JP3117751B2 - Sample flat flow forming device for particle analysis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relating to a sample (sample solution) containing the particle component such as blood or urine, to a sample flat flow forming apparatus for flow in the thin flat flow width is wide thickness. The apparatus of the present invention is suitably used in a particle image analyzer that irradiates a flat flow of a sample with strobe light and captures a still image of particle components.

[0002]

2. Description of the Related Art Japanese Patent Application Laid-Open No. 57-500995 or U.S. Pat.
No. 338,024. The flow cell has a flow channel with a large aspect ratio (tens of times) in the measurement area, forms a flat (flat) sheath flow of the sample in the flow channel, and a still image of the sample flat flow with a video camera. Is imaged. The flow path dimensions in the imaging region are described as 100 μm in width and 5000 μm in height (the aspect ratio is 50 times). In addition, sheath flow (s
The term “heat flow” refers to a flow in which the periphery of a suspension of particles is covered with a laminar sheath liquid in order to accurately pass the particles in a line in the center of the liquid flow.

On the other hand, Japanese Utility Model Laid-Open No. 3-44626 discloses that
For measuring the cleanliness of the clean room used in semiconductor manufacturing, etc., the tip is flat, the nozzle divided into a plurality of nozzle holes along the flat direction, and the discharge hole itself is flat. A nozzle is disclosed. Certainly, these nozzles are for flattening the sample flow, but do not use the sheath flow, but merely for flowing a large amount of sample fluid. With these nozzles alone,
Sufficient flatness required in the present invention (10 μm × 9
(About 00 μm) cannot be realized.

[0004]

SUMMARY OF THE INVENTION The above-mentioned Japanese Patent Application Laid-Open (JP-A) No. 57-5 is disclosed.
In the conventional flow cells described in, for example, Japanese Patent No. 009995 and US Pat. No. 4,338,024, the thickness of the flow channel is about the size of the particles to be analyzed, and precision is required also in the dimensions. For this reason, manufacturing is difficult,
There was a problem that it was expensive. In addition, there is also a problem that it is easily broken due to its small thickness and is difficult to handle. In a normal flow cytometer, a flow cell having an aspect ratio of the flow channel of approximately 1 is used. If such a flow cell is used, the above problem does not occur, but a sample flat flow cannot be formed as it is. Flow Cytometry Handbook Science Forum (1984): 39
Pages 9-400 describe the forces acting on the sample stream. FIG. 15 is a drawing (plan view) of the flow cell portion viewed from the flow direction, which is cited from the document.
Comparing the h and v directions, the force fh in the h direction with a large aperture ratio acts more than the force fv in the v direction with a small aperture ratio. However, this is used to align the orientation of cells in the sample in a certain direction, and is insufficient for forming a flat flow of the sample. If the forces acting on the sample flow are fh and fv, fh: fv = A /
a: B / b, where A / a> B / b. The present invention has been made in view of the above points, and has as its object to provide an apparatus capable of forming a flat flow of a sample while using a flow cell having an aspect ratio of a flow channel of 1 to several times.

[0005]

In order to achieve the above-mentioned object, in the flow channel for introduction of the flow cell, only one width of the flow channel is reduced to be connected to the flow channel for measurement. Then, the discharge ports of the sample nozzle are made flat or small discharge ports are arranged in a horizontal line. Furthermore, the direction in which the width of the introduction channel decreases and the direction in which the width of the sample nozzle orifice is narrow, that is, the direction in which the diameter of the discharge port is short when the discharge port is flat, and the direction in which the width of the discharge port is one line in the vertical direction. (The direction orthogonal to the array direction).

[0006] Sample flat flow forming apparatus for analyzing particles according to claim 1 of the present invention, flow for introduction flow path is narrowed gradually path
When a narrow measuring flow path communicating with the introduction channel, the introduction flow path
To the sheath liquid supply port provided a flow cell having an outlet disposed downstream of the measuring flow path, the introduction flow path of the flow cell, arranged so that the tip is directed to the measuring flow path It has been, and Sanpurunozu Le for the sample discharge, the sample flow forming apparatus for particle analysis consisting, flow cell
The cross section of the measurement flow path is a rectangle having a side ratio of 1 to several times, the cross section of the introduction flow path is rectangular, and the length of the introduction flow path is
The axial direction should be perpendicular to the long axis direction of the measurement channel.
And only the width in the long axis direction of the introduction channel is substantially
Converging to introducing passage is connected to the measuring flow path, the discharge port of the sample Roh <br/>'s Le destination end is opened into a flat shape, the discharge
As long axis direction of the mouth becomes long axis and the same direction of the measurement flow path, it is characterized in that Sanpurunozu Le is located.

[0007] In this case, the discharge port of the sub Npurunozu Le, it is desirable to configure such towards the width of the end portion component has a wider shape than the width of the central portion. Further, in place of the sample discharge nozzles shape is flat, with Sanpurunozu Le the small discharge port provided plurality in a horizontal row, small discharge openings
The arrangement direction long axis and the same as such so that the measuring flow path of, occasionally comprises Sanpurunozu Le. The as an example of the case, the sample inlet of Sa Npurunozuru other end is one, is branched into a plurality of small passages within the sample nozzle, and each small discharge ports, as arranged in a row. the above
In the case of, the number of small discharge ports of the sample nozzle is an even number,
It is desirable to provide these small discharge ports at symmetrical positions at the center of the sample nozzle. Further, it is desirable that the diameter of the small discharge port disposed at the end portion is larger than the diameter of the small discharge port disposed at the center portion.

[0008]

[Function] Long-axis direction of introduction channel and long-axis direction of measurement channel
And the long axis of the introduction channel
Only the width in the direction is substantially converged, and the introduction flow path is
And the outlet at the tip of the sample nozzle is flat.
It is open, and the long axis direction of this discharge port is the length of the measurement channel.
Position the sample nozzle so that it is in the same direction as the axial direction
Flow cell with an aspect ratio of the flow channel of 1 to several times
While using a slab, a sufficient flatness (about 10 μm × 900 μm)
Degree) can form a sample flat flow
You. Oite introduction flow path, since narrowing the only one width, sheath fluid for that direction only, a large force acts inward, in the direction does not change the width force does not act. That is, the sample narrowing action occurs only in one direction. Outlet of Sanpurunozu Le, not as in the prior art circular shape to a flat circle, that is, substantially elliptical. Therefore, Nozzle or al discharged sample liquid, by a synergistic action of both (one direction only sample narrowing effect and flat flow ejected from the nozzle) of an aspect ratio of flow paths for measurement several times 1 Even so, a very flat sample flow can be formed. Similarly, in the case of a plurality of small discharge openings either et discharged sample solution Sanpurunozu Le, it is possible to form an extremely flat sample flow.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below in detail with reference to the drawings. However, the dimensions, materials, shapes, relative positions and the like of the components described in this embodiment are as follows:
Unless there is a specific description, the scope of the present invention is not intended to limit the scope of the invention only to them, but is merely an illustrative example. 1 to 3 show a sample flat flow forming apparatus of the present invention. This apparatus includes a flow cell 10 for forming a sheath flow, and a sample nozzle 12 which is a thin tube for discharging a sample. The flow cell 10 is made of a transparent material such as glass or plastic, and has an introduction flow path 14 in which only the width in one direction is gradually narrowed, and a narrow measurement flow path 16 connected to the introduction flow path 14.
And a sheath liquid supply port 18 provided in the introduction channel 14.
And an outlet 20 provided downstream of the measurement channel 16. The cross section of the measurement channel 16 has a side ratio of 1 to several times, specifically 1 to 10 times, preferably 3 to 10 times.
It is a five-fold rectangle. If the side ratio exceeds 20 times, it approaches the conventional flow cell, making it difficult to manufacture.
And it is easy to be damaged. In this device, the sheath liquid for guiding the sample to the measurement region c while wrapping the sample from the surroundings is
When the sample is poured into the sheath liquid by the nozzle 12 at the same time as flowing into the flow cell 10, the thickness of the sample flow is set to a constant value (about the thickness of the particles to be measured, for example, when measuring red blood cells in blood). Is about 10 μm).

The width a1 of one of the introduction channels 14 (see FIGS. 1 and 3) is constant, for example, 1 mm, so that the width of the sheath liquid flow near the tip of the nozzle 12 does not change. In the direction of the width a1, the narrowing action does not work on the sample flow. On the other hand, the other width b1 of the introduction channel 14 (see FIGS. 2 and 3)
Unlike the case described above, the width gradually decreases from the width b1 to the measurement flow path 16 to become the width b2 (see FIGS. 2 and 3), and the narrowing action of the sample flow works. I have. The width b1 is, for example, 10 mm, and the width b2 is, for example, 0.5 mm. By arranging a conventional circular nozzle in the flow cell 10, the sample flow can be formed in a plate shape, that is, a wide sample flow having a thickness of about 10 μm. However, this method alone cannot produce a sample flow having a sufficient width in the measurement area of the imaging flow cytometer.

Therefore, by using a sample nozzle as shown in FIGS. 4 to 14, the thickness of the sample flow flowing through the measurement flow path 16 of the flow cell 10 can be made even thinner. Hereinafter, this will be described in detail. The imaging area is basically determined by the magnification of the objective lens (not shown) and the size of the imaging device of the video camera (not shown). For example, if the magnification of the objective lens is 1
When the magnification of the CCD image sensor of the video camera is 2/3 inch, the size of the light receiving surface of the CCD device is 8.8 × 6.6 mm.
Since the imaging area is 0.22 × 0.165 mm when the magnification of the objective lens system is 0.66 mm and the magnification of the objective lens system is 40 times, even when the magnification of the objective lens system is 10 times, the sample flow is 0.1 mm. It is only necessary to have a width of about 9 mm. Here, in the sheath flow measurement method, the cross-sectional area of the sample flow flowing in the flow cell is determined by the flow rate of the sample flow and the flow rate of the sheath liquid. For example, a circular conventional nozzle having only one sample outlet is used as the nozzle, and the discharge amount of the sample per unit time is 2.6 μm.
Assuming that the flow rate of the sheath liquid is l / sec and the flow rate of the sheath liquid is 500 μl / sec, the area ratio of the sample flow to the sheath liquid in the cross section of the measurement channel 16 of the flow cell is 1: 187. For this reason, as shown in FIGS.
Is 1 mm × 0.5 mm, the area occupied by the sample flow is 1/187 of 0.5 mm ² , that is, 2.7.
× 10 ⁻³ mm ² .

Now, suppose that the sample flow is halved only in one direction.
Suppose that it was possible to narrow down to zero. The value 1/20 is a value determined by the shape of the flow cell. When the diameter of the sample flow immediately after being discharged from the conventional circular opening nozzle is 0.2 mm, the thickness of the sample flow in the measurement region c is reduced to 1/20, that is, 10 μm. From this result and the result of the area occupied by the sample flow in the measurement flow channel 16 described above, the size of the region occupied by the sample, 0.01 mm × 0.27 mm, is obtained. It can be seen that the diameter of the flat flow is 0.27 mm, and only about 1/3 of the target imaging area width of 0.9 mm can be achieved. To solve this, (a) triple the sample ejection amount. (B) The opening area of the sample discharge port is tripled (the flow rate does not change). The idea of is considered. However, the case (a) has the following problem. That is, the area occupied by the sample flow in the measurement channel 16 is three times the original area. On the other hand, the diameter of the sample flow immediately after the ejection from the nozzle is √3 times the original diameter (three times the area). Therefore, even if the diameter is reduced to 1/20, the thickness of the sample flow becomes √3 times the original thickness. For this reason, the width of the sample flow becomes 3 / √3 = √3 times, and eventually the thickness and the width of the sample flow also become √3 times, and it is necessary to triple the width only with the original thickness. Can not. (B)
In the case of the plan, there are the following problems. That is, the area occupied by the sample flow in the measurement channel 16 remains unchanged. Since the opening area of the nozzle discharge port is three times (√3 times in diameter), the diameter of the sample flow immediately after the discharge is √3 times.
Therefore, the thickness of the sample flow is also √3 times, and the width of the sample flow is conversely 1 / 逆 3 times. In the plan (a), the sheath liquid flow rate may be further increased by three times. However, in this case, the following problem occurs. That is, the area occupied by the sample flow is reduced by a factor of 1, the thickness of the sample flow is reduced by a factor of 1, and the width of the sample flow is increased by a factor of 1. In addition, various combinations in which the sample discharge amount, the sample discharge opening, and the sheath liquid flow rate are changed are conceivable, but in any case, the desired flatness cannot be obtained.

In order to solve such a problem, the present invention provides a substantially elliptical sample discharge port as shown in FIGS. 4 to 9 or a small diameter discharge port as shown in FIGS. Are arranged in a line, and the flatness of the sample flow is further improved by using a sample nozzle having a porous sample discharge port. As described above, the draw-down magnification of the flow path was 1/20, the discharge port diameter at the tip of the nozzle was 0.2 mm, the flow rate of the sheath liquid per unit time was 500 μl / sec, and the sample flow rate was 2.6 μl / sec. In this case, the cross section of the sample flow in the measurement area is 10 μm × 27
0 μm. In order to flow the sample over the entire imaging area, the sample discharge amount per unit time is increased by about 3.3 times to 8.6 μm.
It is necessary to discharge l / sec or more. In the sample nozzle 12 shown in FIG. 4 to FIG.
The width d of the discharge port 22 is 3.3 times as much as 0.66 mm as an example, while the length d in the thickness direction (see FIG. 8) is kept at 0.2 mm as an example. In FIGS. 4 to 8, as an example, the substantially elliptical discharge port 22 is manufactured by providing the tapered portion 24 by a certain length from the tip of the nozzle 12, but instead of the tapered portion. There is no problem even if the stepped portion is provided. 26 is a sample inlet. FIG.
As shown in FIG. 9, if the center of the substantially elliptical discharge port 22 and the sample inlet 26 exist in the same straight line, it is difficult for the flow to branch evenly, so that the shape of the discharge port is as shown in FIG. In addition, it is desirable that the width of the end portion 28 is slightly larger than the width of the central portion.

FIGS. 10 to 14 show a sample nozzle 12a.
2 shows another example. In the sample nozzle 12a of this example, a flow rate per unit time is 3.3 by arranging several 0.2 mm holes in a line at the nozzle tip, for example.
10 μm × 270 per hole
Since a sample flow of μm is created, three or more 0.2 mm holes should be formed at the tip of the nozzle at a pitch of 0.27 mm at the maximum. However, as shown in FIG. 11, when a large number of holes are arranged in a comb shape, if the discharge port exists at the branch of the flow path in the same straight line as the original sample inlet 26, the flow becomes even. Since it is difficult to branch, it is desirable to open four or six small discharge ports 30 symmetrically with respect to the original sample inlet 26. Further, in order to equalize the flow rate from each of the small discharge ports 30, the center hole can be made small (for example, 0.15 mm) and the outside hole can be made large (for example, 0.25 mm). However, this method differs depending on the number of holes formed at the nozzle tip, and the hole diameter is not always this size. In FIG. 11, the sample nozzle 12a is composed of, for example, a main body member 32 and a tip member 34. One sample inflow port 26 is provided in the main body member 32, and six small flow paths 36 are provided in the tip member 34 in a horizontal row as an example.
The main body member 32 and the distal end member 34 are joined and integrated so that their flow paths communicate with each other. In order to provide a plurality of small discharge ports at the tip of the sample nozzle, in addition to the above configuration, a plurality of small pipes may be inserted into the substantially elliptical discharge port 22 shown in FIG. It is also possible to adopt a configuration in which a plurality of partition plates are provided in the device. Further, the shape of the small discharge port may be other shapes such as a quadrangle and a polygon in addition to a circle.

[0015]

As described above, the present invention has the following effects. (1) The major axis direction of the introduction channel and the major axis direction of the measurement channel
And the long axis of the introduction channel
Only the width in the direction is substantially converged, and the introduction flow path is
And the outlet at the tip of the sample nozzle is flat.
It is open, and the long axis direction of this discharge port is the length of the measurement channel.
Position the sample nozzle so that it is in the same direction as the axial direction
Flow cell with an aspect ratio of the flow channel of 1 to several times
While using a slab, a sufficient flatness (about 10 μm × 900 μm)
Degree) can form a sample flat flow
You. ( 2 ) In addition to narrowing the introduction channel only in one direction,
Since the discharge port at the tip of the sample nozzle has a flat elliptical shape or a porous shape, even if it is not a conventional flat measurement flow channel, the aspect ratio is 1 to several times a rectangular or substantially circular measurement flow channel. In addition, a flat sample flow can be easily formed. ( 3 ) Since the flow path of the flow cell can be made nearly square or circular, the production of the flow cell becomes easy and the strength can be improved. For this reason, manufacturing costs can be reduced and breakage is reduced.

[Brief description of the drawings]

FIG. 1 is a front sectional view showing one embodiment of a sample flat flow forming apparatus for particle analysis of the present invention.

FIG. 2 is a right side sectional view of the apparatus shown in FIG.

FIG. 3 is a plan view of the device shown in FIG. 1;

FIG. 4 is a perspective view showing an example of a sample nozzle used in the apparatus of the present invention.

FIG. 5 is a cross-sectional view showing a state in which the nozzle shown in FIG. 4 is cut along a longitudinal direction of a substantially elliptical discharge port.

FIG. 6 is a front view showing a state where the nozzle shown in FIG. 5 is rotated by 90 degrees.

FIG. 7 is a right side view of the nozzle shown in FIG.

FIG. 8 is a left side view of the nozzle shown in FIG.

FIG. 9 is an explanatory view showing another example of the discharge port of the nozzle shown in FIG. 8;

FIG. 10 is a perspective view showing another example of the sample nozzle used in the apparatus of the present invention.

11 is a cross-sectional view showing a state where the nozzle shown in FIG. 10 is cut along a direction in which a plurality of small discharge ports are arranged.

12 is a partially cutaway front view showing a state where the nozzle shown in FIG. 11 is rotated by 90 degrees.

FIG. 13 is a right side view of the nozzle shown in FIG. 11;

FIG. 14 is a left side view of the nozzle shown in FIG.

FIG. 15 is an explanatory plan view showing a force acting on a sample flow in a conventional flow cell.

[Explanation of symbols]

 Reference Signs List 10 flow cell 12 sample nozzle 12a sample nozzle 14 introduction flow path 16 measurement flow path 18 sheath liquid supply port 20 discharge port 22 discharge port 26 sample inlet 28 end portion 30 small discharge port 36 small flow path

Claims (6)

(57) [Claims] 1. An introduction flow path having a flow path gradually narrowed,
Narrow measuring flow path communicating with the introduction channel, flow cell, comprising: a set <br/> vignetting sheath fluid supply port to the introduction flow path, and a discharge port provided downstream of the measuring flow path If, on the introduction flow path of the flow cell, the tip is arranged to face the measuring flow path, and Sanpurunozu Le for the sample discharge, the sample flow forming apparatus for particle analysis consisting, for measurement of the flow cell cross section of the flow path, the ratio of the sides is rectangular several times 1, the cross section of introduction flow passage is rectangular, with the long axis direction of the introduction passage and the long axis direction of the measuring flow path Is a right angle
The width of the introduction channel in the long axis direction
Only substantially converges and the introduction channel is connected to the measurement channel
And, the discharge port of Sanpurunozu Le destination end is opened into a flat shape, as long axis direction of the ejection port becomes the longitudinal direction and the same person <br/> direction of the measuring channel, Sanpurunozu A flat flow forming device for particle analysis, characterized in that a sample is disposed. Wherein Sanpurunozu Le of the discharge port is a sample flat flow for particle analysis according to claim 1, wherein the direction of the width the width of the end component than in the central portion has a wide shape Forming equipment. In place of the sample nozzle is wherein the discharge port shape flat, with Sanpurunozu Le the small discharge port provided plurality in a row, the small nozzle array direction measuring flow path of
The same as such so that the long axis direction of the sample flat flow forming apparatus for analyzing particles according to claim 1, characterized in that a Sanpurunozu Le. Sample inlet 4. A sample nozzle and the other end is <br/> one, is branched into a plurality of small passages within the sample nozzle, characterized in that arranged the small discharge port in a row The sample flat flow forming device for particle analysis according to claim 3. 5. The sample flat for particle analysis according to claim 4, wherein the number of small discharge ports of the sample nozzle is even, and these small discharge ports are provided at symmetrical positions at the center of the sample nozzle. Flow forming device. 6. The flat sample for particle analysis according to claim 4, wherein the diameter of the small discharge port disposed at the end portion is larger than the diameter of the small discharge port disposed at the center portion. Flow forming device.