JP6685057B1 - Imaging flow cytometer, sorting method, and calibration method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an imaging flow cytometer having a simple configuration and having high sort accuracy in consideration of speed variation of an object. An image capturing unit 20 that captures a first visual field 12 and a second visual field 14, a system time management unit 40, an image storage unit 30 that stores an image together with the system time, and particles P1, P2, and P3 are included. A particle detection unit 32 that detects an image, a sort determination unit 34 that determines whether or not the particles P1, P2, and P3 are target particles, and a delay time calculation unit 36 that calculates the delay time of the particles P1, P2, and P3. And a sort signal control unit 38 that issues a sort signal in order to sort the particles P1, P2, and P3 in accordance with the timing calculated by the delay time calculation unit 36 according to the determination result of the sort determination unit 34; An imaging flow cytometer 1 having a sorting unit 50 that sorts target particles based on signals. [Selection diagram] Figure 1

Description

  The present invention relates to an imaging flow cytometer, a sorting method, and a calibration method that sort objects such as cells using images.

  Generally, a flow cytometer that sorts objects such as cells determines whether or not it is a specific object based on a detection unit that detects a signal originating from the object and the signal obtained by the detection unit. And a saw section that sorts objects based on the determination result of the sort determining section (for example, Patent Document 1). In order to improve the sorting accuracy of the objects, it is necessary to operate the sorting unit at the timing when a specific object reaches the sorting unit.

In the conventional flow cytometer, the time required for the signal processing by the sort determination unit is short, and is generally within 1 millisecond. In reality, the target object flowing through the flow path has speed variations depending on the target object. For example, different cell types may have different velocities. Further, even for cells of the same type, the velocity increases as the cells flow near the center of the flow channel.
On the other hand, by shortening the distance from the detection unit to the sorting unit, it is possible to suppress the influence of speed variation of the object. Therefore, in conventional flow cytometers, it is common to operate the sorting unit at a timing when a certain delay time has elapsed after the target passed through the detection unit without considering the speed variation of the target. It was

  On the other hand, in an imaging flow cytometer that determines whether or not it is a specific object using an image, the signal processing time for acquiring necessary information may be long, and for example, about 2 to 32 milliseconds. May take some time. In this case, it is necessary to increase the distance from the detection unit to the sorting unit. If the sorting unit is operated at a timing when a certain delay time has passed after the object passed through the detection unit without considering the speed variation of the target object, the sorting accuracy is lowered due to the influence of the speed variation.

  In order to suppress the reduction in sorting accuracy due to such speed variations, a configuration having a speed measurement unit that measures the speed of each object has been proposed. For example, Patent Document 1 discloses a speed measurement unit using a grating (diffraction grating). Further, in Non-Patent Document 1, laser spots are arranged on the upstream side and the downstream side of a flow path in which an object flows, and the speed of each object is measured from the timing when the object passes each laser spot. Is disclosed.

US Pat. No. 6,532,061

N. Nitta et al., Intelligent Image-Activated Cell Sorting. Cell 175, 266-276 (2018)

In the velocity measurement unit using a grating as in Patent Document 1, it is difficult to measure the velocity of each object when a plurality of objects flow in the flow path in the state of being close to each other.
Further, even if the velocity of each object can be measured, in Patent Document 1, velocity signals and cell classification are calculated independently, and the results are input to a delay processing circuit to control the sorting unit. Is. Therefore, there is no certainty that the individual images captured by the camera and the speeds of the individual objects measured by the speed measuring unit are accurately linked in one-to-one correspondence. If the processing results of the images derived from different objects and the speed information are erroneously linked, it will not be possible to sort correctly, which is a problem. Specifically, when an object is caused to flow through the flow path with a high throughput, the signal intervals of the individual objects may be narrowed, and in some cases, the signals may be redundantly buried and cause an error in the linking. Alternatively, when the object to be measured is small and the object is identified by the camera but cannot be detected by the speed detector, an error may occur in a one-to-one correspondence with the signal.

  According to the configuration using two laser spots as in Non-Patent Document 1, it is possible to measure the velocity of each individual object even for a plurality of adjacent objects. However, in the configuration using two laser spots, laser irradiation to two places for speed detection and detectors of signals emitted from each of these laser spots are required, which makes the apparatus large in size.

  Further, an objective lens is usually used for imaging and detecting particles flowing in a flow cytometer, but the field of view obtained from the objective lens is limited. In the method of Non-Patent Document 1, at least two spots for speed measurement and one spot for image pickup need to be included in the field of view of the objective lens, but there is a certain interval between the spots in order to prevent light leakage. Need to open. Therefore, in particular, when it is desired to set a plurality of spots for image capturing, the location of the laser spots in the field of view of the objective lens becomes a problem. For example, when it is desired to separately capture the fluorescence images obtained by irradiating the excitation lights of different wavelengths, it is conceivable to install the laser spots apart from each other for the excitation lights of different wavelengths. However, in such a case, setting two spots for speed detection is a design burden.

  Further, in Non-Patent Document 1, when a particle is detected in one of the two laser spots for speed measurement, a camera takes an image after a certain period of time, and at that time, the speed measurement value and the image are common. By providing the identification number, the speed information and the image information are linked. However, if the signal detection at the laser spot fails due to insufficient sensitivity, the image is not retained and omission occurs.

  It is an object of the present invention to provide an imaging flow cytometer, a sorting method, and a calibration method that have a simple configuration and have high sorting accuracy in consideration of velocity variations of objects.

A first aspect of the present invention is
An imaging unit for imaging the first visual field and the second visual field on the flow path of the particles;
A system time management unit that issues system time,
An image storage unit that stores the image captured by the image capturing unit together with the system time;
A particle detection unit that detects an image including the particles,
A sort determination unit that determines whether the particles are target particles,
A delay time calculation unit for calculating the delay time of the particles,
A sort signal control unit that issues a sort signal in order to sort the particles in accordance with the timing calculated by the delay time calculation unit according to the determination result of the sort determination unit,
A sorting unit that sorts the target particles based on the sorting signal;
Have
The delay time calculation unit calculates the delay time until the particles reach the sorting unit, based on images of the particles captured in the first field of view and the second field of view,
It is an imaging flow cytometer.

A second aspect of the present invention is
The imaging unit images the first visual field and the second visual field on the flow path of the particles,
The image captured by the image capturing unit is stored together with the system time,
The particle detection unit detects an image containing the particles,
The sorting determination unit determines whether the particles are target particles,
Calculating a delay time until the particles reach the sorting unit, based on the images of the particles captured in the first field of view and the second field of view,
According to the determination result of the sort determination unit, the sorting unit sorts the target particles based on the delay time of the particles,
It is a sorting method.

A third aspect of the present invention is
The imaging unit images the first visual field and the second visual field on the flow path of the particles,
The image captured by the image capturing unit is stored together with the system time,
The first particle detector detects an image containing the particles,
The detector detects the detection position on the flow path,
The detection signal from the detector is stored together with the system time,
The second particle detector detects a detection signal corresponding to the passage of the particles,
Prediction of arrival of the particles at the detection position based on the time taken for the particles to pass through the first field of view and the second field of view, which is obtained from images of the particles captured in the first field of view and the second field of view Calculate the time,
Based on the detection signal, to obtain the arrival time when the particles reached the detection position,
Adjusting the estimated time of arrival calculated from the time that the particles have passed through the first field of view and the second field of view based on the time of arrival;
This is a calibration method.

  According to the present invention, it is possible to provide an imaging flow cytometer, a sorting method, and a calibration method which have a simple configuration and have high sorting accuracy in consideration of speed variations of objects.

Configuration diagram of the imaging flow cytometer of the first embodiment (A) is an image in which the particle with the identification number ID is located at the center of the first visual field at time t1, and (b) is an image in which the particle with the identification number ID is located at the center of the second visual field at time t2. Configuration diagram of an imaging flow cytometer of the second embodiment Diagram showing an example of the calibration method

<First Embodiment>
Hereinafter, the imaging flow cytometer 1 of the first embodiment will be described with reference to the drawings. FIG. 1 is a configuration diagram of an imaging flow cytometer of this embodiment. The imaging flow cytometer 1 of the present embodiment is for determining whether or not the particles P flowing through the flow path 10 are target particles to be sorted and sorting the target particles. The particles P are, for example, beads, cells and cell clusters (blood cells, bone marrow cells, lymphocytes, circulating cancer cells, vascular endothelial cells, platelets, platelet aggregates, eggs, sperm, fertilized eggs, spheroids, organoids, etc.), Organelles (chromosomes, chloroplasts, mitochondria, etc.), microorganisms, parasites, pollen, algae (Chlamydomonas, Euglena, etc.). A large number of particles P flow in the channel 10 in a certain direction. FIG. 1 shows how different types of particles P1, P2, P3 flow through the flow path 10 in this order.

  The imaging flow cytometer 1 includes an imaging unit 20, an image storage unit 30, a particle detection unit 32, a sort determination unit 34, a delay time calculation unit 36, a sort signal control unit 38, and a system time management unit 40. , And a sorting unit 50.

The imaging unit 20 continuously images the first visual field 12 and the second visual field 14 on the flow channel 10. FIG. 1 shows a state in which the particles P3 flow in the first visual field 12 and the particles P do not flow in the second visual field 14. The imaging unit 20 is, for example, a TDI (Time Delay Integration) camera.
Both the first visual field 12 and the second visual field 14 are included in the visual field of the optical system of the imaging unit 20. It should be noted that both the first visual field 12 and the second visual field 14 may be imaged on a single camera, or cameras may be individually assigned to the respective visual fields.

  The TDI camera captures an object that moves at a constant speed while keeping the exposure time and preventing blurring by matching the movement of the imaging target with the movement of the charge on the image sensor of the camera. It is arranged so that the moving direction of the charge on the image sensor of the TDI camera matches the moving direction of the particle P, and further, the moving speed of the charge and the apparent moving speed of the particle P on the image sensor match. An image of the particle P can be picked up by appropriately setting parameters such as the magnification of the optical system, the moving speed of the particle, and the clock of the image sensor. The TDI camera is described in, for example, US Pat. No. 6,211,955 and US Pat. No. 6,249,341.

  Further, the imaging unit 20 is not limited to the TDI camera, and a general line sensor may be used, or an area sensor and a rotating mirror may be combined (for example, DB Kay et al., The Journal of Histochemistry and Cytochemistry 27, 329-334, 1979).

  Further, the imaging unit 20 uses a method that combines a flash lamp and an area sensor (for example, K. Satoh et al., Cytometry 48, 194-201, 2002) and a STEAM method (for example, K. Goda et al., Proc. Natl. Acad. Sci. USA 109, 11630-11635, 2012), FIRE method (for example, E, D. Diebold et al., Nature Photonics 7, 806-810, 2013), Spatial-Temporal Transformation method (for example, Y . Han and Y.-H Lo, Scientific Reports 5, 13267, 2015), FDM method (eg H. Mikami et al., Optica 5, 117-126, 2018) and other imaging flow cytometer technologies May be.

The images of the first visual field 12 and the second visual field 14 captured by the imaging unit 20 are output to the image storage unit 30.
The system time management unit 40 issues the system time. The system time issued by the system time management unit 40 is transmitted to the image storage unit 30 and the sort signal control unit 38.

  The image storage unit 30 stores images of the first visual field 12 and the second visual field 14 that are continuously output from the imaging unit 20. Further, the image storage unit 30 receives the system time issued by the system time management unit 40. Then, the image storage unit 30 stores the system time at the timing when the images of the first visual field 12 and the second visual field 14 are input to the image storage unit 30, together with the images. That is, a time stamp is added to each image.

  The image storage unit 30 processes an image input from the imaging unit 20, an image output to the particle detection unit 32, and a system time input from the system time management unit 40 in real time. The image is stored in the line buffer or the image buffer.

  The image storage unit 30 includes an FPGA (Field Programmable Gate Array) equipped with an internal memory serving as an image buffer, a configuration in which an external memory such as an FPGA and a DRAM (Dynamic Random Access Memory) is combined, or a dedicated or general-purpose FPGA. It may be a computer system in which a logic circuit is replaced and a microprocessor and a memory are combined.

  The particle detector 32 detects an image including the particles P. Specifically, the images stored in the image storage unit 30 include an image containing the particles P and an image not containing the particles P. For example, the particles P are included by the image analysis technique. Images can be detected. Further, the particle detection unit 32 can acquire the area, contour, and position of the particle P by the image analysis technique.

Various image analysis methods are known and can be selected depending on the type of the particles P and the like. For example, as shown in Non-Patent Document 1, the particle detection unit 32 may detect a particle area by binarizing an image of bead particles. In addition, the particle detection unit 32 detects a cell region by extracting a feature amount indicating a cell contour with a filter for an algal cell, binarizing the contour, and concatenating / expanding the contour by a morphological operation. Good.
Further, the cell region may be detected by extracting the contour of the cell, segmenting the cell region, or the like using commercially available image analysis software, open source image analysis software, or the like.

Further, the particle detection unit 32, as shown in FIGS. 2A and 2B, for the detected particle P, an image of the particle P in the first visual field 12 and an image of the second visual field 14 is obtained. The identification numbers are assigned in particle units in a one-to-one correspondence.
The image of the particle P detected by the particle detection unit 32 and given the identification number is output to the sort determination unit 34. In addition, the particle detection unit 32 determines a predetermined feature point (for example, the center, the center of gravity, or the like) of the detected particle P from the image of the particle P to which the identification number is added and the system time is added to the image storage unit 30. The times t1 and t2 when the front end, the rear end, etc. have passed a predetermined reference position (for example, the center) of the first visual field 12 and the second visual field 14 are calculated.

  Here, with reference to FIG. 2, the passage time by the particle detection unit 32 will be described in detail. FIG. 2A shows an image in which the particle P having the identification number ID is located at the center of the first visual field 12. At this time, the particles P do not exist in the second visual field 14. FIG. 2B shows an image in which the particle P having the same identification number ID is located at the center of the second visual field 14. At this time, the particles P do not exist in the first visual field 12.

  When the image capturing unit 20 is a line sensor such as a TDI camera and the system time is given to each line, the particle P indicates the system time given to the line including the feature point of the particle P in the first visual field. 12 and the times t1 and t2 when passing through the second visual field 14.

The imaging unit 20 is an area sensor, system time is given to each image, and as shown in FIG. 2, images at the timing when the particles P reach the centers of the first visual field 12 and the second visual field 14. If it is possible to obtain, let the system time added to the image be t1 and t2.
Here, when the position at which the particle P is imaged in the first visual field 12 and the second visual field 14 is different from the predetermined reference position, from the system time given to the image and the positional information of the characteristic points of the particle P, Estimates of t1 and t2 can be calculated. For example, the system time T1 and T2 given to the image containing the particle P in the first visual field 12 and the second visual field 14, the amount of deviation of the characteristic point of the particle P from the reference position on the image in the flow direction of the flow path. Is Δy1 and Δy2, and the estimated value of the moving speed of the particles in the flow direction is V, it can be calculated as t1 = T1 + Δy1 / V, t2 = T2 + Δy2 / V, and so on.

Although FIG. 2A shows an example in which the particles P are not included in the second visual field 14 at the system time t1, another particle P is included in the second visual field 14 at the system time t1. Good. Further, in FIG. 2B, an example in which the particle P is not included in the first visual field 12 at the system time t2 is shown, but another particle P is included in the first visual field 12 at the system time t2. Good.
The particle detecting unit 32 outputs the system times t1 and t2 calculated for the particle P to the delay time calculating unit 36 together with the identification number given to the particle P.

The sort determination unit 34 determines whether or not the particle P is a target particle. Specifically, the sort determination unit 34 determines whether or not the particle P is a target particle by analyzing the image of the particle P output from the particle detection unit 32. Then, the sorting determination unit 34 outputs the determination result together with the identification number of the particle P to the sorting signal control unit 38.
The sort determination unit 34 can use various image analysis methods depending on the type of target particles. For example, a method of calculating one or more feature quantities from an image and classifying the particles P from the distribution, a method of classifying the distribution of feature quantities by machine learning such as SVM (Support Vector Machine), and an image A method of classifying by applying deep learning is used.

As a classification method based on a feature amount, as shown in Non-Patent Document 1, the sorting determination unit 34 targets beads particles as a target for sorting a specific area with respect to a distribution such as a particle area or brightness of an image. The method of doing may be used. In addition, the sorting determination unit 34 uses a method in which the localization of the brightness of the fluorescence in the image is characterized as a target for algal cells, and a specific region is targeted for sorting with respect to the distribution combined with the cell area. Good.
Further, as a classification method based on deep learning, the sorting determination unit 34 may use a method in which a CNN (Convolutional Neural Network) that classifies two classes by targeting bead particles is configured and one class is targeted for sorting. . Further, the sorting determination unit 34 may use a method of configuring a CNN that classifies three classes with respect to human blood cells and sorting a specific region with respect to the distribution of the certainty factor of each class. .
In addition, the sorting determination unit 34 may use a method of classifying a fluorescent image in the cell nucleus according to the presence or absence of bright spots, as described in US Pat. No. 6,211,955.

The delay time calculation unit 36 causes the particles P to reach the sorting unit 50 based on the times t1 and t2 when the particles P have passed the reference positions of the first visual field 12 and the second visual field 14 calculated by the particle detection unit 32. Calculate the delay time until. Specifically, when the distance between the reference positions of the first visual field 12 and the second visual field 14 is L1 and the distance between the reference position of the first visual field 12 and the sorting unit 50 is L2, the delay time ΔT Is calculated as ΔT = (L2 / L1) × (t2-t1). At this time, if both the values of L1 and L2 are known, those values may be used. When at least one of L1 and L2 is uncertain, the value of the coefficient A = L2 / L1 may be obtained by calibration.
The delay time ΔT is the time from when the particle P passes through the reference position of the first visual field 12 until the particle P reaches the sorting unit 50, but is not limited to this. The delay time may be the time from when the particle P passes through the reference position of the second visual field 14 to when it reaches the sorting unit 50.

  The delay time calculation unit 36 outputs the identification number of the particle P and the delay time of the particle P to the sort signal control unit 38.

The sort signal control unit 38 is required to sort the particles P at the timing calculated based on the delay time of the particles P calculated by the delay time calculation unit 36 according to the determination result of the sort determination unit 34. Emits a sort signal.
Since the system time is transmitted from the system time management unit 40 to the sorting signal control unit 38, the sorting signal control unit 38 sorts the sorting unit 50 appropriately according to the timing when the particles P reach the sorting unit 50. Emit a signal.

The sort signal is, for example, a pulse signal. The pulse signal is emitted at the timing when the target particles reach the sorting unit, and is transmitted to the sorting unit 50. Alternatively, the signal in which the timing information is encoded may be transmitted using a signal line prepared separately.
Further, the sorting unit 50 may sort the target particles into two or more types. For example, there are a case of sorting into two types (2-way sorting) and a case of sorting into four types (4-way sorting). In such a case, the sort determination unit 34 further classifies the target particles into a plurality of different types, and the sorting result is included in the sort signal. In that case, for example, the classification result can be included in the pulse signal by using the amplitude or sign of the pulse signal.
Alternatively, the classification result may be transmitted to the sorting unit 50 as a digital signal or the like by using a signal line separately prepared together with the pulse signal.
Furthermore, the sort signal may convey a time width in which the sort is started with the timing of the sort as a start time. In that case, the time width for sustaining sorting may be transmitted as the pulse width of the pulse signal, or a signal obtained by encoding the time width for sustaining sorting may be transmitted using a separately prepared signal line.

The system time for the particles P to reach the sorting unit 50 may be calculated by either the delay time calculation unit 36 or the sort signal control unit 38.
According to the determination result of the sorting determination unit 34, the sorting signal control unit 38 may issue the sorting signal only when the particle P is the target particle. Further, according to the determination result of the sort determination unit 34, the sort signal control unit 38 may issue different sort signals depending on whether the particle P is a target particle or not.

  The sorting unit 50 sorts the target particles based on the sorting signal issued from the sorting signal control unit 38. Based on the timing indicated by the sort signal, the sorting unit 50 receives, from the sort signal control unit 38 or the sort window information (W1 and W2 in FIG. 4 described later) stored in the sorting unit 50, Controls switching of sorting direction. The sorting unit 50 is, for example, a droplet sorter or an on-chip sorter.

  The sorting unit 50 may be, for example, a droplet sorter disclosed in M. J. Fulwyler, Science 150, 910-911 (1965). In this case, the sorting unit 50 has a nozzle arranged at the tip of the flow path 10, and droplets are formed from the nozzle at a constant cycle. By applying an electric field to the liquid in the vicinity of the target particles at the timing when the droplets containing the target particles are formed, the droplets containing the target particles are selectively charged. By applying an electrostatic field in the vicinity of the trajectory of droplets, it is possible to selectively shift the trajectory of droplets that contain charged target particles. Can be sorted. Furthermore, by controlling the positive / negative of the electric charge charged in the droplet and the amount of the electric charge, and dividing the trajectory of the droplet into different directions and angles, two or more types of cells are divided into two or more individual containers. It can also be collected.

  The sorting unit 50 may be, for example, the on-chip sorter disclosed in Non-Patent Document 1. In this case, by arranging the dual-membrane pump across the flow channel 10, a local flow is generated in the direction orthogonal to the flow channel 10 at the timing when the target particles pass, and the target flow is generated. The target particles are sorted by shifting the trajectory of the particles and distributing them to the flow paths in different directions in the branch flow path arranged downstream. Dual membrane pumps are described, for example, in S. Sakuma et al., Lab on a Chip 17, 2760-2767, 2017.

Hereinafter, a sorting method using the imaging flow cytometer 1 of this embodiment will be described. First, a large number of particles P flow in the flow path 10. The imaging unit 20 continuously images the first visual field 12 and the second visual field 14. The image captured by the image capturing unit 20 is stored in the image storage unit 30 along with the system time. Then, the particle detection unit 32 detects an image including the particles P.
The sort determination unit 34 determines whether or not the particle P is a target particle. Further, the delay time calculation unit 36 calculates the delay time until the particles P reach the sorting unit 50 based on the times t1 and t2 when the particles P pass through the reference positions of the first visual field 12 and the second visual field 14. . Then, the sorting unit 50 sorts the particles P based on the delay time according to the determination result of the sorting determination unit 34.

  As described above, according to the imaging flow cytometer 1 and the sorting method of the present embodiment, the sorting accuracy can be improved in consideration of the speed variation of the particles P with a simpler configuration than the conventional one.

<Second Embodiment>
Hereinafter, the imaging flow cytometer 1 according to the second embodiment will be described with reference to the drawings. FIG. 3 is a configuration diagram of the imaging flow cytometer 1 of this embodiment. The imaging flow cytometer 1 of this embodiment has a delay time calibration function. Specifically, in the imaging flow cytometer 1, the beads (particles P) for calibration are made to flow in the flow channel 10, and the particles P flowing in the first visual field 12 and the second visual field 14 are detected at the detection position X. Measure arrival time. Thereby, the expected arrival time of the particles P calculated from the particles P flowing in the first visual field 12 and the second visual field 14 is calibrated.

  The imaging flow cytometer 1 includes an imaging unit 20, an image storage unit 30, a first particle detection unit 32, a delay time calculation unit 36, a system time management unit 40, a detector 60, and a detection signal storage unit 70. And a second particle training unit 72 and a comparison unit 80. The imaging unit 20, the image storage unit 30, and the delay time calculation unit 36 of this embodiment are the same as those of the first embodiment.

The first particle detector 32 of the present embodiment is the same as the particle detector 32 of the first embodiment. The image of the particle P detected by the first particle detecting unit 32 and given the identification number is output to the comparing unit 80.
The first particle detector 32 also outputs the system times t1 and t2 calculated for the particle P to the delay time calculator 36 together with the identification number assigned to the particle P.

The delay time calculation unit 36 detects the detection position of the particle P described later based on the times t1 and t2 when the particle P passes through the reference positions of the first visual field 12 and the second visual field 14 calculated by the first particle detection unit 32. Calculate the time to reach X (expected arrival time).
The delay time calculation unit 36 outputs the expected arrival time of the particles P together with the identification number of the particles P to the comparison unit 80.

  The system time management unit 40 issues the system time. The system time issued by the system time management unit 40 is transmitted to the image storage unit 30 and the detection signal storage unit 70.

The detector 60 detects the particles P flowing in the detection position X on the flow path 10. The detector 60 is, for example, a camera, a strobe camera, a laser, and a detector. The camera or the strobe camera acquires an image at the detection position X and detects passage of the particles P from the image. In the laser and the detector, the laser is made incident on the detector to acquire the signal of the detector at the detection position X, and the passage of the particle P is detected from the change in the signal waveform when the particle P passes through the detection position X. The detection position X is preferably near the sorting position. The detector 60 outputs the detection signal to the detection signal storage unit 70.
When the detector 60 acquires an image, it is preferable that the first visual field 12, the second visual field 14, and the detection position X can be detected at the same time.

  The detection signal storage unit 70 stores the detection signal output from the detector 60. Further, the detection signal storage unit 70 receives the system time issued by the system time management unit 40. Then, the detection signal storage unit 70 stores the system time (arrival time) at the timing when the detection signal is detected together with the detection signal. That is, a time stamp is added to each detection signal.

The detection signal storage unit 70 processes the input of the detection signal from the detector 60, the output of the detection signal to the second particle detection unit 72, and the input of the system time from the system time management unit 40 in real time.
When the detector 60 acquires an image, the detection signal storage unit 70 may have the same configuration as the image storage unit 30. When the detector 60 acquires a continuous signal in the time direction like a detector, the detection signal storage unit 70 includes an ADC (Analog Digital Converter) that converts the detection signal into a digital signal, an internal memory that serves as a detection signal buffer, and It can be configured by an FPGA (Field Programmable Gate Array) equipped with a processing circuit. Regarding the detection signal buffer and the processing circuit, a configuration in which an external memory such as an FPGA and a DRAM (Dynamic Random Access Memory) is combined, a configuration in which the FPGA is replaced with a dedicated or general-purpose logic circuit, or a microprocessor and a memory are combined. It may be a computer system.

The second particle detection unit 72 detects the particles P by detecting a change in the detection signals caused by the passage of the particles P, and the detection signal storage unit 70 adds the detection signals to the detection signals corresponding to the passage of the particles P. The system time (arrival time) is output to the comparison unit 80.
When the detector 60 acquires an image, the second particle detection unit 72 may employ the same configuration as the first particle detection unit 32. When the detector 60 acquires a continuous signal in the time direction like a detector, for example, by a method of detecting the particle P with the transit time of the particle P being the time when the change of the signal is equal to or more than a certain threshold value, Particles can be detected.

The comparison unit 80 associates the identification number of the particle P output from the delay time calculation unit 36 with the arrival time of the particle P output from the second particle detection unit 72. Specifically, the comparison unit 80 associates the identification number of the particle P with the expected arrival time of the particle P from the delay time calculation unit 36 and the arrival time of the particle P from the second particle detection unit 72. .
The comparison unit 80 outputs the identification number of the associated particle P, the estimated arrival time, and the arrival time as a set.

The comparison unit 80 performs calibration based on the estimated arrival time of the particles P and the arrival time. FIG. 4 shows an example of the calibration method. The horizontal axis represents the visual field passage time ΔT calculated by the delay time calculation unit 36. Here, assuming that the time taken for the particles P to pass through the reference positions of the first visual field 12 and the second visual field 14 is t1 and t2, the visual field passage time ΔT is (t2-t1). The vertical axis represents the arrival time T of the particles P.
If the distance between the reference positions of the first visual field 12 and the second visual field 14 is L1 and the distance between the reference position of the first visual field 12 and the detection position X is L2, the particles P move at a constant velocity. If it is, the expected arrival time is calculated as (L2 / L1) × (t2-t1). Here, by obtaining the value of the coefficient A = L2 / L1 by calibration, it is possible to obtain the estimated arrival time when t1 and t2 are given.

  The comparison unit 80 plots the field-of-view passage time ΔT and the arrival time T of the particle P, and performs fitting to the model formula. The model formula is, for example, a straight line passing through the origin as shown in FIG. 4, but is not limited to this. When a straight line passing through the origin is used as a model formula, the model formula is T = A × ΔT The comparison unit 80 performs the calibration by obtaining the coefficient A from the plot of the transit time ΔT of the particle P and the arrival time T.

  The comparison unit 80 may calibrate the sort window instead of the estimated arrival time of the particles P or in addition to the estimated arrival time of the particles P. Here, the sort window indicates a time zone in which the sorting unit 50 sorts. In the example shown in FIG. 4, when the time of passage of the particle P in the visual field is ΔT1, the sorting is performed in the time zone of the sorting window W1 including the arrival time T1. Further, when the field-of-view transit time of the particles P is ΔT2, it indicates that sorting is performed in the time zone of the sort window W2 including the arrival time T2. The width (length of time) of the sort window may be constant regardless of the delay time of the particles P, or may be variable depending on the delay time of the particles P. In general, when the velocity of the particle P is high, the field-of-view transit time becomes short and the delay time becomes short. On the contrary, when the velocity of the particle P is slow, the time of passing through the visual field becomes long and the delay time becomes long. In the example shown in FIG. 4, the width of the sort window becomes wider as the time for which the particles P pass through the visual field becomes longer and the delay time becomes longer. This is because the larger the delay time is, the larger the error is. Therefore, the sorting accuracy can be improved by increasing the width of the sorting window.

  The sort window does not necessarily have to be centered on the measured arrival time T. The comparison unit 80 can set the sort window based on the plot of the time-of-flight passage ΔT of particles and the arrival time T.

  Although not shown in FIG. 3, the imaging flow cytometer 1 of this embodiment may further include a sort determination unit 34, a sort signal control unit 38, and a sort unit 50 similar to those of the first embodiment. At this time, the detection position X preferably matches the sorting unit 50, but the detection position X does not necessarily have to match the sorting unit 50. For example, the detection position X may be located slightly upstream of the sorting unit 50. In this case, the center position of the sort window is appropriately shifted from the estimated arrival time in accordance with the displacement between the detection position X and the position of the sort unit 50, and the sort window is set at the timing when the particles P reach the sort unit 50. You may adjust to.

Hereinafter, a calibration method and a sorting method using the imaging flow cytometer 1 of this embodiment will be described.
First, a large number of particles P flow in the flow path 10. The imaging unit 20 continuously images the first visual field 12 and the second visual field 14. The image captured by the image capturing unit 20 is stored in the image storage unit 30 along with the system time. Then, the first particle detector 32 detects an image including the particles P. The delay time calculation unit 36 calculates an estimated arrival time for the particle P to reach the detection position X, based on the time taken for the particle P to pass through the first visual field 12 and the second visual field 14.
Further, the detector 60 detects the particles P flowing in the detection position X. The detection signal detected by the detector 60 is stored in the detection signal storage unit 70 together with the system time. Then, the second particle detector 72 detects a detection signal corresponding to the passage of the particle P, and outputs the arrival time when the particle P reaches the detection position X.
The comparison unit 80 calibrates and adjusts the calculation formula of the expected arrival time of the particle P calculated by the delay time calculation unit 36 based on the arrival time. Then, the sorting signal control unit 38 and the sorting unit 50 adjust the timing of sorting the particles P based on the adjusted estimated arrival time.

  As described above, according to the imaging flow cytometer 1, the sorting method, and the calibration method of this embodiment, sorting accuracy can be further improved.

1 Imaging Flow Cytometer 10 Flow Path 12 First Field of View 14 Second Field of View 20 Imaging Section 30 Image Storage Section 32 Particle Detection Section (First Particle Detection Section)
34 Sort determination unit 36 Delay time calculation unit 38 Sort signal control unit 40 System time management unit 50 Sort unit 60 Detector 70 Detection signal storage unit 72 Second particle detection unit 80 Comparison unit P, P1, P2, P3 Particle X detection position

Claims (10)

An imaging unit (20) for imaging the first visual field (12) and the second visual field (14) on the flow path (10) through which the particles (P) flow;
A system time management unit (40) for issuing system time,
An image storage unit (30) for storing the image captured by the image capturing unit (20) together with the system time;
A first particle detector (32) for detecting an image containing the particles (P);
A sort determination unit (34) for determining whether the particles (P) are target particles ,
A detector (60) for detecting the particles (P) at a detection position (X) on the flow channel (10),
A detection signal storage unit (70) for storing the detection signal from the detector (60) together with the system time;
A second particle detector (72) for detecting a detection signal corresponding to passage of the particles (P);
Said first field (12) and said determined by the image of the imaged the particles (P) in the second field of view (14), said particles (P) is the first field of view (12) and said second field of view (14 ), And a calculation unit (36) for calculating an estimated arrival time at which the particle (P) reaches the detection position (X) based on the visual field passage time (ΔT),
A sort signal control unit (38) that issues a sort signal in order to sort the particles (P) at the timing calculated by the calculation unit (36) according to the determination result of the sort determination unit (34). When,
A sorting unit (50) for sorting the target particles based on the sorting signal;
A comparison unit (80) for comparing the estimated arrival time calculated by the calculation unit (36) and the arrival time of the particle (P) detected by the detector (60) to the detection position (X). ), And
The sorting signal control unit (38) controls the sorting unit (50) based on a comparison result between the estimated arrival time and the arrival time and a distance between the detection position (X) and the sorting unit (50). Adjusting the timing for sorting the target particles (P),
Imaging flow cytometer (1). The imaging unit (20) is a TDI camera,
The imaging flow cytometer according to claim 1. The first particle detector (32) gives an identification number to the detected particles (P),
The imaging flow cytometer according to claim 1. The calculation unit (36) calculates the particle (P) based on the system time of the images in the first field (12) and the second field (14) of the particle (P) having the same identification number. ) Arrives at the sorting unit (50),
The imaging flow cytometer according to claim 1. The detection position (X) is located in the sorting unit (50),
The imaging flow cytometer according to claim 1. The detection position (X) is located upstream of the sorting unit (50),
The imaging flow cytometer according to claim 1. The sort signal control section (38) sorts the particles (P) having the first visual field passage time (ΔT1) by a first time width (W1), and obtains the first visual field passage time (ΔT1). The sorting unit (so as to sort the particles (P) having the longer second visual field passage time (ΔT2) by the second time width (W2) longer than the first time width (W1). 50) adjusts the timing of sorting the target particles (P),
The imaging flow cytometer according to claim 1. The imaging unit (20) images the first visual field (12) and the second visual field (14) on the flow path (10) through which the particles (P) flow,
The image captured by the image capturing unit (20) is stored together with the system time,
The first particle detector (32) detects an image including the particles (P),
A detector (60) detects the particles (P) at a detection position (X) on the flow path (10),
Storing the detection signal from the detector (60) with the system time,
The second particle detector (72) detects a detection signal corresponding to the passage of the particles (P),
Said first field (12) and said determined by the image of the imaged the particles (P) in the second field of view (14), said particles (P) is the first field of view (12) and said second field of view (14 ), The estimated arrival time at which the particles (P) reach the detection position (X) is calculated based on the visual field passage time (ΔT),
Acquiring the arrival time at which the particle (P) reaches the detection position (X) based on the detection signal,
Based on the arrival time, and adjust the estimated arrival time calculated from the field of view transit time ([Delta] T),
The detection position (X) is located in the sorting section (50),
Adjusting the timing at which the sorting section (50) sorts the particles (P) based on the adjusted estimated arrival time;
Calibration method. The imaging unit (20) images the first visual field (12) and the second visual field (14) on the flow path (10) through which the particles (P) flow,
The image captured by the image capturing unit (20) is stored together with the system time,
The first particle detector (32) detects an image including the particles (P),
A detector (60) detects the particles (P) at a detection position (X) on the flow path (10),
Storing the detection signal from the detector (60) with the system time,
The second particle detector (72) detects a detection signal corresponding to the passage of the particles (P),
The particles (P) obtained by the images of the particles (P) captured in the first field of view (12) and the second field of view (14) are the first field of view (12) and the second field of view (14). ), The estimated arrival time at which the particles (P) reach the detection position (X) is calculated based on the visual field passage time (ΔT),
Acquiring the arrival time at which the particle (P) reaches the detection position (X) based on the detection signal,
Adjusting the estimated time of arrival calculated from the field-of-view transit time (ΔT) based on the time of arrival;
The detection position (X) is located upstream of the sorting unit (50),
Adjusting the timing at which the sorting unit (50) sorts the particles (P) based on the adjusted estimated arrival time and the distance between the detection position (X) and the sorting unit (50) ,
Calibration method. The sorting unit (50) sorts the particles (P) having the first visual field passage time (ΔT1) by a first time width (W1) and is longer than the first visual field passage time (ΔT1). Sorting said particles (P) having a second said field-of-view transit time (ΔT2) by a second time width (W2) longer than said first time width (W1),
The calibration method according to claim 8 or 9.

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