JP5260949B2 - Optical measuring device and optical measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical measuring instrument which can correctly measure shape information of samples. <P>SOLUTION: This optical measuring instrument is used to measure shape information of the sample dispersed in a fluid flowing inside channel by irradiating light on the above sample. The instrument is provided with a light source section 20 for irradiating irradiated light L on the fluid 11, a light-receiving section 31 which irradiates the irradiated light from the light source section 20 on the fluid and receives optical information of the sample S including the irradiated light transmitted through the fluid to generate light-receiving signal SG1 in a condition that relative position of the sample S varies at the constant speed to the irradiated light, and a measuring section 120 which measures variation in the light-receiving signal caused by the sample. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to an optical measurement device and an optical measurement method, and more particularly to an optical measurement device for irradiating a sample with irradiation light in order to measure optical information of a sample such as a cell dispersed in a liquid flowing in a flow path. The present invention relates to an optical measurement method.

The liquid in which the sample (microscopic object) is dispersed in the liquid flows in the capillary tube, and light from the light source is irradiated to this liquid flow, so that the optical information (fluorescence information) of the sample in the liquid flow It has been proposed to measure. (For example, refer to Patent Document 1).
Japanese Patent No. 2973387

However, in the measuring apparatus disclosed in Patent Document 1, the laser light is irradiated to the sample passing through the liquid in the capillary through the optical fiber, but the shape information about the sample is obtained using the transmitted light. It is difficult to measure accurately.
Accordingly, an object of the present invention is to provide an optical measurement device and an optical measurement method capable of accurately measuring sample shape information in order to solve the above-described problems.

In order to solve the above problems, an optical measuring apparatus of the present invention, by irradiating the single mode light to the sample was flowed into the flow path is dispersed in the liquid body, the light for measuring optical information of the sample A measuring device,
A light source unit for irradiating the liquid with irradiation light having a predetermined wavelength ;
The light source unit is disposed opposite to the flow channel, and the liquid is irradiated with the irradiation light of the light source unit in a state where the relative position of the sample is changed at a constant speed with respect to the irradiation light. An irradiation-light receiving optical unit comprising a light receiving unit that receives light information of the sample including the irradiation light transmitted through the liquid and generates a light reception signal;
A second light source unit that irradiates the liquid with a second irradiation light having a wavelength different from that of the irradiation light;
The second light source that is disposed opposite to the second light source unit via the flow path, irradiates the liquid with the second irradiation light of the second light source unit, and transmits the liquid. A second light-receiving optical unit comprising a second light-receiving unit that receives light information of the sample and generates a light-receiving signal, and
The second irradiation-light receiving optical unit is disposed away from the irradiation-light receiving optical unit in the flow direction of the flow path,
A measurement unit that measures fluctuations of the received light signal due to the sample and a control unit that analyzes the size, shape, and internal structure of the sample from the measurement signal are provided.

The optical measurement device of the present invention is preferably characterized in that the sample is dispersed in the liquid flowing in the flow path.
The optical measurement device of the present invention is preferably characterized in that the irradiation light for measurement is non-condensing light irradiated by an optical fiber.
The optical measurement device of the present invention is preferably characterized in that the irradiation light that has passed through the liquid is received by the optical fiber.

  In the optical measuring device of the present invention, preferably, the fluctuation of the light reception signal due to the sample is a change in the light reception signal due to a relative position of the sample being changed at a constant speed with respect to the irradiation light. And

  The optical measurement device of the present invention preferably analyzes a variation pattern of the received light signal due to the sample.

  The optical measurement device of the present invention is preferably characterized in that it measures the maximum value, width, or area of the pulse shape portion in the variation pattern of the received light signal.

The optical measurement device of the present invention is preferably characterized in that the size of the sample is identified from the maximum value of the pulse shape portion in the variation pattern of the received light signal.

  The optical measurement device of the present invention is preferably characterized in that it is discriminated based on statistical information of an approximate curve of a pulse shape portion in the pattern of fluctuation of the received light signal.

  The optical measurement device of the present invention is preferably characterized in that the received light signal performs a pattern analysis of a pulse shape portion.

  The optical measurement apparatus of the present invention is preferably characterized in that the light reception signal analyzes a single pulse approximate curve by correcting a plurality of peak values from a variation pattern.

  The optical measurement device of the present invention is preferably characterized in that the received light signal is subjected to information analysis on an arbitrary region of a variation pattern to identify the shape of the sample.

  The optical measurement device of the present invention is preferably characterized in that the sample is analyzed by light reception signals from a plurality of wavelengths.

  The optical measurement device of the present invention is preferably characterized in that the light reception signal analyzes a variation pattern to identify a cell type.

  The optical measurement device of the present invention is preferably characterized by recognizing a specific region of an arbitrary phase of the cell cycle or a region of a polyploid nucleus as the sample.

  The optical measurement device of the present invention is preferably characterized in that the size, shape, and internal structure of the sample are identified from a plurality of information obtained by analyzing the variation pattern of the irradiation light.

  The optical measurement device of the present invention preferably includes a plurality of the light receiving units that receive the irradiation light.

  The optical measurement device of the present invention is preferably characterized in that the light receiving unit analyzes the fluorescence information of the sample and a light reception signal of side scattered light.

The measuring device of the present invention is preferably characterized in that an arbitrary sample is taken from the analysis result by the control unit .

The measurement method of the present invention is an optical measurement method for measuring the size, shape, and internal structure light information of the sample by irradiating the sample dispersed in the liquid with a single mode light,
Two light source section for outputting the illumination light of the sample are spaced apart in the flow direction different wavelengths, in a state where the relative position of the sample relative to the previous SL irradiation light is changed at a constant speed, the liquid The sample is irradiated with the irradiation light, and the light information of the sample including the measurement light that has passed through the liquid is received by the two light receiving units disposed opposite to each other through the light source unit and the flow path , Generates a received light signal corresponding to the wavelength ,
A variation of the received light signal due to the sample is measured by a measurement unit.

The measurement method of the present invention preferably measures the optical information of the cells by irradiating the cells in the liquid with irradiation light and receiving the liquid and the transmitted light obtained through the cells. Is an optical measurement method,
Two light source sections or we outputting the irradiation light of the sample are spaced apart in the flow direction different wavelengths, in a state where the relative position of the cell relative to the previous SL irradiation light is changed at a constant speed, before Symbol by irradiating the irradiation light for the liquid, the transmitted light by receiving, before Symbol temporally intensity of the transmitted light by the two light receiving portions that are opposed to each other via the flow path and the light source unit It is characterized by measuring changes.

The optical measurement method of the present invention is preferably characterized in that attenuation and amplification of the intensity of the transmitted light can be measured.
The optical measurement method of the present invention is preferably characterized in that the intensity of the transmitted light changes with time depending on the number of cell types or cell nuclei and has a waveform having two or more attenuation waveform portions. In particular, the state of the corresponding cell is specified by the change in the waveform in the polyploid nucleus region of the cell cycle.

  The optical measurement method of the present invention is preferably characterized in that the intensity of the transmitted light changes with time depending on the number of the cell types or cell nuclei and repeats an attenuation waveform portion and an amplification waveform portion. .

The optical measurement method of the present invention is preferably characterized in that the intensity of the transmitted light varies with time depending on the number and the size of the cell nucleus and the attribute and property of the cell.
The optical measurement method of the present invention preferably measures the optical information of the cells by irradiating the cells in the liquid with irradiation light and receiving the transmitted light obtained by passing through the cells of the liquid. Is an optical measurement method for
Irradiating the cell with the irradiation light from a light source,
In a state where the relative position of the cells is changed at a constant speed with respect to the irradiation light, the liquid is irradiated with the irradiation light of the light source unit, and the transmitted light is received by a light receiving unit,
The waveform shape of the intensity of the transmitted light that changes with time is stratified by approximating one or more standard template waveforms prepared in advance, and the characteristics of the cells are specified. To do.

  The optical measurement method of the present invention preferably uses the intensity of the transmitted light, and changes with time to repeat the attenuation waveform portion and the amplification waveform portion. It is characterized by identifying cancer cells that exhibit different properties.

The optical measurement method of the present invention is preferably characterized in that the intensity of the transmitted light identifies a specific cell including a blood cell.

The optical measurement method of the present invention is preferably characterized in that the wavelength of the transmitted light is 325 nm to 900 nm.
The optical measurement method of the present invention is preferably characterized in that the irradiation light irradiated from the light source unit toward the cell spreads from the light source unit toward the light receiving unit.

  In the optical measurement method of the present invention, preferably, the light receiving unit is a charge coupled device camera, and the cell, the irradiation unit, and the cell even when the cell flows in the flow path or the cell is stationary in the liquid. The strength of the light reception signal and the two-dimensional distribution state of the strength of the light reception signal are measured by resting or changing the relative positions of the three light receiving units.

The optical measurement method of the present invention is preferably configured such that the transmitted light intensity waveform change waveform for the cells of the population is divided into a plurality of groups, the frequency distribution of the groups is different, and the difference of the population of the cells is It is characterized by distinguishing and evaluating.
In the optical measurement method of the present invention, preferably, the measurement result of the transmitted light waveform acquired from the plurality of cells is divided into a plurality of types of waveforms, and the type of the cell is obtained from statistical information for each of the types. And specifying a state.

  In the optical measurement method of the present invention, preferably, the amplification of the transmitted light is measured by the interference phenomenon of the transmitted light being caused by the size and the number of cell nuclei and the cell attributes and properties. It is characterized by a temporal change in intensity of transmitted light.

The optical measurement method of the present invention is preferably characterized in that an arbitrary sample is collected from the result of stratification .
The optical measurement method of the present invention is preferably characterized by dispensing and sorting the specified cells.
The cell evaluation method of the present invention is preferably performed on the selected cells,
The time-dependent change of the selected cells is evaluated by culturing or adding a predetermined reagent.

According to the optical measurement device and the optical measurement method of the present invention, the shape information of the sample can be accurately measured.
According to the optical measurement method of the present invention, the type of cell can be determined.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings.
1 and 2 show a preferred embodiment of the optical measuring device of the present invention. FIG. 1 is a side view of the light measurement device, and FIG. 2 is a cross-sectional view taken along the line KK in FIG. A configuration example of the optical measurement device 1 shown in FIGS. 1 and 2 will be described.

  The optical measurement device 10 shown in FIG. 1 is used as an optical measurement unit of a flow cytometer as an example. The flow cytometer 1 includes the optical measuring device 10, a supply unit 12 for supplying a liquid 11 in which a sample (microscopic object) S is dispersed in a liquid, and a sample dispensing unit 13. .

  The supply unit 12 supplies the liquid 11 in which the sample S is dispersed to the capillary 30 through the tube 14 in the Z1 direction (in the example of FIG. 1, from the upper side) together with the sheath flow 19 as the sample flow 11S. be able to.

The sample S of the liquid 11 that has passed through the capillary 30 of the optical measuring device 10 can be divided into a necessary part and an unnecessary part in the dispensing unit 13.
The optical measurement device 10 shown in FIG. 1 has a light source 20 that irradiates the sample stream 11S of the liquid 11 in the capillary 30 and the sample S with the irradiation light L, and a flow path for flowing the sample stream 11S including the sample S. It has a capillary 30, a light receiving part 31 for transmitted light, a light receiving part 50 for side scattered light, and a control part 100. The control unit 100 is also called an analysis unit, and analyzes the size, shape, and internal structure of the sample from the measurement signal.

  The sample is also called a minute object. Single mode is a Gaussian intensity pattern. An optical measuring device for measuring optical information of a sample is also called a sample measuring device or a minute object measuring device, and is a device that measures the size, shape, and internal structure of a sample.

  The light source unit 20 of the optical measuring device 10 irradiates the liquid 11 in the capillary 30 with the irradiation light L, and the light receiving unit 31 receives the light reception information L1 in an arbitrary region including the irradiation light L by the light receiving unit 31. To do. Thereby, the light receiving unit 31 sends the light reception signal SG1 to the measurement unit 120. The measurement unit 120 is configured to identify shape information such as size information, shape recognition, and cell recognition of the sample S, for example, by analyzing fluctuations in the light reception signal SG1 due to the sample S.

  As an example, the light source unit 20 includes a laser light source 21 such as a laser diode and an optical fiber 22 for irradiation. For example, as shown in FIG. 2, the light receiving unit 13 includes a light receiving optical fiber 32 and a light receiving element 33 such as a photodiode. As shown in FIG. 1, the first optical axis D1 of the light source unit 20 and the second optical axis D2 of the light receiving unit 31 preferably coincide with each other.

  In the optical measuring device 10, single-mode excitation light (irradiation light) L generated by the laser light source 21 is applied to the sample flow 11 S of the liquid 11 in the capillary 30 and the sample sample S passing through the sample flow 11 S of the liquid 11. Irradiate and measure the light information (fluorescence information) of sample S. That is, the light receiving unit 31 irradiates the sample flow 11S of the liquid 11 with the irradiation light L from the light source unit 20 in a state where the relative position of the sample S is changed at a constant speed with respect to the irradiation light L. Optical information including the irradiation light L transmitted through the sample flow 11S is received by the light receiving unit 31. The light receiving unit 31 sends a light reception signal SG1 to the measurement unit 120.

The side scattered light receiving unit 50 is arranged on the side of the capillary 30 and can receive scattered light obtained from the sample flow 11S of the liquid 11 and the sample S.
The light receiving signal SG1 from the light receiving unit 31 and the light receiving signal SG2 from the light receiving unit 50 are processed by the measuring unit 120 of the control unit 100.

  The sample S shown in FIGS. 1 and 2 is, for example, a cell having a size of 5 μm, and the sample flow 11S is passed through the capillary 30 of the optical measuring device 10 in a form of being wrapped by the sheath flow 19. It is supposed to flow. In the sheath flow technique using the sheath flow 19, the width of the sample flow 11S is arbitrarily controlled by the pressure difference between the sample flow 11S and the sheath flow 19, thereby reducing the pressure loss of the sample flow 11S and preventing clogging. .

Here, an operation example of the optical measurement device 10 shown in FIGS. 1 and 2 will be briefly described.
1 supplies the liquid 11 in which the sample S is dispersed in the capillary 30 as the sample flow 11S in the Z1 direction together with the sheath flow 19 (in the example of FIG. 1, from the upper side toward the tube 14). Supply through.

  The irradiation light L generated by the laser light source 21 irradiates the sample flow 11S of the liquid 11 in the capillary 30 and the sample S that passes through the sample flow 11S, and includes an arbitrary region including the irradiation light L that passes through the sample flow 11S. Is received by the light receiving unit 31 as light information (transmitted light information) of the sample S. Further, the side scattered light receiving unit 50 receives scattered light and fluorescence obtained from the sample flow 11S and the sample S. The light receiving signal SG1 from the light receiving unit 31 and the light receiving signal SG2 from the light receiving unit 50 are processed by the measuring unit 120 of the control unit 100. The control unit 100 controls driving of the laser light source 21.

  FIG. 3 shows side scattered light on the vertical axis and transmitted light on the horizontal axis. As shown in FIG. 3, the mound light is not dispersed and has less variation than the side scattered light. For example, FIG. 3 is a scatter diagram showing the results of measuring particles of different sizes, with the vertical axis indicating side scattered light and the horizontal axis indicating transmitted light information, indicating the maximum value (peak value) of fluctuation. It can be confirmed that the distribution is divided for each particle.

  FIG. 4 is a scatter diagram in which the average value of the transmitted light information of each distribution is shown on the vertical axis and the particle diameter on the horizontal axis. The approximate curve M1 obtained from the particle diameter and the measured transmitted light value, the particle diameter, and the particle area. The approximate curve M2 obtained from the above is shown in comparison, and the curve M1 and the curve M2 of the calculated value of transmitted light are very approximate. Therefore, the maximum value of the fluctuation of the transmitted light and the sample diameter (projected area) are shown. ) Has a correlation.

  FIG. 4 shows a comparison between a curve M1 indicating the transmitted light including the irradiation light L1 actually received by the light receiving unit 31 of FIG. 1 and a curve M2 of a calculated value of the transmitted light, and the vertical axis indicates the transmitted light. (Area value), and the horizontal axis is the size of the sample S. In FIG. 4, the curve M1 indicating the transmitted light and the curve M2 of the calculated value of the transmitted light are very close. There is a correlation between the peak value of the transmitted light and the sample diameter (projected area).

  FIG. 5 shows an example of a received light signal SG1 obtained from the transmitted light receiving unit 31 of FIG. 5A shows an example of the skewness of the received light signal SG1 = zero (normal distribution), FIG. 5B shows a distribution example of skewness> zero, and FIG. 5C shows the kurtosis. An example of> zero is shown. FIG. 5D shows an example of fitting analysis of the received light signal SG1, and FIG. 5E shows an example of peak correction of the received light signal SG1. FIG. 5F shows an example of a pulse pattern of the light reception signal SG1, which is an example having two extreme values. FIG. 5G shows an analysis target portion 210 of an arbitrary region in the light reception signal SG1. An example is shown. By fitting, the noise is removed from the light reception signal SG1 and shaped and corrected.

  FIG. 6 shows an enlarged fitting analysis example of the light reception signal SG1 shown in FIG. FIG. 6 shows an example of the shape of the actual received light signal SG1 and an example of the shape of the fitting curve H showing the result of fitting analysis of the received light signal SG1. The actual light reception signal SG1 includes noise, is not in a smooth shape, and the variation is reduced by obtaining the feature amount from the fitting curve H when the peak value is not at the center position of the waveform.

  FIG. 7 shows a case where the pulse shape of the light reception signal SG1 has a first extreme value P1 and a second extreme value P2 instead of a single peak value, and the light reception signal SG1 has a low correlation with the normal distribution. . In this case, the light receiving signal SG1 is obtained by predicting, calculating, and analyzing the peak value 220 indicated by the broken line from the approximate curve of the analysis target portion (from the skirt portion C to the first extreme value P1) 210 excluding the center. Can be reduced.

FIG. 8 shows an example of a spherical cell 300 as an example of the sample S, and FIG. 9 shows an example of a long thin cell 310 as another example of the sample S.
8A shows the light reception signal SG1 when the spherical cell 300 is measured, and FIG. 8B shows the spherical cell 300 and the irradiation region 400 of the irradiation light L. In FIG. 8B, the irradiation region 400 has a circular shape, and the spherical cell 300 passes in the Z1 direction, whereby a light reception signal SG1 shown in FIG. 8A is obtained.

  Similarly, FIG. 9A shows the light reception signal SG1 when the long thin cell 310 is measured, and FIG. 9B shows the long thin cell 310 and the irradiation region 500 of the irradiation light L. Yes. In FIG. 9B, the irradiation region 500 has a circular shape, and a long thin cell 310 passes in the Z1 direction, whereby a light reception signal SG1 shown in FIG. 9A is obtained.

  Compared with the analysis target portion 210 of the light reception signal SG1 shown in FIG. 8A and the analysis target portion 210 of the light reception signal SG1 shown in FIG. 9A, the spherical cells 300 and the long thin cells 310 have the same volume. Even so, since the shape of the spherical cell 300 and the shape of the long and narrow cell 310 are different, the spherical cell 300 and the long and narrow cell 310 enter the irradiation regions 400 and 500, respectively, and from the irradiation regions 400 and 500, respectively. When exiting, the rising and falling slopes of the pattern of the analysis target portion 210 of the received light signal SG1 shown in FIG. 8A and the rising and rising of the pattern of the analysis target portion 210 of the received light signal SG1 shown in FIG. The slope of the fall is different. However, it can be seen that when the spherical cell 300 and the long and narrow cell 310 enter the irradiation regions 400 and 500, respectively, they have almost the same pattern. The sizes of the irradiation areas 400 and 500 are the same.

  FIG. 10 shows another embodiment of the present invention. Unlike the embodiment of FIG. 1, a set 211, 212 of a plurality of light source units 20 and light receiving units 31 is arranged. One set 211 of the light source unit 20 and the light receiving unit 31 and the other set 212 of the light source unit 20 and the light receiving unit 31 can irradiate the sample S of the liquid 11 with, for example, laser light having different wavelengths. . That is, by changing the wavelength of irradiation light generated by a plurality of light source units to two or more, optical information can be obtained independently, and the sample S can be identified by analyzing the change in waveform with respect to the wavelength. it can. Thereby, the kind of sample can be more reliably discriminated.

  In the present invention, the light that the light source unit irradiates the liquid and the cell that is the measurement object (sample) is called irradiation light, and the irradiation light that has passed through the cell that is the measurement object (sample) is transmitted light. Call and distinguish. The wavelength of the transmitted light can be 325 nm to 900 nm, and the wavelength of the transmitted light is particularly preferably 635 nm or 488 nm.

  As shown in FIG. 11, in the embodiment of the present invention, the irradiation light L is irradiated to the sample S such as the liquid and the cell S <b> 1 from a laser light source of the light source unit 20, for example. It spreads from the part 20 toward the light receiving part 31. As the light receiving element of the light receiving unit 31, for example, a photomultiplier tube, a CCD (charge coupled device), a photodiode or the like can be adopted. The irradiation light emitted from the light source unit toward the cell is parallel from the light source unit toward the light receiving unit or spreads from the light source unit toward the light receiving unit.

  In the embodiment of the optical measurement method of the present invention, as shown in FIG. 11, the transmitted light obtained by irradiating the cells S1 in the liquid 11S flowing in the flow path with the irradiation light L and passing through the liquid cells is received. Thus, the optical information of the cell S1 is measured. The light source 20 emits the irradiation light L to the cell S1, and the irradiation light of the light source unit 20 with the relative position of the cell S1 with respect to the irradiation light L is changed at a constant speed along the direction perpendicular to the paper surface of FIG. When the liquid is irradiated with L and the transmitted light is received by the light receiving unit 31 to generate a light reception signal, the measurement unit 120 measures that the intensity of the transmitted light changes with time. Thereby, since the intensity | strength of transmitted light changes temporally, the kind of cell S1 can be discriminate | determined from this temporal change.

  In this measurement, attenuation and amplification of transmitted light intensity can be measured. The intensity of the transmitted light changes with time depending on the number of cell types or cell nuclei and becomes a waveform having two or more attenuation waveform portions. The intensity of transmitted light changes with time depending on the number of cell types or cell nuclei, and the intensity of transmitted light that repeats the attenuation waveform part and the amplification waveform part depends on the number of cell nuclei and the size of the cell nuclei. It changes with time depending on the attributes and properties of the cells that can be expressed.

  By irradiating the cells in the liquid with irradiation light and receiving the transmitted light obtained by passing through the cells in the liquid, when measuring the light information of the cells, the irradiation light is applied to the cells from the light source unit. Irradiation, with the relative position of the cells changing at a constant speed with respect to the irradiation light, irradiating the liquid with the irradiation light of the light source unit, and receiving the transmitted light with the light receiving unit, the transmission changed with time The waveform shape of the light intensity can be stratified by approximating one or more standard template waveforms prepared in advance to specify the properties of the cells.

  Using the intensity of the transmitted light, the type of cell or a cancer cell exhibiting different properties in the same type of cell is specified by a waveform that changes over time and repeats the attenuation waveform portion and the amplification waveform portion. Using the intensity of transmitted light, the B type special cell among the A type special cell, the B type special cell, and the C type special cell is specified, or the abnormal cell in the A type special cell is specified as the cancer cell. . The intensity of the transmitted light identifies specific cells including blood cells. The wavelength of transmitted light is 325 nm to 900 nm.

  The measurement result of the transmitted light waveform acquired from a plurality of cells is stratified into a plurality of types, and the type and state of the cell are specified from statistical information for each type as a whole. Amplification of transmitted light is a temporal change in the intensity of transmitted light that is measured by the interference phenomenon of transmitted light caused by the cell size and the number and size of cell nuclei and the cell attributes and properties. From this identification result, an arbitrary sample can be collected in the cell evaluation method. As a method for obtaining cells, the identified cells are dispensed and selected. The time-dependent change of the selected cells is evaluated by culturing the selected cells or adding a predetermined reagent.

For example, FIG. 12 shows a waveform shape example of the light reception signal SG1 shown in FIG. 1 when transmitted light is received.
FIG. 12A shows a transmitted light pattern waveform shape having a normal single peak. The transmitted light pattern waveform shape having a single peak is attenuation waveform data of transmitted light intensity of non-permeable beads and certain cells, and is a single peak waveform shape having one convex portion on the lower side. In this case, the waveform has one trough and is a signal corresponding to normal forward scattering.

FIGS. 12B and 12C show examples of transmitted light pattern waveform shapes (multi-peaks) confirmed by cells, for example, many confirmed in mouse cells. Therefore, the transmitted light pattern waveform shape when the transmitted light is received differs depending on the type of the cell S1 as the sample S. In this case, there are two troughs in the waveform and one crest, and the waveform as shown in FIG. 12 (A) should appear originally, but such a waveform is changed by the change in the internal state of the cell. Get out.
The transmitted light pattern waveform shape shown in FIG. 12B is a multi-peak waveform shape having two convex portions underneath, and the intensity of transmitted light of a cell showing a change waveform of the degree of attenuation due to an optical phenomenon such as interference. Is shown.

  Further, the transmitted light pattern waveform shape shown in FIG. 12C is a multi-peak waveform shape having two convex portions below, and light such as interference is compared with the transmitted light pattern waveform shape shown in FIG. It shows the intensity of transmitted light of a cell showing a change waveform with a greater degree of attenuation due to the phenomenon.

  The transmitted light pattern waveform shapes shown in FIGS. 12B and 12C are obtained, and the intensity of the transmitted light is formed from two or more attenuated waveforms depending on the number of cell nuclei representing cell types. The intensity of transmitted light is formed by repeating an attenuation waveform and an amplification waveform over time depending on the number of cell nuclei representing the cell type. The intensity of transmitted light varies with time depending on the attributes and properties of cells that can be expressed by the number of cell nuclei, the size of cell nuclei, and the like.

  As a result, by obtaining a transmitted light pattern waveform shape, the transmitted light intensity has a single peak in non-permeable beads or certain types of cells, as in the transmitted light pattern waveform shape shown in FIG. Attenuates as a wave shape. On the other hand, like the transmitted light pattern waveform shape shown in FIG. 12B and FIG. 12C, the intensity of transmitted light shows a repeated behavior of an attenuation waveform and an amplification waveform.

  In the embodiment of the optical measurement method of the present invention, the cell light is received by irradiating the cells in the liquid flowing through the flow path with irradiation light and receiving the transmitted light obtained by passing through the liquid cells. Measure information. At this time, the irradiation light L is irradiated from the light source unit 20 to the cell S1, and the liquid is irradiated with the irradiation light L of the light source unit 20 in a state where the relative position of the cell S1 is changed at a constant speed with respect to the irradiation light L. Then, when receiving the transmitted light and generating a light reception signal, the measurement unit 120 measures that the intensity of the transmitted light changes with time, and the waveform shape of the intensity of the transmitted light that changes with time Specifies the nature of the cell by approximating it to a standard template waveform prepared in advance. The intensity of the transmitted light is formed by repeating an attenuation waveform and an amplification waveform with the passage of time, and identifies the type of cell or cancer cells exhibiting different properties in the same type of cell. The identified cells can be dispensed and sorted. By culturing the selected cell or adding a predetermined reagent or the like, the change with time of the cell can be evaluated.

  Using the intensity of transmitted light, for example, the B type special cell among the A type special cell, the B type special cell, and the C type special cell is specified, or an abnormal cell in the A type cell is specified as a cancer cell. . Moreover, the cell in a blood cell is identified using the intensity of transmitted light.

  FIG. 13 shows another waveform shape example of the transmitted light pattern waveform shape when transmitted light is received. FIGS. 13A and 13B show examples of transmitted light pattern waveform shapes (multi-peaks) D1 and D2 obtained from cells that appear to be certain types of cancer cells, respectively. Each of the shapes D1 and D2 has three or more attenuation waveforms and amplification waveforms. These transmitted light pattern waveform shapes have a plurality of attenuation waveforms and amplification waveforms due to optical phenomena such as interference.

  FIG. 14 shows an example of a standard template waveform (waveform for comparison) D prepared in advance for specifying the property that a certain type of measurement target cell is a cancer cell. The standard template waveform in FIG. 14 includes a plurality of calculation parameters H1, H2, L1, L2, L3, A1, A2, B1 and the like.

  The transmitted light pattern waveform shapes D1 and D2 by certain types of cells shown in FIGS. 13A and 13B are actually measured. For the measured transmitted light pattern waveform shapes D1 and D2, processing belonging to a standard template waveform D prepared in advance is performed at high speed, and the characteristics of cancer cells are specified in this kind of cell. Can do. That is, normal cells and cancer cells of the same cell type can be identified. In the case of FIGS. 13A and 13B, there are three corrugated valleys and two peaks, and the number of peaks and valleys is the same, but they are different. When the frequency analysis of the waveform is used, the peak position of the power spectrum changes and the waveform can be specified in an analog manner. When it is classified by the number of valleys and the number of peaks, it is digital.

The intensity of transmitted light can identify specific cells including blood cells.
The light receiving unit is a charge coupled device camera as described above, and the relative position between the three of the cell, the irradiation unit, and the light receiving unit, even if the cell flows in a thin flow channel or the cell is stationary in the liquid. It is measured by the stationary or changing of. As a result, even if the cells are flowing in a narrow flow path or in a state where the cells are stationary in the liquid, the transmitted light is changed by the stationary or changing of the relative positions of the cells, the irradiation unit, and the light receiving unit. The strength of the can be measured.

  The waveform change waveform of the transmitted light intensity with respect to the cells of the population can be divided into a plurality of groups, and the difference in the population of the cells can be discriminated and evaluated with the frequency distribution for each group. As an example, FIG. 15 shows a population (1) and a population (2) of cells A, B, and C. The distributions of the A, B, and C waveforms in the cells of the two populations (1) and the cells of the population (2) shown in FIG. 15 are different. At this time, the population (1) and the population (2) are in different states and a certain reagent is added to the cells of the population (1), so that the population (1 ) Means that the change of cells can be reflected.

Conventional cell recognition / analysis is performed by nuclear staining of cells or antigen / antibody reactions on the cell surface. However, the nuclear staining of cells has the drawback of killing the cells. In addition to damaging cells due to antigen-antibody reactions on the cell surface, there is a problem that cells cannot be recognized without antibodies. is there.
In the embodiment of the present invention, since cells can be recognized and analyzed without staining, it is indispensable particularly for applications where it is necessary to evaluate changes in cells over time due to cell culture or drugs.

  In the embodiment of the present invention, the transmitted light is light that has passed through the cell, and includes diffracted light and scattered light that are generated during the passage. In the embodiment of the present invention, the waveform of the change in the intensity of transmitted light over time is also referred to as a fluctuation pattern of the intensity of transmitted light. In the embodiment of the present invention, the irradiation light from the light source unit is light that irradiates the cells.

  When measuring cells, the temporal change in the intensity of transmitted light with respect to one cell is recognized as a waveform pattern, and this waveform pattern is characterized by the number of waveforms and the relationship between the waveform valleys and the waveform peaks. be able to. The feature amount of the waveform pattern is determined based on the result of frequency analysis of the waveform pattern. Specifically, the maximum value and peak value of the power spectrum, and the number and ratio of peak values are used as the feature amount of the waveform pattern. When analyzing a cell, it is possible to determine the feature amount of the waveform pattern, the cell type, the internal state of the cell, the number of cell nuclei, and the state of the nucleus.

  A measuring means such as a light receiving unit is provided at a position where the excitation light and at least the light transmitted through the cell (all of which are referred to as transmitted light) can be measured. In this case, a measuring means such as a light receiving unit is provided at a position where the excitation light and the light that has passed through the cell or the light reflected / scattered forward by the cell (in this case, all of them are referred to as transmitted light) can be measured. Provide. This measurement means is an optical fiber, and the core portion of the optical fiber is smaller than the excitation light spot of the measurement means for transmitted light.

  Using information on the waveform pattern measurement of transmitted light, cancer cells that show different types of cells or different properties in the same type of cells are analyzed. In this case, the feature amount of the waveform pattern is extracted from the waveform pattern measurement information of the transmitted light, and the type of cell or cancer cells showing different properties in the same type of cell are analyzed using the feature amount.

  Using the information of the transmitted light waveform pattern measurement, a specific cell is identified from among a plurality of cells, or cells having different intracellular states are identified from among the same cells. For example, using information on the waveform pattern measurement of transmitted light, cancer cells are identified from among a plurality of cells, or cancer cells having different intracellular states are identified from among the same cancer cells.

  The cell state includes not only the cell size and the number and size of cell nuclei but also the cell attributes and properties, for example, the state of proteins in the cytoplasm and the state of other tissues. The interference phenomenon of transmitted light is caused by the size of cells, the number and size of cell nuclei and the state of proteins in the cytoplasm.

  The light receiving unit is, for example, a charge coupled device camera, but the relative position between the three of the cell, the irradiation unit, and the light receiving unit even when the cell flows in the flow path or the cell is stationary in the liquid. The intensity of the received light signal and the two-dimensional distribution state of the intensity are measured.

In the embodiment of the present invention, the irradiation light (incident light) generated by the light source unit is parallel light or light close to parallel light (a range up to the spread angle of the NA (numerical aperture) of the optical fiber). As the detection light detected by the light receiving unit, transmitted light information and scattered light information such as side scattered light and back scattered light can be used. Here, the transmitted light is optical information measured at a position where the irradiation light (incident light) can be directly measured.

  In addition, the fluctuation of the received light signal, that is, the fluctuation of the transmitted light, means that the sample S has passed through the irradiated area with reference to the received light signal value directly receiving the irradiated light L when the sample S does not exist in the irradiated area. This is a change in signal over time. When the sample S is applied to the irradiation region, the amount of light received by the irradiation light L changes due to scattering, absorption, and transmission (including diffraction and interference) by the sample. For example, in the case of uniform material particles, the intensity of the irradiation light is a normally distributed intensity pattern (single mode). Therefore, when the sample is positioned at the center of the irradiation area, the received light signal is the smallest, and a single peak pulse A signal change in shape is obtained. From this peak value, the size of the sample can be identified.

  The sample S, which is an object, is a living body such as cells, particles, and bacteria flowing in the flow path, or an inorganic substance.

  Information on the size and internal structure of the sample S can be obtained by processing the transmitted light information and the scattered light information. As irradiation light, single mode light is irradiated. By detecting light information including irradiation light transmitted through the liquid by the light receiving unit, a change in waveform shape (change in frequency component) or change from the standard waveform of the light reception signal SG1 is detected. The type of sample S is determined.

In the embodiment of the present invention, the liquid 11 in the capillary 30 as the flow cell is irradiated with irradiation light, and an arbitrary region including the irradiation light is received by the light receiving unit, so that the light receiving unit generates a light reception signal SG1 and receives light. By analyzing the variation of the signal SG1 due to the sample S, the accuracy of the size information is improved, the shape is recognized, and the cells are recognized.

  The optical measuring device of the present invention is used to measure the light information of the sample by irradiating the sample dispersed in the liquid flowing in the flow path, and irradiates the liquid 11 with the irradiation light L. In the state where the relative position of the sample S with respect to the light source unit 20 and the irradiation light is changed at a constant speed, the liquid is irradiated with the irradiation light of the light source unit 20 and the sample S including the irradiation light transmitted through the liquid A light receiving unit 31 that receives optical information and generates a light reception signal SG1 and a measurement unit 120 that measures fluctuations in the light reception signal due to the sample are provided. This makes it possible to accurately measure the shape information of the sample (size, cell type, outer shape such as a circle and a long shape). Any sample can be taken from the identification result.

  In the optical measuring device, the sample S is dispersed in the liquid flowing in the flow path. Thereby, the sample S can change the relative position of the sample S with respect to irradiation light at a constant speed only by flowing the liquid through the flow path.

  In the optical measurement device, the measurement irradiation light L is non-condensing light irradiated by the optical fiber 22. Thereby, irradiation light can be irradiated with respect to the sample S in an arbitrary range in the liquid.

  In the optical measurement device, the irradiation light L that has passed through the liquid is received by the optical fiber 32. Thereby, the irradiation light L including the optical information of the sample S can be reliably received by the light receiving unit 31 using the optical fiber 32.

In the optical measurement device, the fluctuation of the light reception signal SG1 due to the sample S is a change in the light reception signal SG1 due to the relative position of the sample S being changed at a constant speed with respect to the irradiation light L. Thereby, the shape of the sample S is obtained accurately.
In the optical measurement device, by analyzing the variation pattern of the light reception signal SG1 due to the sample S, the difference between the samples S can be reliably identified.

  The optical measuring device measures the maximum value, width, or area of the pulse shape portion in the variation pattern of the received light signal.

  The optical measuring device identifies the size of the sample from the maximum value of the pulse shape portion in the variation pattern of the received light signal.

  The optical measuring device discriminates based on the statistical information of the approximate curve of the pulse shape portion in the variation pattern of the received light signal.

The optical measurement device analyzes the pattern of the pulse shape portion of the received light signal.
The optical measuring device analyzes the single pulse approximate curve by correcting a plurality of peak values from the variation pattern of the received light signal.

The optical measuring device analyzes the information of an arbitrary region of the variation pattern of the received light signal, and identifies the shape of the sample.
The optical measuring device analyzes the sample by light reception signals from a plurality of wavelengths.
The optical measurement device analyzes the variation pattern of the received light signal and identifies the cell type.
The optical measurement device recognizes a specific region in an arbitrary period of a cell cycle as a sample.
The optical measurement device identifies the size, shape, and internal structure of the sample from a plurality of information obtained by analyzing the variation pattern of the irradiation light.

The optical measurement device includes a plurality of the light receiving units that receive irradiation light.
In the optical measurement device, the light receiving unit receives the fluorescence information and the side scattered light of the sample. Thereby, the kind of cell can be measured reliably, for example.

  The optical measurement device sorts an arbitrary sample from the identification result. Thereby, arbitrary samples identified from a plurality of samples can be divided and removed.

By the way, this invention is not limited to the said embodiment, A various modified example is employable.
The light source unit 20 illustrated in FIG. 1 may be configured by the optical fiber 22 and the laser light source 21, or may be configured by only the laser light source 21. As shown in FIG. 1, the light receiving unit 31 for transmitted light may be composed of an optical fiber 32 and a light receiving element 33, or may be composed of only the light receiving element 33. The side scattered light receiving unit 50 may be composed of the optical fiber 51 and the light receiving element 52, or may be composed of only the light receiving element 52. Further, the side scattered light receiving unit 50 may use a lens system instead of the optical fiber 51.

The capillary 30 shown in FIGS. 1 and 2 is, for example, a hollow member having a square cross section, but may be, for example, a rectangular cross section or other cross sectional shapes.
The optical measuring device of the present invention is used in fields requiring examination, analysis and analysis of biopolymers of genes, immune systems, proteins, amino acids, and sugars, such as engineering, food, agriculture, fishery processing, etc. It can be applied to all fields such as fields, hygiene, health, immunity, plague, genetic fields such as heredity, and science fields such as chemistry or biology.

It is a perspective view which shows an example of the flow cytometer containing preferable embodiment of the optical measuring device of this invention. It is sectional drawing in the KK line | wire in FIG. It is a top view of the optical measuring device of FIG. It is a figure which shows the side scattered light on the vertical axis | shaft, and has shown the transmitted light on the horizontal axis. It is a figure which compares and shows the curve M1 which shows the transmitted light containing the irradiation light which the light-receiving part of FIG. 1 actually received, and the curve M2 of the calculated value of the transmitted light. It is a figure which shows the example of the light reception signal SG1 obtained from the light-receiving part 31 of the transmitted light of FIG. FIG. FIG. It is a figure which shows the example of a spherical cell as an example of the sample S. It is a figure which shows the example of a long thin cell as another example of the sample. It is a figure which shows other embodiment of this invention. It is a figure which shows a mode that the irradiation light from a light source part has spread. It is a figure which shows the waveform shape example of the received light signal at the time of receiving the transmitted light. It is a figure which shows another waveform shape example of the light reception signal at the time of receiving the transmitted light. It is a figure which shows the example of a standard template waveform prepared beforehand. It is a figure which shows the population (1) and population (2) of cells A, B, and C.

Explanation of symbols

10 Optical measurement device
DESCRIPTION OF SYMBOLS 11 Liquid 11S Sample flow 19 Sheath flow 20 Light source part 30 Capillary (an example of a flow path)
31 Light receiving part 120 Measuring part S Sample L Irradiation light

Claims (40)

An optical measurement device for measuring optical information of the sample by irradiating a sample dispersed in a liquid and flowing in a flow path with a single mode light,
A light source unit for irradiating the liquid with irradiation light having a predetermined wavelength;
The light source unit is disposed opposite to the flow channel, and the liquid is irradiated with the irradiation light of the light source unit in a state where the relative position of the sample is changed at a constant speed with respect to the irradiation light. An irradiation-light-receiving optical unit including a light receiving unit that receives light information of the sample including the irradiation light transmitted through the liquid and the sample and generates a light reception signal;
A second light source unit that irradiates the liquid with a second irradiation light having a wavelength different from that of the irradiation light;
The second light source unit is disposed to face the second light source unit through the flow path, and the second light source unit irradiates the liquid with the second irradiation light of the second light source unit and transmits the liquid and the sample . A second irradiation-light receiving optical unit including a second light receiving unit that receives light information of the sample including irradiation light and generates a light reception signal;
The second irradiation-light receiving optical unit is disposed away from the irradiation-light receiving optical unit in the flow direction of the flow path,
An optical measurement apparatus comprising: a measurement unit that measures fluctuations in the received light signal due to the sample; and a control unit that analyzes the size, shape, and internal structure of the sample from the measurement signal.   The optical measurement device according to claim 1, wherein the sample is dispersed in the liquid flowing in the flow path.   The optical measurement apparatus according to claim 1, wherein the irradiation light for measurement is non-condensing light irradiated by an optical fiber.   The optical measurement apparatus according to claim 3, wherein the irradiation light that has passed through the liquid is received by an optical fiber. The optical measurement device according to claim 4, wherein a core portion of the optical fiber is smaller than a spot of the irradiation light received by the optical fiber.   The optical measurement according to claim 1, wherein the fluctuation of the light reception signal due to the sample is a change in the light reception signal due to a change in relative position of the sample at a constant speed with respect to the irradiation light. apparatus. The optical measurement apparatus according to claim 1, wherein the measurement unit measures attenuation and amplification of the intensity of the transmitted light as the light reception signal. The optical measurement apparatus according to claim 7, wherein the control unit analyzes a sample from an attenuation waveform portion and an amplification waveform portion of the measurement signal. An optical system as claimed in any one of claims 1-8, characterized in that analyzing the pattern of variation of the light receiving signal by the sample. The optical measurement apparatus according to claim 9 , wherein the maximum value, width, or area of a pulse shape portion in the pattern of fluctuation of the received light signal is measured. The optical measurement device according to claim 9 , wherein the size of the sample is identified from the maximum value of the pulse shape portion in the pattern of fluctuation of the received light signal. The optical measurement device according to claim 9 , wherein the determination is made based on statistical information of an approximate curve of a pulse shape portion in the variation pattern of the received light signal. The optical measurement device according to claim 9 , wherein the received light signal performs a pattern analysis of a pulse shape portion.   The optical measurement device according to claim 1, wherein the light reception signal analyzes a single pulse approximate curve by correcting a plurality of peak values from a variation pattern.   The optical measurement apparatus according to claim 1, wherein the received light signal analyzes information on an arbitrary region of a fluctuation pattern and identifies a shape of a sample.   The optical measurement apparatus according to claim 1, wherein the sample is analyzed by light reception signals from a plurality of wavelengths.   The optical measurement apparatus according to claim 1, wherein the light reception signal analyzes a variation pattern to identify a cell type.   The optical measurement apparatus according to claim 1, wherein a specific region or a polyploid nucleus region in an arbitrary phase of the cell cycle as the sample is recognized.   The optical measurement device according to claim 1, wherein the size, shape, and internal structure of the sample are identified from a plurality of pieces of information obtained by analyzing the variation pattern of the irradiation light.   The optical measurement device according to claim 1, further comprising a plurality of the light receiving units that receive the irradiation light. 21. The optical measurement device according to claim 20 , wherein the light receiving unit analyzes the fluorescence information of the sample and a light reception signal of side scattered light.   The optical measurement apparatus according to claim 1, wherein an arbitrary sample is separated from an analysis result by the control unit. It is an optical measurement method for measuring the size, shape, and internal structure light information of the sample by irradiating the sample dispersed in the liquid with single mode light,
From two light source units that are spaced apart in the sample flow direction and output irradiation light of different wavelengths, the relative position of the sample is changed at a constant speed with respect to the irradiation light. wherein by irradiating irradiation light, by receiving the two light receiving portions of the optical information of the sample containing the measurement light spent permeable to the liquid and sample are oppositely arranged through the flow path and the light source unit Te, Generates a received light signal corresponding to each wavelength,
While measuring the attenuation and amplification of the received light signal by the sample by the measurement unit ,
An optical measurement method comprising: analyzing a sample from an attenuation waveform portion and an amplification waveform portion of a measurement signal by a control unit . The irradiation light for measurement is non-collected light irradiated by a first optical fiber, and the irradiation light transmitted through the liquid and the sample is received by a second optical fiber,
  The optical measurement method according to claim 23, wherein a core portion of the second optical fiber is smaller than a spot of the irradiation light received by the second optical fiber. Said sample is a cell, the measurement signal, the number of the cell type or cell nucleus, claim 23 or claim, characterized in that the time varying to waveform having two or more decay waveform 24 The optical measurement method described in 1. 24. The sample according to claim 23, wherein the sample is a cell, and the measurement signal changes with time depending on the number of the cell types or cell nuclei and has a waveform having an attenuation waveform portion and an amplification waveform portion. 24. The optical measurement method according to 24 . 25. The sample according to claim 23 or 24, wherein the sample is a cell, and the measurement signal changes with time according to the attribute and property of the cell expressed by the number of the cell nuclei and the size of the cell nuclei. The optical measurement method described. It is an optical measurement method for measuring optical information of the cells by receiving transmitted light obtained by irradiating the cells in the liquid with irradiation light and passing through the cells of the liquid,
Irradiating the cell with the irradiation light from a light source,
In a state where the relative position of the cells is changed at a constant speed with respect to the irradiation light, the liquid is irradiated with the irradiation light of the light source unit, and the transmitted light is received by a light receiving unit,
The waveform shape of the intensity of the transmitted light that changes with time is stratified by approximating one or more standard template waveforms prepared in advance, and the characteristics of the cells are specified. To measure light. Using the intensity of the transmitted light, the type of the cell or a cancer cell having a different property in the same type of the cell is identified by a waveform that changes with time and repeats the attenuation waveform portion and the amplification waveform portion. The optical measurement method according to claim 28 . 30. The optical measurement method according to claim 29 , wherein the intensity of the transmitted light identifies a specific cell including a blood cell. The optical measurement method according to any one of claims 28 to 30 , wherein the wavelength of the transmitted light is 325 nm to 900 nm. 32. The optical measurement method according to claim 31 , wherein the irradiation light irradiated from the light source unit toward the cell spreads from the light source unit toward the light receiving unit. The light receiving unit is a charge coupled device camera, and the relative position between the cell, the irradiation unit, and the light receiving unit even when the cell flows in the flow path or the cell is stationary in the liquid. 32. The optical measurement method according to claim 31 , wherein the intensity of the received light signal and the two-dimensional distribution state of the intensity are measured by stationary or changing. Dividing the waveform variation waveform of the transmitted light intensity for the cells of the population into a plurality of groups, having a frequency distribution for each group, and distinguishing and evaluating the difference in the population of the cells The optical measurement method according to claim 28 . The measurement result of the transmitted light waveform acquired from the plurality of cells, the waveform is stratified into a plurality of types, and the type and state of the cells are identified from statistical information for each of the types. The optical measurement method according to claim 28 . Amplification of the transmitted light is a temporal change in the intensity of the transmitted light that is measured by the interference phenomenon of the transmitted light caused by the cell size and the number and size of cell nuclei and the attributes and properties of the cells. 29. The optical measurement method according to claim 28 , wherein: The optical measurement method according to any one of claims 28 to 36 , wherein an arbitrary sample is separated from the result of the stratification. 38. The optical measurement method according to claim 37 , wherein the identified cells are dispensed and selected. The cell evaluation method according to claim 38 , wherein the time-dependent change of the selected cells is evaluated by culturing the selected cells or adding a predetermined reagent. An optical measurement device for measuring optical information of the cells by receiving transmitted light obtained by irradiating the cells in the liquid with irradiation light and passing through the cells of the liquid,
A light source unit for irradiating the cell with the irradiation light;
In a state where the relative position of the cell is changed at a constant speed with respect to the irradiation light, a light receiving unit that receives the transmitted light that is irradiated to the liquid and passes through the cell;
The waveform shape of the intensity of the transmitted light that has changed over time is stratified by approximating one or more standard template waveforms prepared in advance, and a control unit that identifies the properties of the cells is provided. An optical measuring device comprising:

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