial direction with the functional light. The optical measurement method according to claim 12 , wherein the scattered light is side scattered light scattered in a direction substantially perpendicular to an irradiation direction of the functional light. 13. The optical measurement method according to claim 12 , wherein when measuring the fluorescence having different wavelengths, the fluorescence on the long wavelength side is measured first. A stimulus-sensitive substance is added to at least one of the fine particle-containing solution or the sheath solution, and the flow of the irradiated solution is controlled by applying a predetermined stimulus to the stimulus-sensitive substance with the other functional light. The optical measurement method according to any one of claims 1 to 15 . The optical measurement method according to claim 11 , wherein an optical fiber is connected to the light incident / exit point, and the measurement light is incident and the functional light is emitted. The optical measurement method according to claim 17 , wherein two or more optical fibers are arranged in parallel at the light incident / exit point. The optical measurement method according to claim 10, wherein the functional light is irradiated from a plurality of optical fibers arranged so that optical axes thereof are parallel to each other along the flow path. According to claim 18 or 19, characterized in that the measuring light emitted from the functional point by the irradiation of the functional light, the functional light to guide the optical fiber identical to that guided Light measurement method. The optical measurement method according to claim 18, wherein the two or more optical fibers are connected to the optical path forming unit using an optical connector. Provided the two or more functional point in the flow direction of the irradiation target solution, the two from the above measurement light incident from the function point of claims 1 to 21, characterized in that to estimate the flow rate of the irradiation target solution The optical measurement method according to any one of claims. 23. The optical measurement method according to claim 22 , wherein the backscattered light is received at the two or more functional points to estimate a flow rate of the irradiated solution. 23. The optical measurement method according to claim 22 , wherein the side scattered light is received at the two or more functional points to determine a flow rate of the irradiated solution. Determined individually the flow rate of the fine particles contained in the said fine particles free solution, according to any one of claims 22 to 24, characterized in that to each time calculated timing from flow velocity performing fractionation of the particulates Light measurement method. The fine particle-containing solution or the sheath solution to which the stimulus-sensitive substance is added is irradiated with a predetermined functional light and heated, thereby changing the flow velocity resistance to adjust the interval between the fine particles, or from the flow path The optical measurement method according to claim 16 , wherein the fine particles are selectively allowed to flow through another branched flow path. 27. The optical measurement method according to claim 26 , wherein infrared light is used as the functional light. 28. The optical measurement method according to claim 27 , wherein a position away from a straight line connecting the two or more measurement points is heated with the infrared light. 28. The optical measurement method according to claim 27 , wherein a position on a straight line connecting the two or more measurement points is heated with the infrared light. Optical measurement method according to any one of claims 27 to 29, characterized in that heating the downstream side in the infrared light from the measuring point. The fine particles, optical measurement method according to any one of claims 1 to 30, characterized in that a cell.

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