Patent Description:
In a cell observation system (flow cytometer or imaging flow cytometer) that observes a cell moving in a flow path with a fluid, focusing of an imaging apparatus is important to acquire a clear image of the cell being an observation object. By acquiring the clear image of the cell, for example, it is possible to identify whether or not the cell is a cancer cell (circulating tumor cell) included in a cancer patient.

The flow cytometer uses a point sensor type photodetector such as a photomultiplier tube and a photodiode. In the cell observation using the flow cytometer, cells are caused to flow at a flow speed of several m/s to <NUM>/s.

The imaging flow cytometer uses a linear array sensor type or two-dimensional sensor type photodetector. In the cell observation using the imaging flow cytometer, it is said that cells are caused to flow at a flow speed of several mm/s to several <NUM>/s. This flow speed is about <NUM>/<NUM><NUM> of that of the cell observation using the flow cytometer. The imaging flow cytometers include a two-dimensional imaging flow cytometer capable of acquiring a quantitative phase image of the cell, and a three-dimensional imaging flow cytometer capable of acquiring a three-dimensional image for a refractive index distribution of the cell.

In general, in the flow cytometer, the cells are caused to flow with the fluid such as a culture solution, and thus, by a hydrodynamic focusing effect, the cells can be substantially aligned along a flow direction. By the hydrodynamic focusing effect, even when the focus of the optical system of the imaging apparatus is fixed, it is possible to acquire the image of the cell in focus.

The degree of alignment of the cells by the hydrodynamic focusing effect depends on a volume ratio of a sample liquid (cell suspension) and a sheath liquid (culture solution). This volume ratio is, in general, controlled by a pressure applied to extrude these liquids. By reducing the volume ratio, a diameter of the sample liquid column is reduced, and as a result, the cells are more truly aligned. On the other hand, by increasing the volume ratio, the diameter Δd of the sample liquid column is increased, and as a result, the cells do not flow with true alignment. In this case, the cells flow over a range having a thickness in an optical axis direction of the optical system of the imaging apparatus, and the image of the cell not in focus is acquired.

On the other hand, when the diameter Δd of the sample liquid column is smaller than a depth of focus (DOF) of the optical system of the imaging apparatus (that is, DOF > Δd), the image of the cell in focus is obtained. Further, in the flow cytometer, when an adjustment of an instrument that affects a position of a sample nozzle is performed, a position of the cells flowing substantially in a line in the flow path cross section changes, and as a result, the image of the cell not in focus is acquired. Further, at the current technical level, a time required for the focus adjustment of the imaging optical system is in general said to be at least <NUM>, and a sufficient response speed cannot be obtained.

In order to avoid these two technical problems, a preliminary sample or a dummy sample such as a bead is caused to flow before the main measurement, the focus adjustment is performed to determine a focus plane, and then a test sample is caused to flow. When the cell flows in a time shorter than a time required for the focus adjustment of the imaging optical system, focus is not readjusted while the test sample is flowing.

In the imaging flow cytometer, a linear array sensor (including TDI sensor) is often used by using the movement of the cell of an imaging object. In the linear array sensor, the object is scanned in the flow direction for each line (L1, L2, L3,. ) in the direction perpendicular to the flow direction to acquire the image. When a spherical object such as the cell is imaged by scanning in the flow direction, it is necessary to estimate the focus of the entire cell on the first line (L1). However, this is difficult.

In the inventions described in Patent Documents <NUM> and <NUM>, the imaging apparatus measures a focus deviation amount by an optical system provided separately from the imaging optical system, and adjusts the focus of the imaging optical system based on a measurement result. This imaging apparatus performs a measurement of the focus deviation amount and imaging at a common position in the moving direction of the cell in the flow path.

Patent Document <NUM> discloses a flow cytometric system and method for observing, analyzing and/or separating objects in a liquid sample, comprising a digital holographic microscope (DHM) and at least one fluidic system, whereby the DHM comprises illumination means, an interferometric system and digital recording means, whereby the fluidic system is capable of guiding said objects through an illumination beam of the illumination means of said DHM, whereby the fluidic system comprises a mechanism for inducing a liquid sample stream through the fluidic system, whereby preferably the fluidic system comprises a stream size controlling device for controlling the transverse dimensions of a liquid sample stream inside said fluidic system, preferably said stream size controlling device is capable of lining up the objects one-by-one or multiple objects at a time in said liquid sample stream.

Patent Document <NUM> discloses apparatus, systems, compositions, and methods for analyzing a sample containing particles. A particle imaging system or analyzer can include a flowcell through which a urine sample containing particles is caused to flow, and a high optical resolution imaging device which captures images for image analysis. A contrast pattern for autofocusing is provided on the flowcell. The image processor assesses focus accuracy from pixel data contrast. A positioning motor moves the microscope and/or flowcell along the optical axis for autofocusing on the contrast pattern target. The processor then displaces microscope and flowcell by a known distance between the contrast pattern and the sample stream, thus focusing on the sample stream. Cell or particle images are collected from that position until autofocus is reinitiated, periodically, by input signal, or when detecting temperature changes or focus inaccuracy in the image data.

In the inventions described in Patent Documents <NUM> and <NUM>, a speed at which the cells flow in the flow path is restricted by a time required for the measurement of the focus deviation amount and the focus adjustment of the imaging optical system. Since there is no guarantee that the position of the next flowing cell in the optical axis direction is the same as that of the previous cell, in the above invention, the flow speed of the cell is restricted by the focusing speed (time required for the measurement of the focus deviation amount and the focus adjustment of the imaging optical system).

An object of an embodiment is to provide a cell observation system and a cell observation method capable of relaxing restriction of a flow speed of a cell due to a focusing speed.

An embodiment is a cell observation system. The cell observation system is a cell observation system for observing a cell moving in a flow path with a fluid, and includes (<NUM>) a first imaging apparatus including a first optical system and a first imaging element, the first imaging apparatus arranged to capture an image of the cell by receiving, by the first imaging element, light reaching the first imaging element from the cell at a first position in a moving direction of the cell in the flow path through the first optical system; (<NUM>) a second imaging apparatus including a second optical system, in which a focus is adjusted based on a focus adjustment signal, and a second imaging element, the second imaging apparatus capture an image of the cell by receiving, by the second imaging element, light reaching the second imaging element from the cell at a second position downstream of the first position in the moving direction of the cell in the flow path through the second optical system; and (<NUM>) a control device arranged to input the image obtained by imaging by the first imaging element of the first imaging apparatus, analyze the image to obtain a passing position of the cell in a cross section of the flow path, and generate the focus adjustment signal based on the obtained passing position to provide the focus adjustment signal to the second optical system of the second imaging apparatus, wherein the cell is focused on a region near a center in the cross section of the flow path in advance upstream of the first position and the second position for restricting a focus deviation amount of the passing position of the cell.

An embodiment is a cell observation method. The cell observation method is a cell observation method for observing a cell moving in a flow path with a fluid, and includes (<NUM>) a first imaging step of, using a first imaging apparatus including a first optical system and a first imaging element, capturing an image of the cell by receiving, by the first imaging element, light reaching the first imaging element from the cell at a first position in a moving direction of the cell in the flow path through the first optical system; (<NUM>) a second imaging step of, using a second imaging apparatus including a second optical system, in which a focus is adjusted based on a focus adjustment signal, and a second imaging element, capturing an image of the cell by receiving, by the second imaging element, light reaching the second imaging element from the cell at a second position downstream of the first position in the moving direction of the cell in the flow path through the second optical system; and (<NUM>) a focus adjustment instruction step of, after the first imaging step and before the second imaging step, inputting the image obtained by imaging by the first imaging element of the first imaging apparatus, analyzing the image to obtain a passing position of the cell in a cross section of the flow path, and generating the focus adjustment signal based on the obtained passing position to provide the signal to the second optical system of the second imaging apparatus, wherein the cell is focused on a region near a center in the cross section of the flow path in advance upstream of the first position and the second position for restricting a focus deviation amount of the passing position of the cell.

According to the cell observation system and the cell observation method of the embodiments, restriction of a flow speed of a cell due to a focusing speed can be relaxed.

Hereinafter, embodiments of a cell observation system and a cell observation method will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference signs, and redundant description will be omitted. The present invention is not limited to these examples.

<FIG> is a diagram illustrating a configuration of a cell observation system <NUM>. The cell observation system <NUM> observes a cell <NUM> moving in a flow path <NUM> with a fluid <NUM>. The cell observation system <NUM> includes a first imaging apparatus <NUM>, a second imaging apparatus <NUM>, a control device <NUM>, and an analysis device <NUM>.

For example, the flow path <NUM> is a flow cell, the fluid <NUM> is a culture solution, and the cell <NUM> is a red blood cell, a white blood cell, a CTC, or the like. The cell <NUM> is prepared in a state of being suspended in the culture solution. A suspension concentration is, for example, <NUM><NUM> cells/mL. The cell suspension is guided to the flow path <NUM>. The cell <NUM> is caused to move substantially on a straight line in the flow path <NUM> by the hydrodynamic focusing effect.

The first imaging apparatus <NUM> includes a first optical system <NUM> and a first imaging element <NUM>. The first optical system <NUM> guides light reaching from the cell <NUM> at a first position in a moving direction of the cell <NUM> in the flow path <NUM> to a light receiving plane of the first imaging element <NUM>. The first imaging element <NUM> is optically coupled to the first optical system <NUM>, and captures an image of the cell <NUM> by receiving the light reaching the light receiving plane (first imaging step).

The second imaging apparatus <NUM> includes a second optical system <NUM> and a second imaging element <NUM>. The second optical system <NUM> guides light reaching from the cell <NUM> at a second position in the moving direction of the cell <NUM> in the flow path <NUM> to a light receiving plane of the second imaging element <NUM>. The second imaging element <NUM> is optically coupled to the second optical system <NUM>, and captures an image of the cell <NUM> by receiving the light reaching the light receiving plane (second imaging step). The second position is downstream of the first position.

The second optical system <NUM> adjusts the focus based on a focus adjustment signal provided from the control device <NUM>. After the focus adjustment, the second imaging element <NUM> captures the image of the cell <NUM>, thereby acquiring a clear image of the cell <NUM> in focus. The imaging operation by the second imaging element <NUM> may be performed at a timing instructed by a camera trigger signal provided from the control device <NUM>, or may be continuously performed with a constant frame rate.

The control device <NUM> is electrically coupled to the first imaging element <NUM> of the first imaging apparatus <NUM>, and is electrically coupled to the second optical system <NUM> and the second imaging element <NUM> of the second imaging apparatus <NUM>. The control device <NUM> inputs the image obtained by imaging by the first imaging element <NUM>, and analyzes the image to obtain a passing position of the cell <NUM> in a cross section of the flow path <NUM>. Further, the control device <NUM> generates the focus adjustment signal based on the obtained passing position, and provides the focus adjustment signal to the second optical system <NUM> (focus adjustment instruction step). Further, the control device <NUM> provides the camera trigger signal for instructing the timing of imaging by the second imaging element <NUM> to the second imaging element <NUM>.

The control device <NUM> may be a general-purpose computer including a central processing unit (CPU) as a processor, a random access memory (RAM) or a read only memory (ROM) as a storage medium, an input unit such as a keyboard or a mouse, a display unit such as a liquid crystal display, and an input-output module. The control device <NUM> may be configured as a dedicated device using, for example, a microcomputer or a field programmable gate array (FPGA).

The analysis device <NUM> is electrically coupled to the second imaging element <NUM> of the second imaging apparatus <NUM>. The analysis device <NUM> inputs the image obtained by imaging by the second imaging element <NUM>, and analyzes the image to identify the type of the cell <NUM> in the image. The analysis device <NUM> may be a general-purpose computer including a CPU as a processor, a RAM or a ROM as a storage medium, an input unit such as a keyboard or a mouse, a display unit such as a liquid crystal display, and an input-output module.

The flow path <NUM> has a pipe structure made of a transparent material such as a glass, and the cell <NUM> is caused to flow therein with the fluid <NUM>. The fluid <NUM> is, for example, a cell culture solution. In the imaging position (first position, second position), the wall surface of the flow path <NUM> is preferably flat, and the cross sectional shape of the flow path <NUM> is preferably rectangular (including square). In this way, it is possible to acquire the image for the cell with small distortion.

When the fluid <NUM> is caused to flow in the flow path <NUM>, the fluid <NUM> is preferably caused to flow under a laminar flow condition. When a turbulent flow condition is used instead of the laminar flow condition, a flow line of the fluid <NUM> such as a culture solution does not fall within a plane perpendicular to the optical axis in many cases, and therefore, a position in the optical axis direction is different in the imaging position (first position, second position) of the cell <NUM> flowing along the flow line, and thus, an effect of the focus adjustment is not easily exhibited.

When the fluid <NUM> is caused to flow in the flow path <NUM>, it is preferable to focus the cell <NUM> on a certain region (for example, a region near the center) in the cross section of the flow path <NUM> in advance upstream of the imaging position (first position, second position). As a method of realizing the focusing, hydrodynamic focusing, acoustic focusing, inertial focusing, and the like are known. By focusing the cell <NUM> near the center of the flow path <NUM> in advance in this way, the focus deviation amount can be restricted to a certain width (Δd).

The cell observation method of the present embodiment is to observe the cell <NUM> moving in the flow path <NUM> with the fluid <NUM> by using the first imaging apparatus <NUM> and the second imaging apparatus <NUM> described above.

In the first imaging step, the first imaging apparatus <NUM> including the first optical system <NUM> and the first imaging element <NUM> is used to capture the image of the cell by receiving, by the first imaging element <NUM>, light reaching the first imaging element <NUM> from the cell <NUM> at a first position in the moving direction of the cell <NUM> in the flow path <NUM> through the first optical system <NUM>. The image data obtained by the imaging is sent to the control device <NUM>.

In the second imaging step, the second imaging apparatus <NUM> including the second optical system <NUM>, in which the focus is adjusted based on the focus adjustment signal, and the second imaging element <NUM> is used to capture the image of the cell by receiving, by the second imaging element <NUM>, light reaching the second imaging element <NUM> from the cell <NUM> at a second position downstream of the first position through the second optical system <NUM>. The image of the cell obtained by the imaging is analyzed by the analysis device <NUM> to identify the type of the cell, and is displayed on the display unit.

In the focus adjustment instruction step after the first imaging step and before the second imaging step, the control device <NUM> obtains the passing position of the cell <NUM> in the cross section of the flow path <NUM> based on the image obtained by imaging by the first imaging element <NUM> of the first imaging apparatus <NUM>. Then, the focus adjustment signal is generated based on the obtained passing position and provided to the second optical system <NUM> of the second imaging apparatus <NUM>.

<FIG> is a diagram illustrating an example of a structure of the flow path <NUM>. In this structure, the fluid <NUM> is caused to flow as a laminar flow in the flow path <NUM> by utilizing the hydrodynamic focusing effect. A nozzle <NUM> is inserted into the flow path <NUM> upstream of the imaging position (first position, second position). The cell suspension (sample liquid) is supplied to the flow path <NUM> from the nozzle <NUM>, and the sheath liquid is supplied to the flow path <NUM> from a concentric tube around the nozzle <NUM>.

The flow rate can be adjusted by the pressure applied to each of the sample liquid and the sheath liquid, and the cell <NUM> contained in the sample liquid can be focused around the center of the flow path <NUM>. When the flow rate of the sample liquid is Qsa and the flow rate of the sheath liquid is Qsh, the focusing width Δd depends on the flow rate ratio (Qsh / Qsa) of the two liquids. Further, the average flow speed V of the cell <NUM> is determined by the flow paths of the two liquids, and is expressed by V = (Qsh + Qsa) / S, where S is the flow path cross sectional area. For example, when Qsh = <NUM>µL/min, Qsa = <NUM>µL/min, and S = <NUM><NUM>mm<NUM>, the average flow speed V is <NUM>/s.

The initial cell suspension concentration before being supplied from the nozzle <NUM> is, in general, <NUM> × <NUM><NUM> cells/mL. The volume fraction in this case is about <NUM>%, and the presence of the cell <NUM> in the cell suspension is extremely sparse. Therefore, even when the cell concentration is locally increased by focusing the cell <NUM> near the center of the flow path <NUM>, the distance between the cells <NUM> is sufficiently larger than the size of the cell <NUM> (in general, about <NUM>).

<FIG> shows an example of a histogram of the distance L between the cells <NUM>. This histogram is obtained under the conditions of the above flow speed and the cell suspension concentration using the hydrodynamic focusing effect. In this example, the average distance between the cells <NUM> is <NUM>.

<FIG> is a diagram illustrating a configuration of an imaging apparatus 100A. The imaging apparatus 100A is a configuration having a trans-illumination optical system, and can be used as the first imaging apparatus <NUM>. The imaging apparatus 100A includes an imaging optical system <NUM> including an objective lens <NUM> and an imaging lens <NUM>, an imaging element <NUM>, a light source <NUM>, and an irradiation optical system <NUM>.

The light source <NUM> outputs light with which the cell <NUM> is irradiated. The light source <NUM> may be a laser light source (for example, a HeNe laser light source). The irradiation optical system <NUM> is optically coupled to the light source <NUM>, and irradiates the cell <NUM> in the flow path <NUM> with the light output from the light source <NUM>.

The irradiation optical system <NUM> is preferably a beam forming optical system that irradiates the cell <NUM> with line-shaped light having a beam cross section long in the vertical direction with respect to the moving direction of the cell <NUM> in the flow path <NUM>, in terms of light energy efficiency. Further, when the cell <NUM> is irradiated with the line-shaped light, a linear array sensor can be used as the imaging element <NUM>, and the exposure time of the linear array sensor can be shortened, so that the clear image can be acquired also for the cell <NUM> flowing faster.

The irradiation optical system <NUM> may be configured to include a focusing lens, a single mode optical fiber, a collimator lens, and a cylindrical lens to irradiate the cell <NUM> with the line-shaped light. In this case, the light output from the light source <NUM> is focused on an input end of the optical fiber by the focusing lens, and guided by the optical fiber. The guided light is output from an output end of the optical fiber, and converted into parallel light by the collimator lens provided at the output end. Then, the parallel light is focused only in one axis direction by the cylindrical lens, thereby forming the line-shaped beam near the cell focus.

The imaging optical system <NUM> corresponds to the first optical system <NUM> of the first imaging apparatus <NUM>, and includes the objective lens <NUM> and the imaging lens <NUM>. The objective lens <NUM> and the imaging lens <NUM> guide light from the cell <NUM> to the light receiving plane of the imaging element <NUM>. For example, the magnification of the objective lens <NUM> is <NUM>×, the NA of the objective lens <NUM> is <NUM>, and the focal length of the imaging lens <NUM> is <NUM>. A position of the objective lens <NUM> in the optical axis direction is adjusted, and a focus is set to the center of the flow path <NUM>. The imaging optical system <NUM> becomes a fixed focus.

The imaging element <NUM> corresponds to the first imaging element <NUM> of the first imaging apparatus <NUM>. The imaging element <NUM> may be a linear array sensor. For example, a line sensor (model number raL2048-<NUM>) manufactured by Basler is preferably used as the imaging element <NUM>. The size of one pixel of the line sensor is <NUM> × <NUM>. The imaging element <NUM> may continuously perform an imaging operation at a predetermined line rate.

The control device <NUM> obtains the focus deviation amount (deviation amount of the passing position of the cell <NUM> with respect to the reference position in the cross section of the flow path <NUM>) as follows, based on the image acquired by the imaging apparatus 100A as the first imaging apparatus <NUM>. The control device <NUM> creates a two-dimensional image by arranging one-dimensional images output one after another from the imaging element <NUM> being the line sensor in that order. For example, the two-dimensional image (M rows and N columns) can be obtained by arranging the M one-dimensional images (<NUM> row and N columns). As long as the output of the one-dimensional image continues from the imaging element <NUM>, the number of pixels of the two-dimensional image is increasing, and in addition, when a FIFO (first-in first-out) memory is used, old image data can be erased, and the latest two-dimensional image having a predetermined number of pixels can be stored.

The control device <NUM> detects whether or not there is a partial image indicating a desired cell in the two-dimensional image, and cuts out the partial image indicating the cell. In this case, whether or not there is the partial image indicating the desired cell is detected based on whether or not an intensity of each pixel exceeds a threshold value. Then, a rectangular image including the partial image indicating the cell is cut out.

The control device <NUM> obtains the deviation amount ΔZ of the cell <NUM> from the focus plane (conjugate plane with respect to the light receiving plane of the imaging element <NUM>) of the imaging optical system <NUM> based on the rectangular image by a phase difference autofocus technique, an image plane phase difference focus technique, or an autofocus technique using digital holography. Since the rectangular image is blurred according to the deviation amount ΔZ, the deviation amount ΔZ can be obtained from the blur amount (see Non Patent Document <NUM>). The deviation amount ΔZ can take positive and negative values. The control device <NUM> generates the focus adjustment signal based on the deviation amount ΔZ, and provides the focus adjustment signal to the second optical system <NUM> of the second imaging apparatus <NUM>.

The control device <NUM> may determine whether to adjust the focus of the second optical system <NUM> based on the passing position (deviation amount ΔZ) of the cell, and provide the focus adjustment signal to the second imaging apparatus <NUM> when it is determined that the focus of the second optical system <NUM> is to be adjusted. For example, when the depth of focus of the second optical system <NUM> is large and the deviation amount ΔZ is small, the control device <NUM> may not provide the focus adjustment signal to the second imaging apparatus <NUM>. Further, when the deviation amount ΔZ of the cell with which the image is to be captured this time is the same as the deviation amount of the cell with which the image was captured last time, it is not necessary to move the focus position of the second optical system <NUM>, and thus, the control device <NUM> may not provide the focus adjustment signal to the second imaging apparatus <NUM>.

The second imaging apparatus <NUM> can also have substantially the same configuration as the imaging apparatus 100A. In addition, when the imaging apparatus 100A is used as the second imaging apparatus <NUM>, it is preferable that the irradiation optical system <NUM> irradiates the cell <NUM> with light not in a line shape but in a plane shape, and it is preferable that the imaging element <NUM> is a two-dimensional array sensor. Further, the imaging optical system <NUM> may adjust the focus in response to the focus adjustment signal provided from the control device <NUM>. At this time, it is preferable to move the objective lens <NUM> in the optical axis direction according to the deviation amount ΔZ. As a result, the imaging optical system <NUM> is focused, and the clear image of the cell <NUM> in focus is acquired by the imaging element <NUM>.

When the imaging apparatus 100A is used as the second imaging apparatus <NUM>, the imaging element <NUM> may continuously perform imaging at a constant frame rate regardless of the focus adjustment operation of the imaging optical system <NUM>. Further, the imaging element <NUM> may perform imaging at a timing instructed by the camera trigger signal provided from the control device <NUM>.

<FIG> is a timing chart when the imaging element <NUM> performs imaging at a timing instructed by the camera trigger signal provided from the control device <NUM>. The distance along the cell moving direction between the first position where the first imaging apparatus <NUM> captures the image of the cell and the second position where the second imaging apparatus <NUM> captures the image of the cell is set to L. The average flow speed of the cell is set to V. A time difference Td between imaging by the first imaging apparatus <NUM> and imaging by the second imaging apparatus <NUM> is expressed by Td = L / V. The time required for imaging and sending image data in the first imaging apparatus <NUM> is set to Ta. The time required for calculating the deviation amount ΔZ based on the image data and generating the focus adjustment signal in the control device <NUM> (time required for the measurement of the focus deviation amount) is set to Tb.

The control device <NUM> outputs the focus adjustment signal to the second imaging apparatus <NUM> after the lapse of time (Ta + Tb) from the time when the first imaging apparatus <NUM> captures the image of the cell. Further, the control device <NUM> outputs the camera trigger signal to the second imaging apparatus <NUM> after the lapse of time (Td - Ta - Tb) from the time when the focus adjustment signal is output in consideration of the time Tc required for the focus adjustment in the second optical system <NUM> of the second imaging apparatus <NUM>. There is a relationship of Ta + Tb + Tc < Td in these parameters.

Each of the first imaging apparatus <NUM> and the second imaging apparatus <NUM> may have various configurations in addition to the configuration having the trans-illumination optical system shown in <FIG>. Each of the first imaging apparatus <NUM> and the second imaging apparatus <NUM> may be a configuration having an epi-illumination optical system. The first imaging apparatus <NUM> may be a configuration having an interference optical system such as a Mach-Zehnder interferometer, a Michelson interferometer, or the like (for example, a quantitative phase microscope (see Non Patent Document <NUM>)). The second imaging apparatus <NUM> may be a quantitative phase microscope or a phase tomographic microscope (see Non Patent Document <NUM>). The first imaging apparatus <NUM> may be a quantitative phase microscope, and the second imaging apparatus <NUM> may be a phase tomographic microscope.

As modifications of the imaging apparatus 100A, an imaging apparatus 100B (<FIG>) having an epi-illumination optical system, an imaging apparatus 100C (<FIG>) having an interference optical system, and an imaging apparatus 100D (<FIG>) having a separator lens as the imaging lens in the imaging optical system will be described below.

<FIG> is a diagram illustrating a configuration of the imaging apparatus 100B. The imaging apparatus 100B is a configuration having an epi-illumination optical system, and includes the imaging optical system <NUM> including the objective lens <NUM> and the imaging lens <NUM>, the imaging element <NUM>, the light source <NUM>, and the irradiation optical system <NUM>, and further includes a mirror <NUM> and a beam splitter <NUM>.

The mirror <NUM> reflects the light output from the light source <NUM> and beam-formed by the irradiation optical system <NUM> to the beam splitter <NUM>. The beam splitter <NUM> is provided on an optical path between the objective lens <NUM> and the imaging lens <NUM>. The beam splitter <NUM> reflects a part of the light reaching from the mirror <NUM> and inputs it to the objective lens <NUM>, and transmits a part of the light reaching from the objective lens <NUM> and inputs it to the imaging lens <NUM>. In this case, a dark field illumination image of the cell can be acquired.

A dichroic mirror that reflects excitation light and transmits fluorescence may be provided instead of the beam splitter <NUM>. The light source <NUM> outputs the excitation light, and the fluorescence is generated from the cell irradiated with the excitation light. The dichroic mirror reflects the excitation light reaching from the mirror <NUM> and inputs it to the objective lens <NUM>, and transmits the fluorescence reaching from the objective lens <NUM> and inputs it to the imaging lens <NUM>. In this case, a fluorescence image of the cell can be acquired.

<FIG> is a diagram illustrating a configuration of the imaging apparatus 100C. The imaging apparatus 100C is a configuration having an interference optical system, and includes the imaging optical system <NUM> including the objective lens <NUM> and the imaging lens <NUM>, the imaging element <NUM>, the light source <NUM>, and the irradiation optical system <NUM>, and further includes a beam splitter <NUM>, a mirror <NUM>, a mirror <NUM>, and a beam splitter <NUM>.

The beam splitter <NUM> is provided on an optical path between the light source <NUM> and the irradiation optical system <NUM>. The beam splitter <NUM> inputs the light output from the light source <NUM>, reflects a part of the light, and transmits the remaining part, thereby splitting the light into two beams. The irradiation optical system <NUM> inputs the light transmitted through the beam splitter <NUM>.

The beam splitter <NUM> is provided on an optical path between the imaging lens <NUM> and the imaging element <NUM>. The beam splitter <NUM> inputs the light reaching from the imaging lens <NUM>, inputs the light reaching after being reflected by the beam splitter <NUM> and further reflected by the mirrors <NUM>, <NUM>, combines the light beams, and outputs the light to the imaging element <NUM>.

The optical system from the beam splitter <NUM> to the beam splitter <NUM> constitutes a Mach-Zehnder interferometer. The imaging element <NUM> can acquire an interference image of the cell. Further, a phase image of the cell can be acquired by changing the optical path difference between the two optical paths from the beam splitter <NUM> to the beam splitter <NUM>, acquiring the interference image at each optical path difference, and analyzing the plurality of interference images.

In addition, the optical system illustrated in this diagram is a Mach-Zehnder interferometer, but may be an optical system of a Michelson interferometer.

In the configuration examples described above, when the imaging apparatus 100A, 100B, or 100C is used as the first imaging apparatus <NUM>, the imaging element <NUM> may be a linear array sensor in which pixels are arrayed one-dimensionally, or may be a two-dimensional array sensor in which pixels are arrayed two-dimensionally. In the latter case, the cross sectional shape of the light output from the irradiation optical system <NUM> is not a line shape, but a circular shape, quadrangular shape, or the like.

When the imaging apparatus 100A, 100B or 100C is used as the second imaging apparatus <NUM>, the imaging element <NUM> may be a linear array sensor in which pixels are arrayed one-dimensionally, or may be a two-dimensional array sensor in which pixels are arrayed two-dimensionally. In the former case, the cross sectional shape of the light output from the irradiation optical system <NUM> is preferably a line shape.

Further, the control device <NUM> may perform digital refocusing processing (see Non Patent Documents <NUM> to <NUM>) on the image of the cell acquired by the first imaging apparatus <NUM>, determine whether to perform imaging by the second imaging apparatus <NUM> based on the image in focus, and provide the camera trigger signal to the second imaging apparatus <NUM> when it is determined to perform imaging by the second imaging apparatus <NUM>.

As a method of autofocus (AF), contrast AF, phase difference AF, and image plane phase difference AF are well known (Non Patent Document <NUM>). In these, the contrast AF is not suitable for use in the present embodiment because it includes mechanical driving to move the lens back and forth. On the other hand, the phase difference AF and the image plane phase difference AF do not require mechanical driving of the lens, and the deviation amount ΔZ and its direction (positive or negative of ΔZ) can be known, and therefore can be suitably used in the present embodiment.

In the configuration illustrated in <FIG>, the deviation amount ΔZ can be obtained by the phase difference AF. <FIG> is a diagram illustrating a configuration of the imaging apparatus 100D. The imaging apparatus 100D is different from the imaging apparatus 100A of the configuration having the trans-illumination optical system illustrated in <FIG> in that the imaging optical system <NUM> includes a separator lens <NUM> as the imaging lens.

In this configuration, the above-described digital refocusing processing cannot be performed. Therefore, it cannot be determined whether to perform imaging by the second imaging apparatus <NUM> based on the image of the cell acquired by the first imaging apparatus <NUM>, and the camera trigger signal cannot be provided to the second imaging apparatus <NUM>.

In the present embodiment, by relaxing restriction of the flow speed of the cell due to the focusing speed, it is possible to acquire a clear (small blur) image of the cell even when the cell is moving at high speed. Therefore, it is possible to improve the throughput of the inspection of the cell flowing in the flow path. Even when the fluid flow in the flow path is not a complete laminar flow and the cell moves spatially randomly, a clear (small blur) image of the cell can be acquired.

A line sensor can be used as the imaging element in both or one of the first imaging apparatus and the second imaging apparatus, and in this case, the system can be configured at low cost compared to the case where a two-dimensional sensor is used as the imaging element in both of the first imaging apparatus and the second imaging apparatus.

The cell observation system and the cell observation method are not limited to the embodiments and configuration examples described above, and various other modifications are possible.

The cell observation system of the above embodiment is a cell observation system for observing a cell moving in a flow path with a fluid, and includes (<NUM>) a first imaging apparatus including a first optical system and a first imaging element, and for capturing an image of the cell by receiving, by the first imaging element, light reaching the first imaging element from the cell at a first position in a moving direction of the cell in the flow path through the first optical system; (<NUM>) a second imaging apparatus including a second optical system, in which a focus is adjusted based on a focus adjustment signal, and a second imaging element, and for capturing an image of the cell by receiving, by the second imaging element, light reaching the second imaging element from the cell at a second position downstream of the first position in the moving direction of the cell in the flow path through the second optical system; and (<NUM>) a control device for obtaining a passing position of the cell in a cross section of the flow path based on the image obtained by imaging by the first imaging element of the first imaging apparatus, and generating the focus adjustment signal based on the obtained passing position to provide the signal to the second optical system of the second imaging apparatus.

The cell observation method of the above embodiment is a cell observation method for observing a cell moving in a flow path with a fluid, and includes (<NUM>) a first imaging step of, using a first imaging apparatus including a first optical system and a first imaging element, capturing an image of the cell by receiving, by the first imaging element, light reaching the first imaging element from the cell at a first position in a moving direction of the cell in the flow path through the first optical system; (<NUM>) a second imaging step of, using a second imaging apparatus including a second optical system, in which a focus is adjusted based on a focus adjustment signal, and a second imaging element, capturing an image of the cell by receiving, by the second imaging element, light reaching the second imaging element from the cell at a second position downstream of the first position in the moving direction of the cell in the flow path through the second optical system; and (<NUM>) a focus adjustment instruction step of, after the first imaging step and before the second imaging step, obtaining a passing position of the cell in a cross section of the flow path based on the image obtained by imaging by the first imaging element of the first imaging apparatus, and generating the focus adjustment signal based on the obtained passing position to provide the signal to the second optical system of the second imaging apparatus.

In the above cell observation system, the control device may determine whether to adjust the focus of the second optical system based on the passing position, and may provide the focus adjustment signal to the second imaging apparatus when it is determined to adjust the focus of the second optical system. Further, in the above cell observation method, in the focus adjustment instruction step, whether to adjust the focus of the second optical system may be determined based on the passing position, and the focus adjustment signal may be provided to the second imaging apparatus when it is determined to adjust the focus of the second optical system.

In the above cell observation system and method, the first imaging apparatus may be a quantitative phase microscope. Further, the second imaging apparatus may be a quantitative phase microscope or a phase tomographic microscope. Further, the first imaging apparatus may be a quantitative phase microscope, and the second imaging apparatus may be a phase tomographic microscope.

In the above cell observation system, the control device may obtain the passing position based on the image by a phase difference autofocus technique, an image plane phase difference focus technique, or an autofocus technique using digital holography. Further, in the above cell observation method, in the focus adjustment instruction step, the passing position may be obtained based on the image by a phase difference autofocus technique, an image plane phase difference focus technique, or an autofocus technique using digital holography.

In the above cell observation system and method, the fluid may be caused to flow as a laminar flow in the flow path by using a hydrodynamic focusing effect.

The embodiments can be used as a cell observation system and a cell observation method capable of relaxing restriction of a flow speed of a cell due to a focusing speed.

Claim 1:
A cell observation system (<NUM>) for observing a cell (<NUM>) moving in a flow path (<NUM>) with a fluid (<NUM>), the system (<NUM>) comprising:
a first imaging apparatus (<NUM>) including a first optical system (<NUM>) and a first imaging element (<NUM>), the first imaging apparatus (<NUM>) arranged to capture an image of the cell (<NUM>) by receiving, by the first imaging element (<NUM>), light reaching the first imaging element (<NUM>) from the cell (<NUM>) at a first position in a moving direction of the cell (<NUM>) in the flow path (<NUM>) through the first optical system (<NUM>);
a second imaging apparatus (<NUM>) including a second optical system (<NUM>), in which a focus is adjusted based on a focus adjustment signal, and a second imaging element (<NUM>), the second imaging apparatus (<NUM>) arranged to capture an image of the cell (<NUM>) by receiving, by the second imaging element (<NUM>), light reaching the second imaging element (<NUM>) from the cell at a second position downstream of the first position in the moving direction of the cell (<NUM>) in the flow path (<NUM>) through the second optical system (<NUM>); and
a control device (<NUM>) arranged to input the image obtained by imaging by the first imaging element (<NUM>) of the first imaging apparatus (<NUM>), analyze the image to obtain a passing position of the cell (<NUM>) in a cross section of the flow path (<NUM>), and generate the focus adjustment signal based on the obtained passing position to provide the focus adjustment signal to the second optical system (<NUM>) of the second imaging apparatus (<NUM>), wherein
the cell (<NUM>) is focused on a region near a center in the cross section of the flow path (<NUM>) in advance upstream of the first position and the second position for restricting a focus deviation amount of the passing position of the cell.