JP6238272B2 - Biological particle counter and biological particle counting method - Google Patents

Description

  The present invention relates to a biological particle counter and a biological particle counting method for detecting biological particles in a liquid in real time.

  Conventionally, in the detection of biological particles, a culture method (official method), a microcolony method, an ATP (luciferase) method, a fluorescent dye method, an autofluorescence method, and the like are known. Among these detection methods, the autofluorescence method is a detection method that can obtain the result of the presence or absence of biological particles in real time. This autofluorescence method irradiates a certain substance with light of a predetermined wavelength, excites the energy state of the substance (absorption of irradiation light), and then fluoresces extra energy when returning to the ground state. This is a method of knowing the presence or absence of biological particles based on the presence or absence of fluorescence detection. Moreover, by irradiating the wavelength peculiar to the substance, the substance easily absorbs light, and the self-fluorescence is easily emitted to the outside.

  The prior art which confirms the presence or absence of the biological particle in water using this autofluorescence phenomenon is known (for example, refer patent document 1). This prior art confirms the presence or absence of biological particles by irradiating an aqueous medium containing biological particles with ultraviolet rays to detect autofluorescence. In particular, this prior art uses a filter that selects a specific portion (wavelength region) of autofluorescence to be measured.

Special table 2009-501907

  Here, in the detection of biological particles using the autofluorescence phenomenon in water as in the technique of Patent Document 1, when ultraviolet light is irradiated to water, the irradiated ultraviolet light is scattered by water (Raman scattering), and from the ultraviolet light In other words, Raman scattered light having a long wavelength is generated, and in addition to autofluorescence, Raman scattered light due to water is also detected. Therefore, it is difficult to detect the presence or absence of only biological particles using autofluorescence as an index. In addition, even if a specific part of autofluorescence is selected, depending on the wavelength of the ultraviolet light to be irradiated, Raman scattered light from a liquid having the same wavelength as the autofluorescence may be detected at the same time. It is difficult to do.

  Therefore, the present invention provides a technique capable of reducing the influence of Raman scattered light caused by water, detecting biological particles in a liquid, and counting the number of biological particles.

In order to solve the above problems, the present invention employs the following solutions.
Solution 1: The biological particle counter of the present invention includes a light emitting means for irradiating a liquid containing a target object to be detected with light of a predetermined wavelength, and a light irradiated by the target object or the liquid and the light emitting means. Autofluorescence selection optical means for reducing Raman scattered light emitted from the liquid that is transmitted through the interaction and transmitting the autofluorescence emitted from the object, and the autofluorescence Biological particle determination means for determining whether or not the object contained in the liquid is a biological particle based on the light after the Raman scattered light is reduced by the selection optical means, and the light emitting means The biological particle counter is characterized by irradiating light having a predetermined wavelength that makes a peak wavelength of the autofluorescence emitted after irradiation different from a peak wavelength of the Raman scattered light.

The counting of biological particles by the biological particle counter of the present invention has, for example, the following characteristics and proceeds according to a predetermined procedure.
(1) Assuming that the liquid containing the object flows in the flow cell, the liquid containing the object flowing in the flow cell is irradiated with light of a specific wavelength. For example, laser light with a single wavelength is emitted from a laser diode. Here, the object represents biological particles, non-biological particles, or the like. Moreover, the liquid represents water, for example.

(2) When the laser beam is irradiated according to the above (1), light is emitted by the interaction between the laser beam and the object or the interaction between the laser beam and the liquid. Specifically, the main light emitted is scattered light emitted by reflection from biological particles, etc., autofluorescence emitted from laser light absorbed by biological particles, and reflection from non-biological particles. Scattered light emitted by the same, or Raman scattered light emitted by converting the wavelength of light incident on the liquid (molecule). In addition, the wavelength of the laser beam to be irradiated is set so that the peak wavelength of the autofluorescence spectrum is different from the peak wavelength of the scattered light and the Raman scattered light.

(3) The wavelengths of the light emitted by the above (2) are different. For example, the scattered light from biological particles and non-biological particles is approximately the same as the wavelength of the laser light, whereas Raman scattered light or The wavelength of light such as autofluorescence is longer than the wavelength of the laser light. In addition, for the wavelength of Raman scattered light and autofluorescence, the respective wavelength distribution regions may overlap. For example, the peak value of the wavelength distribution (peak wavelength) in autofluorescence is the peak value of the wavelength distribution of Raman scattered light. The wavelength may be longer than (peak wavelength). Utilizing the fact that the peak wavelength of autofluorescence and Raman scattered light differs depending on the setting of the wavelength of the laser light to be irradiated, the amount of Raman scattered light using an optical separator based on a specific wavelength (cutoff wavelength) Can be reduced more than the amount of autofluorescence. For example, using a long-pass filter or a band-pass filter with a specific wavelength as a reference, Raman scattered light is reduced, and almost all autofluorescence is transmitted.

(4) It is determined whether or not the object contained in the liquid is a biological particle based on the light transmitted in (3) above, that is, autofluorescence from the biological particle. And when it determines with it being a biological particle in the determination, the number of biological particles will be counted by counting the number.

  In this way, according to the present solution, the Raman scattered light is optically suppressed by using irradiation light that makes the peak wavelength of the Raman scattered light and the autofluorescence different from each other. Since fluorescence can be transmitted, it is possible to determine the presence or absence of biological particles in the liquid by reducing the influence of Raman scattered light from the liquid. Thereby, the counting precision with respect to a biological particle can be improved.

  Solution 2: The biological particle counter according to the present invention is the scattered light selection optical means that reflects scattered light emitted from the object and transmits light including the autofluorescence and the Raman scattered light in Solution 1. The biological particle determination means further determines whether the object contained in the liquid is a biological particle based on the light after passing through the scattered light selection optical means and the autofluorescence selection optical means. It is a biological particle counter characterized by determining.

The counting of biological particles by the biological particle counter of the present solution has the following characteristics, for example, and proceeds according to a predetermined procedure.
(5) According to the above (3), the wavelength of the light emitted by the above (2) is about the same as the wavelength of the laser light from the scattered light from the biological particles and the non-biological particles. The wavelength of light such as scattered light and autofluorescence was longer than the wavelength of the laser light. Therefore, the wavelength of scattered light from biological particles and non-biological particles is shorter than Raman scattered light and autofluorescence. Using this wavelength relationship, scattered light from biological particles or non-biological particles can be selected using an optical separator based on a specific wavelength. For example, it is possible to transmit only scattered light from biological particles or non-biological particles using a short-pass filter based on a specific wavelength (cutoff wavelength). In addition, by using a dichroic mirror that separates based on the cutoff wavelength, scattered light from biological and non-biological particles with wavelengths shorter than the separation wavelength is reflected, and light with a wavelength longer than the separation wavelength ( Raman scattered light and autofluorescence) can be transmitted.

(6) Included in the liquid based on the correlation between the light separated by (3) (autofluorescence) and the light separated by (5) (scattered light from biological or non-biological particles) It is determined whether the target object is a biological particle. And when it is determined that it is a biological particle in the determination, the number of biological particles is counted by counting the number, and when it is determined that it is a non-biological particle, the number is also counted. Also good.

(7) Here, the long pass filter of (3) may be installed on the transmission side downstream of the dichroic mirror of (5). Regarding the light separation according to the above (3) and (5), first, the separation according to the above (5) may be performed, and the separation according to the above (3) may be performed on the light after the separation. Specifically, first, scattered light from biological particles or non-biological particles shorter than the cutoff wavelength is reflected by the separation according to (5) above, and light including Raman scattered light or autofluorescence longer than the cutoff wavelength. Then, the separation according to the above (3) is performed on the light including the Raman scattered light and the autofluorescence transmitted earlier, and the light including the autofluorescence longer than the cutoff wavelength is transmitted.

(8) Correlation between light (autofluorescence) finally separated (transmitted) by (7) above and light (scattered light from biological or non-biological particles) separated (reflected) in the middle Based on the relationship, it is determined whether or not the object contained in the liquid is a biological particle. And when it is determined that it is a biological particle in the determination, the number of biological particles is counted by counting the number, and when it is determined that it is a non-biological particle, the number is also counted. Also good.

  Thus, according to this solution, since the transmission of Raman scattered light is suppressed, while the autofluorescence from biological particles is transmitted, it is included in the liquid due to the correlation with the scattered light from the object. It can be determined whether the target object is a biological particle. For example, when there is scattered light and transmitted light, it is determined that the object is a biological particle. When there is only scattered light, it is determined that the object is a non-biological particle that is not a biological particle, and there is transmitted light. Can be determined to be Raman scattered light by water. Thereby, the counting accuracy with respect to the biological particles can be further improved. Also, the use of optical separators such as dichroic mirrors, long pass filters, band pass filters, and short pass filters can be selected according to the installation environment (conditions).

  Solution 3: The biological particle counter according to the present invention receives the light after passing through the autofluorescence selection optical means in the solution 2, and outputs a first signal having a magnitude corresponding to the amount of light when the light is received. Autofluorescence light receiving means for outputting, light after passing through the scattered light selecting optical means, receiving scattered light, and outputting a second signal having a magnitude corresponding to the amount of light when received, and the scattering light When the magnitude of the second signal output by the light receiving means is greater than or equal to a predetermined threshold value, a scattered light detection signal output that outputs a detection signal as detecting scattered light emitted from the object contained in the liquid And a light shielding wall that prevents light incident from other than the optical path from entering the optical path from the scattered light selection optical means to the autofluorescence light receiving means through the autofluorescence selection optical means. Judgment means When the detection signal is output by the scattered light detection signal output means, and at the same time as the scattered light emitted from the object contained in the liquid is received by the scattered light receiving means. When light is received by the autofluorescence light receiving means, and the magnitude of the first signal corresponding to the light received by the autofluorescence light receiving means at the same time as the time is greater than or equal to a predetermined threshold, the scattering It is a biological particle counter characterized by determining that the object contained in the liquid corresponding to the detection signal output by the light detection signal output means is a biological particle.

The counting of biological particles by the biological particle counter of the present solution has the following characteristics, for example, and proceeds according to a predetermined procedure.
(9) The light (autofluorescence) transmitted in (3) is received, and a first signal having a magnitude corresponding to the received light amount is output. For example, a first signal corresponding to the amount of received light is output by a light detection device such as a photomultitube or a photodiode.

(10) Receives the light reflected in (5) (scattered light of the object) or the light reflected in (7) (scattered light of the object), and the size according to the received light quantity The second signal is output. For example, a second signal corresponding to the amount of received light is output by a photodetector such as a photodiode.

(11) It is determined whether or not the magnitude of the second signal output in (10) is equal to or greater than a predetermined threshold value. A detection signal (for example, a detection flag) indicating that light has been detected is output.

(12) When the detection signal is output in (11) above, it is separated in (3) at the time corresponding to the detection signal, that is, at the same time as the time of receiving light in (10) above Alternatively, when the light transmitted in (8) is received and the first signal is output, it is determined in the determination in (4) that the object is a biological particle. In addition, when the detection signal is output and the 1st signal is not output, you may determine with a target object being a non-living particle. And when it is determined that it is a biological particle in the determination, the number of biological particles is counted by counting the number, and when it is determined that it is a non-biological particle, the number is also counted. Also good. Further, the size of the biological particles (or non-biological particles) may be determined based on the size of the second signal.

  As described above, according to this solution, a predetermined threshold value is provided for the scattered light with the object, so that a signal larger than the threshold value can be determined as the scattered light of the object.

  Solution 4: The biological particle counter according to the present invention is the biological particle counter according to any one of Solution 1 to Solution 3, wherein the predetermined wavelength of the light emitted from the light-emitting device is 375 nm to 420 nm. In this case, the biological particle counter is characterized in that the reference cutoff wavelength for transmitting light by the autofluorescence selective optical means is 450 nm to 490 nm.

The counting of biological particles by the biological particle counter of the present solution has the following characteristics, for example, and proceeds according to a predetermined procedure.
(13) The wavelength of the laser beam irradiated by the above (1) is set to 375 nm to 420 nm, and the cutoff wavelength for reducing the Raman scattered light by the water according to the above (3) or (8) and transmitting the autofluorescence is 450 nm. Set to a predetermined wavelength between ˜490 nm.

  Thus, according to this solution, for example, by irradiating laser light of 405 nm and using autofluorescence from intracellular riboflavin of biological particles in water as an index, the energy state of riboflavin can be easily excited. Furthermore, since the peak wavelength of the auto-fluorescence is about 520 nm, the peak wavelength of the Raman scattered light of water is about 465 nm, so that the cut-off used as a reference for the auto-fluorescence selection optical means (long-pass filter) If the wavelength is set to 490 nm, the separation can be efficiently performed, so that the counting accuracy with respect to the biological particles can be further improved.

  According to the biological particle counter and the biological particle counting method of the present invention, autofluorescence emitted from biological particles can be efficiently detected even in a state where Raman scattering light of water is generated in the flowing liquid. Counting with high counting accuracy can be performed.

It is a schematic block diagram which shows one Embodiment of a biological particle counter system. It is a figure which shows the autofluorescence spectrum from the excitation absorption spectrum of the riboflavin which is an example of an autofluorescent substance, and NAD (P) H, and the substance. It is the schematic of the Raman scattered light spectrum by water at the time of irradiating the light whose wavelength is 405 nm. It is a figure which shows an example of the time change intensity distribution of the Raman scattered light by the water before optical filter entrance. It is a figure which shows an example of the time change intensity distribution of the Raman scattered light by the water after permeation | transmission of an optical filter. It is a figure which shows an example of the time change intensity distribution of all the lights before an optical filter incident. It is a figure which shows an example of the time change intensity distribution of all the lights after optical filter transmission. It is a flowchart which shows the example of a procedure of an autofluorescence count process. It is a flowchart which shows the example of a procedure of a data collection process. It is a flowchart which shows the example of a procedure of a data analysis process. It is a flowchart which shows the example of a procedure of an analysis process. It is a figure which shows an example of the output signal from the light-receiving device for fluorescence and the light-receiving device for scattering. It is a flowchart which shows the example of a procedure of an analysis result output process. It is a flowchart which shows the example of a procedure of alerting | reporting process. It is a figure which shows an example of the alerting | reporting of the count result of a biological particle.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a schematic configuration diagram showing an embodiment of a biological particle counter system.
As shown in FIG. 1, the biological particle counter system irradiates a target with light, detects a scattered light or autofluorescence from the target, and a signal output from the light detection system 1. The autofluorescence counting system 2 is configured to count the autofluorescence number based on the autofluorescence count. With these systems, it is possible to detect and count biological particles (hereinafter referred to as biological particles) among objects in water. The bioparticles that can be detected (counted) in the present embodiment are, for example, bioparticles having a size of 0.1 μm to several hundreds of μm, and specifically, bacteria, yeasts, molds, and the like. The light irradiated to the biological particles is a laser beam in the ultraviolet region, and is a substance necessary for metabolism (riboflavin, NAD (P) H (nicotinamide adenine dinucleotide (phosphorus) in the body (cells) of the biological particles). The autofluorescence emitted from the acid))) is detected as an index.

[Light detection system]
The light detection system 1 includes, for example, a light emitting device 10, an irradiation optical lens system 20, a target flow device 30, a first condensing optical lens system 40, a light shielding device 50, a scattered light selection optical device 60, a light shielding wall 65, and autofluorescence selection. The optical device 70 includes a second condensing optical lens system 80, a fluorescence light receiving device 90, a third condensing optical lens system 100, and a scattering light receiving device 110. With these components, the object can be irradiated with light, and scattered light and autofluorescence from the object can be detected. Hereinafter, each component will be specifically described.

[Light emitting device]
The light emitting device 10 includes, for example, a semiconductor laser diode (including a semiconductor LED element; hereinafter referred to as a laser diode). Laser light is oscillated by the laser diode 10 to irradiate a liquid containing biological particles. The wavelength of the laser light oscillated by the laser diode 10 is determined corresponding to a substance that can emit autofluorescence existing in the cells of the biological particles (hereinafter referred to as autofluorescent substance). Here, the autofluorescent material has an excitation wavelength that easily absorbs the energy of the irradiated light and is excited to an excited state. The excitation wavelength differs depending on the substance, and the wavelength of the autofluorescence emitted when returning from the excited state to the ground state also differs depending on the autofluorescent substance. The excitation wavelength and autofluorescence wavelength of the autofluorescent material will be described with specific examples.

[Excitation wavelength and autofluorescence wavelength]
FIG. 2 is a diagram illustrating an example of an excitation absorption spectrum of an autofluorescent substance and an autofluorescence spectrum from the substance.
As shown in FIG. 2, each distribution shows an excitation wavelength spectrum of NAD (P) H, an excitation wavelength spectrum of riboflavin, an autofluorescence spectrum from NAD (P) H, and an autofluorescence spectrum from riboflavin. For example, the excitation wavelength spectrum of NAD (P) H has a distribution with a peak at a wavelength of about 340 nm. Moreover, the excitation wavelength spectrum of riboflavin has a distribution with a peak at a wavelength of about 375 nm, and expresses that it is suitable to irradiate laser light having a wavelength of 375 nm to 420 nm in order to facilitate excitation of riboflavin. ing.

  Therefore, in order to release a lot of autofluorescence from the biological particles, the wavelength of the laser light oscillated from the laser diode 10 corresponds to the excitation wavelength of NAD (P) H or riboflavin present in the cells of the biological particles. It is determined. In the present embodiment, it is assumed that laser light having a wavelength of 405 nm is oscillated from the laser diode 10. By irradiating the laser beam having a wavelength of 405 nm, autofluorescence due to riboflavin is released from the biological particles.

[Irradiation optical lens system]
The irradiation optical lens system 20 is composed of, for example, a plurality of types of optical lenses. For example, it is composed of a collimator lens, a biconvex lens, and a cylindrical lens, and the laser beam oscillated from the laser diode 10 is adjusted to a parallel light beam and irradiated onto the object.

[Target flow equipment]
The target flow device 30 is composed of, for example, a hollow quadrangular cylindrical portion 32 made of synthetic quartz, sapphire, or the like, and a liquid 33 containing a target (biological particles 35 or non-biological particles 37) is on the top. It has a structure that flows from the bottom to the bottom. The laser beam 31 oscillated from the laser diode 10 is applied to the hollow region where the liquid in the cylindrical portion 32 flows to form a detection region.

  In this detection region, the laser beam 31 interacts with water (water molecules) of the liquid 33 flowing through the flow cell 30 and an object (biological particles 35 or non-biological particles 37).

  Since the wavelength of the laser beam 31 incident on the biological particle 35 is 405 nm, the scattered light from the biological particle 35 is also emitted at a wavelength of 405 nm. As shown in FIG. 2, when the laser beam 31 is absorbed by riboflavin in the cells of the biological particles 35, the wavelength has a distribution with a peak at about 520 nm. Here, the scattered light or autofluorescence emitted from the biological particles 35 is emitted to the surroundings.

  Further, the scattered light by the laser light 31 incident on the non-biological particle 37 is the same as the scattered light emitted from the biological particle 35.

  As described above, when the biological particles 35 and the non-biological particles 37 interact with the laser light 31, the scattered light from the biological particles 35 and the non-biological particles 37 or the autofluorescence from the biological particles 35 is emitted. . These lights are detected by the light receiving device through a plurality of condensing lens systems and wavelength selection optical devices. The intensity of the scattered light, that is, the amount of scattered light depends on the size of the biological particles 35 and the non-biological particles 37, and the larger the amount, the larger the amount of light. Here, autofluorescence from the biological particle 35 depends on the amount of riboflavin in the cell of the biological particle 35. Further, depending on the amount of light (intensity) of the laser beam 31, if the laser output is increased and the flow cell 30 is irradiated with a large amount of the laser beam 31, the scattered light from the biological particles 35 and the non-biological particles 37, the biological particles 35. Autofluorescence from will also increase. However, scattered light from biological particles 35 and non-biological particles 37 and light other than autofluorescence from biological particles 35 also increase, and specifically, light due to the interaction (Raman scattering) between laser light 31 and water 33. (Raman scattered light) will also increase. Next, the Raman scattered light by water is demonstrated concretely.

[Raman scattering by water]
FIG. 3 is a diagram illustrating an example of a Raman scattered light spectrum caused by water when light having a wavelength of 405 nm is irradiated. As shown in FIG. 3, when water is irradiated with laser light 31 having a wavelength of 405 nm, Raman scattered light having a wavelength distribution having a peak at a wavelength of about 465 nm is emitted by the interaction between water and the laser light 31.

[Shading device]
The light shielding device 50 is composed of, for example, a laser trap. The laser trap 50 oscillates from the laser diode 10 and shields the laser beam 31 that has passed through the flow cell 30 without causing interaction. By shielding the light, the laser beam 31 that has passed therethrough is prevented from being reflected at various places and the like to become noise in detecting scattered light from the biological particles 35 and autofluorescence.

[First condensing optical lens system]
The 1st condensing optical lens system 40 is comprised from the some optical lens, for example. The first condensing optical lens system 40 is installed at a position at an angle of about 90 degrees with respect to the traveling direction (optical axis) of the laser light 31. The first condensing optical lens system 40 collects the scattered light from the biological particles 35 and the non-biological particles 37 in the flow cell 30 and the autofluorescence from the biological particles 35. In order to collect as much side scattered light and autofluorescence as possible from these biological particles 35, it is preferable that the lens aperture is large, and a detection device that detects scattered light and autofluorescence from the biological particles 35 is provided. It is determined corresponding to the position (distance).

[Scattered light selective optical device]
The scattered light selection optical device 60 is composed of, for example, a dichroic mirror. The dichroic mirror 60 of the present embodiment transmits light having a wavelength longer than 410 nm and reflects light having a wavelength shorter than 410 nm. A specific wavelength that serves as a reference for separation based on the wavelength of light is referred to as a cutoff wavelength. Therefore, since the wavelength of the scattered light from the biological particles 35 and the non-biological particles 37 scattered by the laser beam 31 of 405 nm in the flow cell 30 is mainly 405 nm, the biological particles 35 and the non-biological particles are caused by the dichroic mirror 60. The scattered light from 37 can be reflected. The scattered light from the reflected biological particles 35 and non-biological particles 37 is then condensed on the third condensing optical lens system 100 and imaged on the scattering light receiving device 110.

  On the other hand, the autofluorescence emitted from the biological particles 35 flowing in the flow cell 30 has a wavelength distribution peaking at about 520 nm, as shown in FIG. 2, so that it is not reflected by the dichroic mirror 60. Almost everything will be transmitted. Similarly, as shown in FIG. 3, Raman scattered light due to water has a wavelength distribution with a peak at about 465 nm, and a wavelength longer than the cut-off wavelength of 410 nm occupies the most part. Most of the light passes through the dichroic mirror 60 except for. The transmitted autofluorescence and the Raman scattered light due to water then proceed to the autofluorescence selection optical device.

  In addition, the cutoff wavelength used as the reference | standard of the dichroic mirror 60 is not limited to 410 nm, the scattered light from the biological particle 35 or the non-biological particle 37 scattered by the laser beam 31 is reflected, and autofluorescence is reflected from the biological particle 35. Any wavelength can be used as long as it is transmitted.

[Auto fluorescence selection optical device]
The autofluorescence selection optical device 70 is composed of, for example, an optical filter. In the present embodiment, a long pass filter 70 that transmits light having a wavelength longer than a wavelength of 490 nm (cutoff wavelength) is provided.

  On the other hand, as shown in FIG. 3, most of the Raman scattered light by water has a wavelength shorter than the cut-off wavelength of 490 nm and can be reduced by about 90%.

[Intensity distribution change of Raman scattering light of water by long pass filter]
FIG. 4 is a diagram showing an example of a time-varying intensity distribution of Raman scattered light due to water before entering the long-pass filter, and FIG. 5 is an example of a time-varying intensity distribution of Raman scattered light due to water after passing through the long-pass filter. FIG. 4 and 5, it is shown that the intensity distribution of the Raman scattered light 33s due to water is changed by the long-pass filter.

[Intensity distribution of Raman scattered light by water before entering the long pass filter]
As shown in FIG. 4, the Raman scattered light 33s due to water has a distribution that repeats random increase and decrease, with the horizontal axis representing time and the vertical axis representing intensity in arbitrary units of light. For example, there is a distribution in which the intensity of Raman scattered light 33s due to water has a peak of 0.5, there is also a distribution in which 0.3 is the peak, and a large amount of Raman scattered light 33s due to water may be incident. It may be incident in a small amount, and is incident randomly regardless of time.

[Distribution of Raman scattered light in water after passing through long-pass filter]
Further, as shown in FIG. 5, the Raman scattered light 33 s by water after passing through the long pass filter 70 with the horizontal axis representing time and the vertical axis representing intensity in arbitrary units of light is due to the water shown in FIG. 4. It shows that the magnitude is changed to about 1/10 with respect to the temporal change distribution of the Raman scattered light 33s.

  Next, not only the Raman scattered light 33s caused by water but also the light Lt that passes through the dichroic mirror, that is, the light Lt that is a total of the Raman scattered light 33s caused by water and the autofluorescence 35e emitted from the biological particles 35 will be described.

[Change of light intensity distribution by long pass filter]
FIG. 6 is a diagram illustrating an example of a time variation distribution of all light before incidence of the optical filter, and FIG. 7 is a diagram illustrating an example of a time variation distribution of all light after transmission through the optical filter. 6 and 7, it is shown that the intensity distribution of all the light Lt that has passed through the dichroic mirror 60 is separated by the long pass filter 70.

[Intensity distribution of light before entering the long pass filter]
As shown in FIG. 6, with the horizontal axis representing time and the vertical axis representing the relative intensity of light, the distribution of light incident on the long pass filter 70 is the distribution of Raman scattered light 33s and autofluorescence 35e due to water. It consists of the distribution by the combined (total) light Lt. For example, from a certain time to a time Δt, Raman scattered light 33s with water having a distribution with a relative intensity peaking at 0.5 and light intensity having a distribution with a relative intensity peaking at 0.2 Assuming that the self-fluorescent light 35e is incident, during the time Δt, the amount of light Lt synthesized from these amounts of light is incident, and the relative intensity distribution due to the light Lt peaks at 0.7. Distribution.

[Intensity distribution of light after passing through long pass filter]
Further, as shown in FIG. 7, the temporal change distribution of all the transmitted light Lt is almost the same as the temporal change intensity distribution of the amount of Raman scattered light 33s of water, which is about 10% of the incident time, and the incident time. This represents a distribution obtained by combining the time-varying intensity distribution of the light amount of the autofluorescence 35e that has not changed. For example, from a certain time to a time Δt, Raman scattered light 33s with water having a distribution with a relative intensity peaking at 0.05 and light intensity having a distribution with a relative intensity peaking at 0.2 Assuming that the self-fluorescent light 35e is transmitted, during the time Δt, the amount of light Lt synthesized is transmitted, and the relative intensity distribution by the light Lt peaks at 0.25. Distribution.

  The wavelength of the light that is separated by the autofluorescence selection optical device 70 is selected so that the Raman scattered light 33s due to water is smaller than the autofluorescence 35e emitted from the biological particles 35. Specifically, the cutoff wavelength is not limited to 490 nm, and may be a wavelength having a value of 450 nm to 520 nm, preferably 450 nm to 490 nm. Further, the present invention is not limited to a long pass filter that transmits a wavelength longer than 490 nm, and a band pass filter that transmits light in a wavelength region of 490 nm to 600 nm may be provided.

  Here, as the autofluorescence selection optical device 70, in order to use the autofluorescence 35e from the riboflavin in the cells of the biological particles 35 as an index, a long-pass filter based on the cutoff wavelength (for example, 490 nm) is provided. However, when the intracellular NAD (P) H of the biological particle 35 is used as an index, a long path such that any wavelength from 410 nm to 470 nm (for example, 450 nm) is set as a cutoff wavelength and light having a longer wavelength is transmitted. A filter or a bandpass filter that transmits light in a wavelength range of 450 nm to 600 nm as a cutoff wavelength may be provided. This is because when the laser beam 31 of about 350 nm is irradiated, the Raman scattered light 33s due to water has a distribution with a peak at 400 nm, and the detection of the autofluorescence 35e from riboflavin is performed with a long-pass filter based on 450 nm as an index. This is because most of the Raman scattered light 33s of water can be shielded in the same manner as in the above case.

[Second condensing optical lens system: see FIG. 1]
The second condensing optical lens system 80 is composed of, for example, a plurality of optical lenses.
The second condensing optical lens system 80 is installed on the traveling direction (optical axis) of the light transmitted through the long pass filter 70. By the second condensing optical lens system 80, the auto-fluorescence 35e transmitted through the long pass filter 70 and the Raman scattered light 33s by water are condensed and imaged on the incident surface of the fluorescence light receiving device 90.

[Fluorescence receiver]
The fluorescence light receiving device 90 includes, for example, a semiconductor light receiving element (photodiode Photo Diode: PD) or a photomultiplier tube (Photo Multiplier Tube: PMT) having higher sensitivity than the photodiode. These photodiodes and photomultiplier tubes (hereinafter referred to as photomultipliers) use the received light as a current, and output a current corresponding to the received light quantity. Note that the magnitude of the output current varies depending on the amount of received light, and the greater the amount of received light, the greater the current. Note that the electrical signal output from the photomultiplier 90 is then input to the autofluorescence counting system 2.

[Shading wall]
The light shielding wall 65 is composed of a cylindrical structure that surrounds the optical path from the transmission side of the dichroic mirror 60 to the photomultiplier 90. The light shielding wall 65 can prevent light other than the light (autofluorescence 35e) transmitted through the dichroic mirror 60 from entering the photomultiplier 90. For example, the Raman scattered light 33 s and the scattered light 35 s and 37 s from the object are reflected in the light detection system 1 and can be shielded from entering the optical path. Although not shown, a light shielding wall may be similarly provided on the optical path from the reflection side of the dichroic mirror 60 to the light receiving device 110 for scattering.

[Third condensing optical lens system]
The third condensing optical lens system 100 is composed of, for example, a plurality of optical lenses. The third condensing optical lens system 100 is installed on the traveling direction (optical axis) of the light reflected by the dichroic mirror 60.

[Light scattering device]
The scattering light receiving device 110 is composed of, for example, a photodiode or a photomultiplier. Here, the light incident on the scattering light receiving device 110 is light having a wavelength shorter than 410 nm reflected by the dichroic mirror 60, and specifically, biological particles 35 and non-biological particles 37 that flow in the flow cell 30. Scattered light scattered by. Since the scattered light from these biological particles 35 and non-biological particles 37 has a larger amount of light than the autofluorescence 35e emitted from the biological particles 35, it can be sufficiently detected not by a photomultiplier but also by an inexpensive photodiode. In the present embodiment, this photodiode 110 is provided, and the scattered light from the biological particles 35 and the non-biological particles 37 reflected by the dichroic mirror 60 is received. The light received by the photodiode 110 is converted into an electric signal corresponding to the light amount, and the electric signal is output from the photodiode 110. The output signal from the photodiode 110 is then input to the autofluorescence counting system 2.

[Autofluorescence counting system: see Fig. 1]
The autofluorescence counting system 2 includes, for example, a detection signal processing unit 200, a data processing unit 300, and a notification unit 400. FIG. 8 is a flowchart showing an example of the procedure of the autofluorescence counting process.

  For example, the detection signal processing unit 200 receives an output signal from the light detection system 1, that is, an output signal from the fluorescence light receiving device (photomal) 90 and an output signal from the scattering light receiving device photodiode 110. Then, the received signal is amplified and AD conversion from an analog signal to a digital signal is performed (data collection processing step S200 in FIG. 8).

  For example, the data processing unit 300 receives and stores the autofluorescence signal (signal A) and the scattered light signal (signal B) subjected to AD conversion processing by the detection signal processing unit 200 (data analysis processing step S300 in FIG. 8). Then, it is determined from the stored signals A and B whether or not a signal derived from the biological particles 35 is contained in the liquid, that is, a signal from the autofluorescence 35e, and the determination result is output (step in FIG. 8). Analysis result output processing S400) and the like are performed.

For example, the notification unit 400 notifies the result determined by the data processing unit 300 to the outside or outputs a notification signal to the outside (notification processing step S500 in FIG. 8).
Hereinafter, each component and its process are demonstrated concretely.

[Detection signal processor]
The detection signal processing unit 200 includes, for example, a fluorescence output signal processing device 210 and a scattering output signal processing device 220. Further, the fluorescence output signal processing device 210 includes, for example, a first amplifier 212 and a first analog / digital converter 214, and the scattering output signal processing device 220 includes, for example, a second amplifier 222, a second analog / digital converter. It consists of a digital converter 224.

[Data collection processing]
FIG. 9 is a flowchart illustrating an example of a procedure of data collection processing.
First, when the fluorescence output signal processing device 210 receives an output signal from the fluorescence light receiving device (photomultiplier) 90 (output signal reception processing step S210), the first amplifier 212 outputs the output signal output from the photomultiplier 90. (Output signal amplification processing step S220). Then, the first analog / digital converter 214 converts the analog signal amplified by the first amplifier 212 into a digital signal (signal A) (output signal A / D conversion processing step S230).

  Similarly, when the output signal processing device for scattering 220 receives the output signal from the light receiving device for scattering (photodiode) 110 (output signal reception processing step S210), the output output from the photodiode 110 by the second amplifier 222 is output. The signal is amplified (output signal amplification processing step S220). Then, the second analog / digital converter 224 converts the analog signal amplified by the second amplifier 222 into a digital signal (signal B) (output signal A / D conversion processing step S230).

  Thereafter, the signals A and B converted into digital signals are output from the fluorescence output signal processing device 210 and the scattering output signal processing device 220 (converted signal output processing step S240). The output signals A and B are input to the data processing unit 300 next.

[Data processing section]
The data processing unit 300 includes, for example, a data collection device 310, a data analysis device 320, and an analysis result output device 330. In addition, the data collection device 310 includes, for example, a memory (RAM) 310 that stores data.

[Data analysis processing]
FIG. 10 is a flowchart illustrating an exemplary procedure of data analysis processing.
First, the data processing unit 300 receives the signals A and B output from the fluorescence output signal processing device 210 and the scattering output signal processing device 220 (conversion signal reception processing step S310). The received signals A and B are stored in the storage area of the memory 310 as they are.

  When the storage of the signal A and the signal B in the memory 310 is completed, an analysis process (analysis process step S340) is performed using the signal A and the signal B.

[Data analysis equipment]
The data analysis device 320 prestores (saves), for example, a calculation circuit (for example, the CPU 322) that analyzes the data (signal A and signal B) stored in the memory 310, calculation processing contents (program, threshold data), and the like. It consists of a memory 324 (ROM).

[Analysis processing]
FIG. 11 is a flowchart illustrating an exemplary procedure of analysis processing.
First, the signal B stored in the memory 310 is compared with threshold data (voltage value) stored in the memory 324 in advance by the CPU 322. Specifically, it is determined whether or not the voltage value of the stored signal B is greater than or equal to a threshold value B (V _thB ) (step S342). As a result of this determination, when it is determined that the voltage value of the signal B is greater than or equal to the threshold value B (step S342: Yes), the scattered light from the biological particle 35 or the non-biological particle 37 is incident on the light receiving photodiode 110 for scattering. This means that it has been detected. Here, a process of turning on a scattered light detection flag indicating that scattered light from the biological particles 35 or the non-biological particles 37 has been detected may be performed.

Next, the signal A stored in the memory 310 is compared with threshold data (voltage value) stored in the memory 324 in advance by the CPU 322. Specifically, it is determined whether or not the voltage value of the stored signal A is equal to or greater than a threshold value A (V _thA ) (step S344). As a result of this determination, when it is determined that the voltage value of the signal A is greater than or equal to the threshold value A (step S344: Yes), the autofluorescence 35e emitted from the biological particle 35 is incident on the fluorescence light receiving device Photomal 90 and detected. It represents that it was done. And the process which turns ON the detection flag which shows that the autofluorescence 35e was detected is performed (step S346). This detection flag (ON) is then transmitted as a flag signal to the analysis result output processing device 330.

  On the other hand, as a result of these determinations, when it is determined that the voltage value of the signal B is not equal to or greater than the threshold value B (step S342: No), or when it is determined that the voltage value of the signal A is not equal to or greater than the threshold value A ( Step S344: No), a process of turning off the detection flag is performed (Step S348), indicating that the autofluorescence 35e has not been detected. Here, when the scattered light detection flag is ON and the detection flag is OFF, a process of turning ON the non-biological detection flag indicating that the non-biological particle 37 that is not the biological particle 35 has been detected. It may be done. This detection flag (OFF) is then transmitted as a flag signal to the analysis result output processing device 330. Also, an abiotic detection flag may be transmitted.

  The analysis process will be specifically described with reference to the diagrams of the signals A and B corresponding to the output signals output from the respective light receiving devices.

[Example of output signals from fluorescent light receiving device and scattering light receiving device]
FIG. 12 is a diagram illustrating an example of output signals from the fluorescence light receiving device and the scattering light receiving device.
The upper signal in FIG. 12 is a temporal change distribution of the signal A corresponding to the detection signal output from the photomultiplier 90 of the fluorescence light receiving device, and the lower signal in FIG. 12 is output from the photodiode 110 of the scattering light receiving device. The time change distribution of the signal B corresponding to the detected signal is shown. Here, it is assumed that the distribution of the signals A and B shown in the upper and lower stages in FIG. 12 is a timing-adjusted distribution. Moreover, about the time of a horizontal axis, it has shown that time has passed in order of time t1, t2, t3, ....

For example, at time t1, the threshold _{B (in V thB} (Figure _{V THB1))} and the voltage value of the large signal B than is input to the data processing unit 300, CPU 322 is a voltage value of the signal B is equal to or greater than the threshold B It is determined that there is (step S342: Yes). That is, it represents that the scattered light from the biological particle 35 or the non-biological particle 37 is incident on the light-receiving device photodiode 110 for scattering and detected at time t1.

Then, the threshold value A (V _thA ) stored in the memory 324 in advance by the CPU ₃₂₂ is compared with the voltage value of the signal A (step S344). Since the signal A at the time t1 is not a signal larger than the threshold value A (step S344: No), the signal B at the time t1 becomes scattered light from the non-living particles 37, and the detection flag is set to OFF (step) S348).

  Next, at time t2, the CPU 322 determines that the voltage value of the signal B is greater than or equal to the threshold value B (step S342: Yes).

Then, the CPU 322 compares the threshold value A (V _thA ) with the voltage value of the signal A (step S344), and as a result, the CPU 322 determines that the voltage value of the signal A is equal to or greater than the threshold value A (step S344: Yes). . Therefore, the signal A and the signal B at the time t2 represent the autofluorescence 35e and the scattered light from the biological particle 35, and the detection flag is set to ON (step S346).

  As described above, the result of the presence / absence of the biological particle 35 is obtained in real time. Here, the magnitude of the signal A or the signal B depends on the amount of light incident on the fluorescence light receiving device photomultiplier 90 or the scattering light receiving device photodiode 110, and further, the magnitude of the scattered light depends on the biological particles 35 or non-living matter. It depends on the size of the particles 37. Therefore, not only the presence / absence of the biological particle 35 but also the size of the biological particle 35 or the non-biological particle 37 can be measured based on the magnitudes of the signal A and the signal B.

Here, a plurality of threshold values B corresponding to the sizes (0.1 μm to 0.3 μm, 0.3 μm to 0.5 μm, 0.5 μm to 1.0 μm,...) Of the biological particles 35 are previously stored in the memory 324 (V It is assumed that _thB1 , _VthB2 , _VthB3 , _VthB4,. For example, for the signal B at time _t2, since less than larger _{V THB2} than _{V THB1,} the size of the biological particles 35 can be determined to be 0.1Myuemu～0.3Myuemu.

  In this embodiment, the analysis processing S340 is performed on the data stored in the signal storage processing step S310, but the biological particles 35 or the non-biological particles 37 are detected by sequentially comparing with the threshold values B and A without storing them. Alternatively, the peak of the signal B may be detected in real time to determine the particle size classification. Further, since the magnitude of the signal A corresponding to the light amount of the autofluorescence 35e also corresponds to the type and the active state of the biological particles, even if the information is obtained by detecting the peak of the signal A. Good.

  As described above, the presence or absence of the biological particles 35 can be detected in real time by the signals A and B, and the size of the biological particles 35 can also be measured. When the detection flag is set to ON by detecting the presence / absence of the biological particle 35, the biological particle 35 is counted in an analysis result output processing step S400.

[Analysis result output device]
The analysis result output device 330 is a device that counts the number of biological particles 35 analyzed by the data analysis device 320 and transmits the count value to the notification unit 400.

[Analysis result output processing]
FIG. 13 is a flowchart illustrating a procedure example of the analysis result output process.
First, the analysis result output device 330 receives a flag signal (detection flag) from the data analysis device 320. Then, it is determined whether or not the detection flag is set to ON (step S410). As a result, when the detection flag is ON (step S410: Yes), the biological particle 35 is detected, and the count number is incremented by 1, and the count value is calculated (step S420). And a count value (count number) is transmitted to the alerting | reporting part 400 (step S430). Although not shown here, when a reset button (not shown) is pressed or when a start button (not shown) is pressed, a process of resetting the count is executed before step S410. May be. Further, the number of biological particles 35 may be counted according to the size of the biological particles 35 based on the received flag signal (size flag).

[Notification Department]
The alerting | reporting part 400 is comprised from the display apparatus 410 and the speaker 420, for example.

[Informing process]
FIG. 14 is a flowchart illustrating a procedure example of the notification process.
First, the notification unit 400 receives the count value transmitted by the analysis result output device 330 of the data processing unit 300 (step S510). Next, the display device 410 updates the received count value and displays the number of detected biological particles 35 (step S520). In addition, a notification sound is output from the speaker (step S530). Here, the number of biological particles 35 detected for each size of the biological particles 35 may be displayed on the display device 410. Further, a display form in which the count is incremented by 1 in real time may be used, or the updated count value may be displayed after a predetermined time (for example, every 5 seconds).

  For the notification sound, the output mode (the number of output of the notification sound, the level of the notification sound) may be changed in accordance with the increasing frequency of the count value. Moreover, you may change the output aspect of an alerting sound according to the magnitude | size of the biological particle 35. FIG. For example, when the count value of the biological particles 35 of 0.5 μm to 1.0 μm per unit time is 1 to 9, a single sound such as “pi!” Is output once, and the count value is 10 to 99. In this case, a single sound such as “Pi! Pi!” May be output twice, and when the counted value is 100 or more, an output mode in which a single sound such as “Pi! Pi! Pi!” Is output three times may be used.

  FIG. 15 is a diagram illustrating an example of a display device and a speaker that notify the counting result of the biological particles 35. As a display device, there are provided a display panel 410 for notifying the counting result according to the size of the biological particles 35 and a speaker 420 for notifying that the biological particles 35 are detected. For example, the display panel 410 displays “Size (μm)” indicating the size reference of the biological particle 35 and “Count” indicating the number (count value) of the detected biological particles 35 corresponding to each size. The display part. For example, six values “0.1”, “0.3”, “0.5”, “1.0”, “2.” are displayed on the display portion of “Size (μm)” indicating the reference of the size of the biological particle 35. “0” and “5.0” are displayed in advance. For each value, “0.1” is the size of the biological particle 35 of 0.1 μm to 0.3 μm, “0.3” is the size of the biological particle 35 of 0.3 μm to 0.5 μm, “ “0.5” is the size of the biological particle 35 of 0.5 μm to 1.0 μm, “1.0” is the size of the biological particle 35 of 1.0 μm to 2.0 μm, and “2.0” The size of the biological particle 35 of 2.0 μm to 5.0 μm, and “5.0” correspond to the size of the biological particle 35 of 5.0 μm to 5.0 μm, respectively.

  Accordingly, 50,609 biological particles 35 having a size of 0.1 μm to 0.3 μm, 3,621 biological particles 35 having a size of 0.3 μm to 0.5 μm, and 0.5 μm to 1.0 μm. 287 biological particles 35 having a size, 31 biological particles 35 having a size of 1.0 μm to 2.0 μm, 12 biological particles 35 having a size of 2.0 μm to 5.0 μm, and 5.0 μm to 5.0 μm This indicates that one biological particle 35 having a size of 1 is counted as one.

  As described above, the notification unit 400 can notify the count value of the biological particles 35 from the display device 410 in real time, and can output a notification sound when the biological particles 35 are detected from the speaker 420. In addition, the notification unit 400 may include an external output terminal, and may output data to another device through the terminal.

  Note that the autofluorescence selection optical device of the light detection system 1 is not limited to the long pass filter 70, and may be composed of a dichroic mirror. For example, by providing a dichroic mirror that transmits light having a wavelength longer than the cutoff wavelength 490 nm and reflects light having a wavelength shorter than the cutoff wavelength 490 nm, light having a wavelength shorter than 410 nm (mainly biological particles 35 and (Scattered light from the biological particles 37) is received by the light receiving photodiode 110 for scattering, and light having a wavelength longer than 490 nm (mainly autofluorescence 35e from the biological particles 35) is received by the fluorescent light receiving device photomultitube 90. Will be.

In addition, for the scattered light selection optical device and the autofluorescence selection optical device of the light detection system 1,
Do not install an autofluorescence selection optical device behind the scattered light selection optical device, but install the scattered light selection optical device and the autofluorescence selection optical device in parallel so that the optical path of the scattered light and the autofluorescence is in a separate system. Also good. In this case, a short pass filter that transmits only light having a wavelength shorter than the cutoff wavelength of 410 nm is used as the scattered light selection optical device. A condensing lens optical system for condensing scattered light from the flow cell and autofluorescence 35e before and after each optical device, a light receiving device for scattering 110, and a light receiving device for fluorescence 90 are provided in each optical device. Install against. Thereby, for example, from the biological particles 35 and the non-biological particles 37 mainly by the scattered light selection optical device (short pass filter) based on 410 nm installed at a position 90 degrees (horizontal plane) from the optical axis of the laser light 31. Light can be detected by the light receiving photodiode 110 for scattering, and is an auto-fluorescence selection optical device (cut-off wavelength of 490 nm, which is installed at a position (vertical surface) 90 degrees from the optical axis of the laser light 31). The self-fluorescence 35e mainly from the biological particles 35 can be detected by the fluorescence light receiving device Photomal 90 by the long pass filter.

  As described above, according to the present embodiment, the biological particles 35 to be detected (measured) are detected from substances necessary for metabolism of biological activities performed in vivo, such as riboflavin and NAD (P) H in the cell. The detection of the autofluorescence 35e is used as an index, the laser light 31 having a wavelength corresponding to the substance is irradiated, the dichroic mirror for reflecting the scattered light from the object, the Raman scattered light 33s due to water or the like is reduced, and the biological particles 35 The counting accuracy with respect to the biological particles 35 can be improved by providing a long-pass filter that transmits the autofluorescence 35e from.

DESCRIPTION OF SYMBOLS 1 Light detection system 2 Auto-fluorescence counting system 10 Light-emitting device 20 Irradiation optical lens system 30 Target flow apparatus 40 1st condensing optical lens system 50 Light-shielding device 60 Scattered light selection optical device 65 Light-shielding wall 70 Auto-fluorescence selection optical device 80 2nd Condensing optical lens system 90 Fluorescence light receiving device 100 Third condensing optical lens system 110 Scattering light receiving device 200 Detection signal processing unit 300 Data processing unit 400 Notification unit

Claims (4)

A light emitting means for irradiating light having a predetermined wavelength from a single light source toward a liquid containing an object to be detected;
Among the light emitted by the interaction between the object or the liquid and the light irradiated by the light emitting means, the scattered light emitted from the object is separated , and autofluorescence emitted from the object Scattered light selection optical means for transmitting light including Raman scattered light emitted from the liquid;
Autofluorescence selection optical means for reducing the Raman scattered light having a wavelength overlapping a part of the wavelength range of the autofluorescence from the light transmitted through the scattered light selection optical means and transmitting the autofluorescence;
Biological particle determination means for determining whether or not the object contained in the liquid is a biological particle based on the light after the Raman scattered light is reduced by the autofluorescence selection optical means;
Autofluorescence light receiving means for receiving the light after passing through the autofluorescence selection optical means and outputting a first signal having a magnitude corresponding to the amount of light when the light is received;
Scattered light receiving means for receiving scattered light separated by the scattered light selecting optical means and outputting a second signal having a magnitude corresponding to the amount of light when received.
Scattered light detection that outputs a detection signal when detecting the scattered light emitted from the object contained in the liquid when the magnitude of the second signal output by the scattered light receiving means is greater than or equal to a predetermined threshold value Signal output means;
A light shielding wall that prevents light incident from other than the optical path from entering the optical path from the scattered light selection optical means to the autofluorescence light receiving means through the autofluorescence selection optical means,
The light emitting means includes
Irradiating light of the predetermined wavelength that makes the peak wavelength of the autofluorescence emitted after irradiation different from the peak wavelength of the Raman scattered light,
The biological particle determination means includes
The detection signal is output by the scattered light detection signal output means, and at the same time as the scattered light emitted from the object contained in the liquid is received by the scattered light receiving means. When light is received by the autofluorescence light receiving means, and the magnitude of the first signal corresponding to the light received by the autofluorescence light receiving means at the same time as the time is greater than or equal to a predetermined threshold, the scattered light A biological particle counter that determines that the object contained in the liquid corresponding to the detection signal output by the detection signal output means is a biological particle. The biological particle counter of claim 1.
The predetermined wavelength of light irradiated by the light emitting means is 375 nm to 420 nm;
The biological particle counter according to claim 1, wherein a cutoff wavelength which is a reference for transmitting light by the autofluorescence selective optical means is 450 nm to 490 nm. A light emitting step of irradiating light of a predetermined wavelength from one light source toward the liquid containing the object to be detected;
Of the light emitted by the interaction between the object or the liquid and the light irradiated in the light emitting step, the scattered light emitted from the object is separated , and the autofluorescence emitted from the object and Scattered light selective optical process for transmitting light including Raman scattered light emitted from the liquid;
An autofluorescence selection optical step of reducing the Raman scattered light having a wavelength overlapping a part of the wavelength range of the autofluorescence from the light transmitted by the scattered light selection optical step, and transmitting the autofluorescence;
A bioparticle determination step for determining whether or not the object contained in the liquid is a bioparticle based on the light after the Raman scattered light is reduced by the autofluorescence selection optical step; and the autofluorescence selection An autofluorescence light receiving step for receiving light after passing through an optical step and outputting a first signal having a magnitude corresponding to the amount of light when the light is received;
A scattered light receiving step of receiving the scattered light separated by the scattered light selection optical step, and outputting a second signal having a magnitude corresponding to the amount of the light when received;
When the magnitude of the second signal output by the scattered light receiving step is greater than or equal to a predetermined threshold, the scattered light detection outputs a detection signal as detecting the scattered light emitted from the object contained in the liquid A signal output process;
Including a light shielding step for preventing light incident from other than the optical path from entering the optical path from the scattered light selection optical step to the autofluorescence light receiving step through the autofluorescence selection optical step,
The light emitting step includes
Irradiate light of a wavelength that makes the peak wavelength of the autofluorescence emitted after irradiation different from the peak wavelength of the Raman scattered light,
The biological particle determination step includes
The detection signal is output by the scattered light detection signal output step, and at the same time as the scattered light emitted from the object contained in the liquid is received by the scattered light reception step. When light is received by the autofluorescence light receiving step, and the magnitude of the first signal corresponding to the light received by the autofluorescence light receiving step at the same time as the time point is equal to or greater than a predetermined threshold, the scattered light A biological particle counting method characterized in that the object contained in the liquid corresponding to the detection signal output in the detection signal output step is determined as a biological particle. The biological particle counting method according to claim 3,
The predetermined wavelength of the light irradiated in the light emitting step is 375 nm to 420 nm,
The biological particle counting method, wherein a cutoff wavelength that is a reference for transmitting light in the autofluorescence selective optical step is 450 nm to 490 nm.

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