JP7006360B2 - Sampling device and sampling method - Google Patents

Description

The present invention relates to a sampling device and a sampling method.

Conventionally, for example, in the field of drug discovery screening, a compound that is a candidate for a drug is given to a cell that is a basic component of an organism, and a sample of the cell or intracellular substance that has reacted with the compound has been collected. It has been. Further, there is known a nanospray chip that can directly convert a collected sample into a nanospray and introduce it into a sample introduction port of a mass spectrometer (see, for example, Patent Documents 1 to 3). Further, there is a device that automates the work of collecting the sample on the nanospray chip (see, for example, Patent Document 4) and a technique for improving the accuracy of the position control of the nanospray tip in the device (for example, see Patent Document 5). Has been developed.

Japanese Patent No. 5317983 Japanese Unexamined Patent Publication No. 2016-1942 Japanese Patent No. 4370510 Japanese Patent No. 6066110 Japanese Patent No. 6090387

By the way, when the sample is subjected to analysis such as mass spectrometry, it is required to perform quantitative analysis. However, since the sample is extremely small, it is difficult to accurately determine the volume of the sample.

An object of the present invention is to provide a sampling device and a sampling method capable of accurately determining the volume of a sample.

In the sampling device as the first aspect of the present invention, a sampling tube for partitioning an opening and a hollow portion capable of holding a sample collected through the opening, and the sampling tube at a sampling position where the sample can be collected. A movable carrier, a digital phase difference image acquisition unit capable of acquiring a digital phase difference image of the sample collected in the collection tube by moving the collection tube with the transfer body, and the digital phase difference. A volume determination unit for determining the volume of the sample collected in the collection tube based on the digital phase difference image acquired by the image acquisition unit is provided.

The sampling apparatus as one embodiment of the present invention includes an opening image acquisition unit capable of acquiring an image of the opening of the sampling tube, and the volume determining unit is an image of the opening acquired by the opening image acquisition unit. And the volume of the sample collected in the collection tube is determined based on the digital phase difference image acquired by the digital phase difference image acquisition unit.

In the sampling device as one embodiment of the present invention, the aperture image acquisition unit is composed of an optical system for observing the sample and an imaging device.

In the sampling apparatus as one embodiment of the present invention, the sampling tube is a nanospray chip capable of selectively collecting a specific cell or a specific intracellular substance as the sample.

In the sampling method as the second aspect of the present invention, the sampling tube for partitioning the opening and the hollow portion connected to the opening is moved to the sampling position by the carrier, and the sample is collected in the hollow portion through the opening. Based on the step, the digital phase difference image acquisition step of acquiring the digital phase difference image of the sample collected in the collection tube by moving the collection tube with the carrier, and the acquired digital phase difference image. The volume determination step of determining the volume of the sample collected in the collection tube is included.

According to the present invention, it is possible to provide a sampling device and a sampling method capable of accurately determining the volume of a sample.

It is a figure which shows the structure of the sampling apparatus as one Embodiment of this invention. It is a vertical cross-sectional view which shows the tip part of a nanospray tip enlarged. It is a figure which shows the state of acquiring the digital phase difference image by using the digital phase difference image acquisition part of the sampling apparatus shown in FIG. It is a flowchart which shows the sampling method as one Embodiment of this invention. It is a flowchart explaining the operation of the sampling apparatus shown in FIG. It is a figure which shows an example of the aperture image acquired by the aperture image acquisition part of the sampling apparatus shown in FIG. It is a figure which shows an example of the digital phase difference image acquired by the digital phase difference image acquisition part of the sampling apparatus shown in FIG.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The same reference numerals are given to the common elements in each figure.

[Sampling device]
FIG. 1 is a diagram showing a configuration of a sampling device 1 as an embodiment of the present invention. As shown in FIG. 1, the sampling device 1 includes a suction operation unit 300 including a nanospray chip 10 as a sampling tube and a carrier 20, a first optical system unit 40, and a second opening image acquisition unit 70. It includes an optical system unit 200 and an information processing device 400 including a volume determination unit 470.

The second optical system unit 200 includes an XY stage 201 on which a microplate 110 as a cell culture container in which a plurality of wells are formed is placed, a microscope 210 including an objective lens 211, a bright field illumination source 213, and a dichroic filter. It includes a mirror 220, a variable magnification lens 221, a bright-field observation camera 222, and a confocal scanner unit 230.

The XY stage 201 is movable along the X-axis and the Y-axis. Here, the X-axis and the Y-axis are orthogonal to each other in the horizontal plane. That is, the XY stage 201 is movable along the horizontal plane. The cell culture vessel placed on the XY stage 201 is not limited to the microplate 110, and may be a cell culture dish, a cover glass chamber, a petri dish, or the like.

In the present embodiment, the second optical system unit 200 will be described as having a configuration capable of fluorescence observation and bright field observation of two confocal colors. The second optical system unit 200 is not limited to such a configuration, and may be, for example, good as long as it enables bright field observation.

The confocal scanner unit 230 includes a dichroic mirror 231, a pinhole array disk (nipo disk) 232, a microlens array disk 233, a relay lens 234, a dichroic mirror 235, a first band pass filter 236a, and a first lens. It includes a 237a, a first camera for fluorescence observation 238a, a second band pass filter 236b, a second lens 237b, a second camera for fluorescence observation 238b, and an excitation light source unit 239.

In brightfield observation, the brightfield illumination source 213 irradiates the brightfield signal light toward the microplate 110. The irradiated bright-field signal light is reflected by the dichroic mirror 220 through the microscope 210, and is imaged by the bright-field observation camera 222 by the variable magnification lens 221. The bright field illumination source 213 partitions the hole 214 communicating in the vertical direction, and is, for example, a torus (doughnut shape). The bright field illumination source 213 is not limited to the torus, and the central portion may be a space in the top view.

In fluorescence observation, an excitation light flux having a specific wavelength is emitted from the excitation light source unit 239 toward the microplate 110. After that, a fluorescence signal having a wavelength longer than that of the excitation light flux is emitted from the sample excited by the excitation light flux, and the fluorescence signal passes through the pinhole array disk 232 to become a cofocal image. After that, the fluorescence signal is reflected by the dichroic mirror 231 and is imaged by the fluorescence observation first camera 238a and the fluorescence observation second camera 238b via the relay lens 234.

The dichroic mirror 235 has a property of dispersing a fluorescent signal and is used to cope with the simultaneous use of excitation light sources having a plurality of wavelengths. Further, the first bandpass filter 236a and the second bandpass filter 236b are installed to improve the S / N ratio of the image and to pass only the required wavelength band of the fluorescent signal. Since the fluorescence wavelength emitted by the sample varies, each of the first bandpass filter 236a and the second bandpass filter 236b includes, for example, a plurality of bandpass filters, and a band corresponding to the required wavelength is used by using a filter wheel or the like. It is desirable to configure the path filter so that it can be switched.

In the present embodiment, the microscope 210, the bright field illumination source 213, the dichroic mirror 220, the variable magnification lens 221 and the bright field observation camera 222 constitute an aperture image acquisition unit 70. The aperture image acquisition unit 70 acquires an image (hereinafter, also referred to as “aperture image”) of the opening 12 (see FIG. 2 and the like) partitioned by the tip portion 11 (see FIG. 2 and the like) of the nanospray chip 10 described later. It is possible. As described above, the aperture image acquisition unit 70 includes a bright field illumination source 213 as an optical system for observing a sample, a dichroic mirror 220, a variable magnification lens 221 and a bright field observation camera 222 as an image pickup device. It is composed of. The aperture image acquisition unit 70 can take an image from the axial direction of the nanospray chip 10.

The suction operation unit 300 includes an XYZ stage 310, a transport body 20, a chip rack 341, and a sample rack 342. One or more nanospray chips 10 for selectively collecting a specific cell or a specific intracellular substance as a sample are arranged in the chip rack 341. The sample rack 342 stores a nanospray chip 10 holding a specific cell collected as a sample or a specific intracellular substance.

The nanospray chip 10 can be nanosprayed as it is from the collected sample and introduced into the sample introduction port of the mass spectrometer, and is detachably attached to the carrier 20. As shown in FIG. 1, the nanospray tip 10 has a tip portion 11. FIG. 2 is an enlarged vertical sectional view showing the tip portion 11 of the nanospray tip 10. As shown in FIG. 2, the tip portion 11 of the nanospray tip 10 partitions the opening 12 and the hollow portion 13 and has a cylindrical shape. The tip portion 11 of the nanospray chip 10 can collect a sample through the opening 12 and hold the sample in the hollow portion 13. Although not shown, the nanospray chip 10 has an electrode for nanospraying a sample on the introduction port of a mass spectrometer used in a subsequent step on the outer surface or the inner surface thereof. Here, the electrode is formed by, for example, vapor deposition of metal. However, the electrode is arranged at a position that does not interfere with the acquisition of the digital phase difference image by the digital phase difference image acquisition unit 30 (see FIG. 3) described later.

The carrier 20 shown in FIG. 1 can move the nanospray chip 10 to a collection position where a sample can be collected. Specifically, the carrier 20 can detachably support the nanospray tip 10 and can be moved along the X-axis, Y-axis, and Z-axis by the XYZ stage 310. Here, as described above, the X-axis and the Y-axis are orthogonal to each other in the horizontal plane. Further, the Z axis is an axis along the vertical direction orthogonal to each of the X axis and the Y axis. That is, the carrier 20 can move in the space by the XYZ stage 310. The carrier 20 can control the position of the nanospray tip 10 with high accuracy.

The carrier 20 moves directly above the chip rack 341 and any nanospray chip from the chip rack 341 when the nanospray chip 10 selectively collects a specific cell or a specific intracellular substance as a sample. Select 10 and attach it. After that, the carrier 20 moves the nanospray tip 10 directly above the microplate 110, and applies a suction force to the nanospray tip 10 at a predetermined sampling position to collect a sample from the well. The sampling position can be, for example, on the optical axis of the microscope 210. At this time, the XY stage 201 moves the microplate 110 so that the sample to be collected is located at the collection position. As a result, the nanospray chip 10 and the sample to be collected overlap in the vertical direction. By setting the sampling position to the position corresponding to the hole 214 of the bright field illumination source 213, it is possible to prevent the nanospray tip 10 and the carrier 20 from interfering with the bright field illumination source 213 when collecting the sample. Can be done.

After collecting a sample on the nanospray chip 10, the carrier 20 moves the nanospray chip 10 directly above the sample rack 342 and releases the nanospray chip 10 to store the nanospray chip 10 in the sample rack 342. can do.

The information processing device 400 can be configured by using an information processing device such as a personal computer (PC), and includes an image acquisition unit 410, an image processing unit 420, a display unit 430, an operation reception unit 440, and operation control. A unit 450, a storage unit 460, and a volume determination unit 470 are provided.

The image acquisition unit 410 acquires images from the image pickup device 50, the bright-field observation camera 222, the fluorescence observation first camera 238a, and the fluorescence observation second camera 238b. The image acquisition unit 410 can control the image acquisition device 50, the bright-field observation camera 222, the fluorescence observation first camera 238a, and the fluorescence observation second camera 238b, respectively, to control the timing of image acquisition. May be good.

The image processing unit 420 includes a processor and the like, and performs image processing and various analyzes on the image acquired by the image acquisition unit 410. Specifically, the image processing unit 420 generates a digital phase difference image based on a plurality of images acquired from the image pickup apparatus 50. Further, the image processing unit 420 refers to the images acquired from the bright-field observation camera 222, the fluorescence observation first camera 238a, and the fluorescence observation second camera 238b by template matching or the like to obtain cells / cell organs. Recognizing is performed, and characteristic quantities such as size, brightness, protein amount, and ion amount are calculated for each identified cell. In addition, the image processing unit 420 performs processing such as listing and graphing information on cells using the calculated feature amount.

The display unit 430 is configured to include an arbitrary display device, and performs image processing acquired by the image acquisition unit 410, image processing of the image processing unit 420, and display processing of analysis results. The operation reception unit 440 is configured to include an arbitrary input device such as a keyboard and a mouse, and receives various operations from the operator.

The operation control unit 450 is configured to include a processor and the like, and can control the operation of the XY stage 201 to move the microplate 110. Further, the motion control unit 450 can control the motion of the XYZ stage 310 to move the carrier 20. Further, the motion control unit 450 can control the motion of the carrier 20 to switch between mounting and detaching of the nanospray tip 10, and applies suction force to the mounted nanospray tip 10 to collect a sample. Can be made to.

The storage unit 460 stores the image acquired by the image acquisition unit 410, the analysis result of the image processing unit 420, and the like. Further, the storage unit 460 stores in advance programs and data for each unit of the information processing apparatus 400 to execute various processes.

The volume determination unit 470 includes a processor and the like, and is collected by the tip portion 11 of the nanospray chip 10 based on the digital phase difference image acquired by the digital phase difference image acquisition unit 30 (see FIG. 3) described later. Determine the volume of the sample.

As shown in FIG. 1 and FIG. 3 described later, the first optical system unit 40 includes an image pickup device 50 and an optical system 60. The image pickup apparatus 50 includes a camera for capturing an image of the tip portion 11 of the nanospray chip 10 from which the sample was taken. The optical system 60 includes a light source 61, an objective lens 62, and an imaging lens 63, and the tip portion 11 of the nanospray chip 10 arranged on the optical axis 64 between the light source 61 and the objective lens 62. This is an optical system for magnifying an image with a microscope composed of an objective lens 62 and an imaging lens 63 and forming an image on the image pickup apparatus 50. The light source 61 irradiates the image pickup apparatus 50 with the emitted bright-field illumination through the objective lens 62 and the imaging lens 63. The magnification of the microscope composed of the objective lens 62 and the imaging lens 63 is preferably 20 to 40 times.

FIG. 3 is a diagram showing a state in which a digital phase difference image is acquired by using the digital phase difference image acquisition unit 30 of the sampling device 1. As shown in FIG. 3, the digital phase difference image acquisition unit 30 includes a first optical system unit 40, an image acquisition unit 410 of the information processing device 400, and an image processing unit 420 of the information processing device 400. .. The digital phase difference image acquisition unit 30 was collected by the nanospray chip 10 by moving the nanospray chip 10 after collecting the sample and before being stored in the sample rack 342 by the carrier 20. A digital phase difference image of the sample can be acquired. As shown in FIG. 3, the digital phase difference image is captured from the radial direction of the nanospray chip 10. The digital phase contrast image is an image having higher contrast than a normal image, whereby the boundary between the sample and the nanospray chip 10 is further emphasized. The details of the method for the digital phase difference image acquisition unit 30 to acquire the digital phase difference image will be described later.

[Sample collection method]
Next, a sampling method as an embodiment of the present invention will be described. FIG. 4 is a flowchart showing a sampling method as an embodiment of the present invention. As shown in FIG. 4, the sampling method of the present embodiment includes a sampling step S1, a digital phase difference image acquisition step S2, and a volume determination step S3.

As an example of the sampling method of the present embodiment, an example using the sampling device 1 will be described. However, the sampling method of the present embodiment may be carried out using an apparatus other than the sampling apparatus 1. FIG. 5 is a flowchart illustrating the operation of the sampling device 1. As shown in FIG. 5, the sampling device 1 first moves the nanospray chip 10 as a sampling tube to the sampling position by the carrier 20 (step S101). Specifically, the operation control unit 450 controls the operation of the XYZ stage 310 based on the operation received by the operation reception unit 440, so that the carrier 20 selects one nanospray chip 10 from the chip rack 341. The nanospray tip 10 is moved to a collection position where the sample of the microplate 110 can be collected. At this time, the XY stage 201 moves the microplate 110 so that the sample to be collected is located at the collection position.

Next, the sample sampling device 1 hollows out the sample at the sampling position through the opening 12 of the nanospray tip 10 as a sampling tube by the motion control by the motion control unit 450 based on the operation received by the operation reception unit 440. It is collected in the unit 13 (step S102). The above-mentioned steps S101 to S102 constitute the sampling step S1 (see FIG. 4) of the sampling method of the present embodiment.

Next, the sampling device 1 acquires an aperture image of the opening 12 of the nanospray chip 10 by using the aperture image acquisition unit 70 based on the operation received by the operation reception unit 440 (step S103). The order of the process of step S102 and the process of step S103 may be reversed or may be simultaneous. The sampling device 1 may display the acquired aperture image using the display unit 430.

FIG. 6 is a diagram showing an example of an aperture image acquired by the aperture image acquisition unit 70 of the sampling device 1. As shown in FIG. 6, the cross-sectional area of the opening 12 of the nanospray tip 10 perpendicular to the axial direction of the nanospray tip 10 can be determined from the aperture image.

Next, in the sampling device 1, the sample collected by the nanospray chip 10 (hereinafter, also referred to as “collected sample”) is located on the optical axis 64 of the optical system 60 included in the digital phase contrast image acquisition unit 30. The nanospray chip 10 is moved by the carrier 20 (step S104). Specifically, the operation control unit 450 controls the operation of the XYZ stage 310 based on the operation received by the operation reception unit 440, so that the sample is conveyed so as to be located on the optical axis 64 of the optical system 60. The body 20 moves the nanospray chip 10.

After the sample to be collected moves on the optical axis 64, the sample collection device 1 acquires an image of the sample by the image acquisition unit 410 using the image pickup device 50 (step S105).

Subsequently, the sampling device 1 moves the nanospray tip 10 by the transport body 20 so that the sample is moved by a predetermined distance along the optical axis 64 (step S106). Specifically, the operation control unit 450 controls the operation of the XYZ stage 310 based on the operation received by the operation reception unit 440 so that the sample is moved by a predetermined distance along the optical axis 64 of the optical system 60. In addition, the carrier 20 moves the nanospray chip 10.

After the sample is moved along the optical axis 64, the sample collection device 1 acquires an image of the sample by the image acquisition unit 410 using the image pickup device 50 (step S107). When the sampling device 1 determines that the acquisition of the image of the sample by the imaging device 50 has not been completed (No in step S108), the sample collecting device 1 returns to the process of step S106. On the other hand, when the sampling device 1 determines that the acquisition of the image of the sample by the imaging device 50 is completed (Yes in step S108), the sample collecting device 1 proceeds to the process of step S109. The above-mentioned steps S104 to S108 constitute the digital phase difference image acquisition step S2 (see FIG. 4) of the sampling method of the present embodiment.

In the process of step S109, the sampling device 1 takes a digital phase difference image based on the images of the plurality of samples acquired in the processes of steps S105 to S108 and the information of the predetermined distance moved in the process of step S106. A digital phase difference image is generated using the image processing unit 420 as the acquisition unit 30 (step S109). Digital phase contrast images can be generated using, for example, the method introduced in Mustafa Mir et al., “Quantitative Phase Imaging”, Progress in Optics, Vol. 57, p. 133-217. The sampling device 1 may display the generated digital phase difference image by using the display unit 430.

FIG. 7 is a diagram showing an example of a digital phase difference image acquired by the digital phase difference image acquisition unit 30 of the sampling device 1. As shown in FIG. 7, in the high-contrast digital phase contrast image, the boundary of the sample S is emphasized at the tip portion 11 of the nanospray chip 10, so that the length of the nanospray chip 10 is longer than that of a normal image. The total length L of the collected sample S along the direction can be determined more accurately.

After that, the sampling apparatus 1 uses the volume determination unit 470 to determine the volume of the sample to be collected based on the digital phase contrast image generated in the process of step S109 and the aperture image acquired in the process of step S103. (Step S110). Specifically, based on the information of the total length L (see FIG. 7) of the sample S determined from the digital phase difference image and the information of the cross-sectional area of the opening 12 of the nanospray chip 10 determined from the aperture image. Determine the volume of the sample to be collected. For example, when the cross-sectional area is 7 pm ² and the total length L of the collected sample S is 30 μm, the volume of the collected sample is about 212 fL when calculated assuming that the cross-sectional area is constant in the axial direction of the tip portion 11. Is determined as. The sampling device 1 may display the value of the determined volume of the sample to be sampled by using the display unit 430. The above-mentioned step S110 constitutes the volume determination step S3 (see FIG. 4) of the sampling method of the present embodiment.

As described above, the sampling device 1 of the present embodiment acquires a digital phase difference image by utilizing the movement of the carrier 20 capable of highly accurate position control, and collects the sample based on the digital phase difference image. The volume of the sample can be determined. Therefore, the volume of a very small amount of sample can be measured with high accuracy with a simple configuration. Therefore, for example, when mass spectrometry is performed, not only the identification of the collected sample but also its quantification can be realized.

Further, the sampling device 1 of the present embodiment includes an image of the opening 12 of the nanospray chip 10 as a sampling tube acquired by the aperture image acquisition unit 70 and a digital phase difference image acquired by the digital phase difference image acquisition unit 30. , The volume of the sample to be collected can be determined. Therefore, since the volume of the collected sample can be determined based on the actually measured value, the volume of the collected sample can be determined accurately.

By the way, the sampling device 1 does not have to use the image of the opening 12 as the measured data. Specifically, as shown in FIG. 7, the sampling apparatus 1 determines the total length L of the collected sample S and the width of the collected sample S (inner diameter φ of the tip portion 11) from the digital phase contrast image. The volume of the sample S may be determined based on these. Further, the sampling device 1 may determine the longitudinal cross-sectional area of the sampling sample S along the axial direction of the nanospray chip 10 from the digital phase contrast image, and determine the volume of the sampling sample S based on the longitudinal cross-sectional area. .. Further, the sampling device 1 is based on the total length L of the sample S collected from the digital phase contrast image (see FIG. 7) and the inner diameter φ of the opening 12 from the aperture image (see FIG. 6). The volume may be determined.

Alternatively, although the accuracy of the sampling device 1 is lower than when the measured value is used as described above, the design value of the width (inner diameter φ) or the cross-sectional area of the hollow portion 13 of the nanospray chip 10 is previously set in the storage unit 460. The volume may be determined using the total length L (see FIG. 7) of the sample to be stored and determined from the digital retardation image, and the design value of the stored inner diameter φ or cross-sectional area.

The present invention is not limited to the configuration specified in each of the above-described embodiments, and various modifications can be made without departing from the contents described in the claims.

For example, the tip portion 11 of the nanospray chip 10 is not limited to a cylindrical shape. Further, in the above embodiment, the collection tube is configured as a nanospray chip 10. However, the collection tube does not have to be the nanospray chip 10. Further, in the above embodiment, the collection tube is configured to be able to selectively collect a specific cell or a specific intracellular substance. However, the collection tube is not limited to such a configuration.

In the above embodiment, specific cells or a specific intracellular substance is selectively collected in a collection tube, but cells and substances other than the intracellular substance may be collected as a sample.

The present invention relates to a sampling device and a sampling method.

1: Sampling device 10: Nanospray chip 11: Tip part 12: Opening 13: Hollow part 20: Transporter 30: Digital phase difference image acquisition part 40: First optical system part 50: Image pickup device 60: Optical system 61: Light source 62: Objective lens 63: Imaging lens 64: Optical axis 70: Aperture image acquisition unit 110: Microplate 200: Second optical system unit 201: XY stage 210: Microscope 211: Objective lens 213: Bright field illumination source 214: Hole 220: Dycroic mirror 221: Variable magnification lens 222: Bright field observation camera 230: Cofocal scanner 231: Dycroic mirror 232: Pinhole array disk 233: Microlens array disk 234: Relay lens 235: Dycroic mirror 236a: 1st band pass filter 236b: 2nd band pass filter 237a: 1st lens 237b: 2nd lens 238a: 1st camera for fluorescence observation 238b: 2nd camera for fluorescence observation 239: Excitation light source unit 300: Suction operation unit 310 : XYZ stage 341: Chip rack 342: Specimen rack 400: Information processing device 410: Image acquisition unit 420: Image processing unit 430: Display unit 440: Operation reception unit 450: Operation control unit 460: Storage unit 470: Volume determination unit φ : Inner diameter of the opening (tip) of the nanospray tip L: Total length of the sample to be collected S: Sample to be collected

Claims (5)

A collection tube that partitions the opening and the hollow portion that can hold the sample collected through the opening,
A carrier that can move the collection tube to a collection position where the sample can be collected, and
A digital phase difference image acquisition unit capable of acquiring a digital phase difference image of the sample collected in the collection tube by moving the collection tube with the carrier.
A volume determination unit that determines the volume of the sample collected in the collection tube based on the digital phase difference image acquired by the digital phase difference image acquisition unit.
A sampling device. An opening image acquisition unit capable of acquiring an image of the opening of the collection tube is provided.
The volume determination unit is a sample of the sample collected in the collection tube based on the image of the aperture acquired by the aperture image acquisition unit and the digital phase difference image acquired by the digital phase difference image acquisition unit. The sampling device according to claim 1, wherein the volume is determined. The sampling device according to claim 2, wherein the aperture image acquisition unit includes an optical system for observing the sample and an imaging device. The sampling device according to any one of claims 1 to 3, wherein the sampling tube is a nanospray chip capable of selectively collecting a specific cell or a specific intracellular substance as the sample. A collection step in which a collection tube for partitioning an opening and a hollow portion connected to the opening is moved to a collection position by a transporter, and a sample is collected in the hollow portion through the opening.
A digital phase difference image acquisition step of acquiring a digital phase difference image of the sample collected in the collection tube by moving the collection tube with the carrier.
A volume determination step for determining the volume of the sample collected in the collection tube based on the acquired digital phase difference image, and a volume determination step.
Sampling methods, including.

Images