Method for counting fine particles using a multi-channel sample chip and a fine particle counter that realizes this method

Abstract

A method for counting fine particles using a multi-channel sample chip and a fine particle counting device implementing the method are disclosed. According to the method for counting fine particles using a multi-channel sample chip and a fine particle counting device implementing the method, a large number of samples can be observed in a short time, and even if warping occurs during the manufacturing process due to the large size of the sample chip, this can be corrected to enable accurate counting of fine particles. In addition, the problem that the focal length for each channel is not constant due to warping that occurs in a chip having multiple channels, and therefore it takes a long time to obtain the focal value for each channel and acquire an image, can be solved. Furthermore, since the amount of sample injected into the channel is small, it is possible to prevent the sample from drying out and the resulting measurement error from occurring if the image is not quickly acquired and counted.

Description

[Technical Field]

[0001] The present invention relates to a method for counting fine particles using a multi-channel sample chip, and a fine particle counting device that realizes this method.More specifically, the present invention relates to a method for counting fine particles using a multi-channel sample chip that can observe a large number of samples in a short period of time, and that can correct warping that occurs during the manufacturing process due to the large size of the sample chip, thereby enabling accurate counting of fine particles, and a fine particle counting device that realizes this method. [Background technology]

[0002] In order to diagnose patients with diseases such as AIDS, leukemia, and anemia, monitor their progression, and understand the effectiveness of treatment, it is necessary to count the number of white blood cells or red blood cells related to the disease in the patient's blood and understand their distribution.

[0003] In particular, blood tests are being increasingly used to diagnose these diseases as well as to monitor patients who have been determined to have said diseases.

[0004] Previously, most hospitals had clinical pathology technicians manually count white blood cells or red blood cells in the blood, but this manual counting resulted in many errors in the test results and took a long time to complete.

[0005] In order to solve this problem, the applicant developed a fine particle counting device that can automatically and quickly count fine particles such as cells, and applied for and registered it as Korean Patent No. 10-0608498.

[0006] In recent years, the application of such fine particle counting devices has expanded beyond being limited to the diagnosis and treatment of human diseases to include testing raw milk extracted from cows, and as a result, there is a growing need for devices that can quickly and accurately test and count large quantities of a wide variety of samples.

[0007] However, in the case of a sample chip with multiple channels manufactured to observe a large number of samples in a short period of time, its size becomes large, and therefore it is not possible to keep it flat overall during the manufacturing process, resulting in warping.

[0008] As a result of the warping that occurs in a chip having such a large number of channels, the height of each channel is not uniform, which causes the problem that it takes a long time to obtain the focusing value for each channel and acquire an image.

[0009] Furthermore, since the amount of sample injected into each channel is small, about 10 μl, there is a problem in that the sample will dry out if the image is not captured and counted quickly, resulting in measurement errors.

[0010] Therefore, there is an emerging need for a fine particle counting method using a multi-channel sample chip that can solve these problems, allowing a large number of samples to be observed in a short period of time, and that can correct for warping that occurs during the manufacturing process due to the larger sample chip, thereby enabling accurate counting of fine particles, as well as a fine particle counting device that realizes this method. Summary of the Invention [Problem to be solved by the invention]

[0011] An embodiment of the present invention is capable of observing a large number of samples in a short period of time, and even if warping occurs during the manufacturing process due to the increased size of the sample chip, this can be corrected to enable accurate counting of fine particles.

[0012] In addition, embodiments of the present invention are intended to solve the problem that the focal length for each channel is not constant due to warping that occurs in chips with multiple channels, thereby taking a long time to obtain the focal value for each channel and acquire an image.

[0013] Furthermore, embodiments of the present invention are intended to prevent the problem that, because the amount of sample injected into the channel is small, if the image is not captured and counted quickly, the sample will dry out, resulting in measurement errors. [Means for solving the problem]

[0014] According to one aspect of the present invention, a method for counting microparticles in a sample contained in each channel on a sample chip having a plurality of channels and focusing marks includes the steps of: calculating a difference between a focus value for a specific focus mark among the plurality of focusing marks and a focus value of a channel adjacent to the specific focus mark to obtain a warp correction value; measuring each focus value for the plurality of focusing marks; and, while moving sequentially between each of the channels, For each channel Through the focus value obtained by substituting the warp correction value into the focus value of the adjacent focus mark For each channel A method for counting fine particles using a multi-channel sample chip is provided, which includes the steps of acquiring an image and counting fine particles in a sample contained in each channel via the acquired image.

[0015] The number of focus marks may be less than the number of channels.

[0016] The focal mark may have a shape in which a large number of protrusions are clustered together.

[0017] The focus marks may be in the form of a grid.

[0018] The grid pattern of the grid-shaped focus marks may be used to determine the distance per unit pixel, and the distance may be substituted into the image resolution to determine an area, which may then be applied to the area of ​​the adjacent channel image.

[0019] The step of determining the warp correction value may be configured to determine a focus value for the particular focus mark by automatic focus adjustment, and to determine a focus value for a channel adjacent to the particular focus mark by manual focus adjustment or automatic focus adjustment.

[0020] The step of measuring each focus value for the plurality of focus marks may be configured to determine the focus values ​​by automatic focus adjustment.

[0021] According to another aspect of the present invention, a sample chip having a plurality of channels for accommodating a sample containing fine particles, a stage on which the sample chip is placed, a light source unit for irradiating light onto the channels on the sample chip placed on the stage, an objective lens unit for enlarging an image of the sample accommodated in the channel through which the light has passed, an image sensor unit for acquiring an image related to the image enlarged by the objective lens unit, a fine particle counting unit for counting fine particles in the sample accommodated in each channel through the acquired image, and a stage moving unit for moving the stage, wherein the sample chip further includes a plurality of focus marks for determining focusing information for correcting a warpage height deviation of each channel. Each of the channels and the focal mark are adjacent to each other. A microparticle counting device using a multi-channel sample chip is provided.

[0022] Correcting the warp height deviation of each channel may be performed by calculating the difference between the focus value for a specific focus mark among the multiple focus marks and the focus value of a channel adjacent to the specific focus mark, and substituting the obtained warp correction value for the focus value of each focus mark.

[0023] The number of focus marks may be less than the number of channels.

[0024] The focal mark may have a shape in which a large number of protrusions are clustered together.

[0025] The focus marks may be in the form of a grid.

[0026] According to another aspect of the present invention, there is provided a method for counting fine particles in a sample contained in each channel on a sample chip having a plurality of channels and focal marks, the method including the steps of measuring each focal value for the plurality of focal marks, calculating the difference between each focal value for a specific focal mark among the plurality of focal marks and the focal value of a channel adjacent to the specific focal mark to obtain a warp correction value, acquiring an image through the focus value obtained by substituting the warp correction value for the focus value of the adjacent focal mark while moving sequentially between each of the channels, and counting fine particles in the sample contained in each channel through the acquired image.

[0027] The focus values ​​of the plurality of focus marks may be configured to measure the focus values ​​of some of the focus marks among all the focus marks, and then use the measured focus values ​​to perform topology mapping of the degree of warpage of the entire sample chip to determine the focus values ​​of the remaining focus marks.

[0028] The grid-shaped focus marks may be engraved.

[0029] The step of determining the warpage correction value, the step of measuring the focus value for the focus mark, and the step of acquiring the image may be repeated for each group formed by dividing the sample chip.

[0030] Groups to which the same warp correction value is applied may be designated in advance. [Effects of the Invention]

[0031] An embodiment of the present invention may be capable of observing a large number of samples in a short period of time, and may also be capable of correcting warping that occurs during the manufacturing process due to the increased size of the sample chip, thereby enabling accurate counting of fine particles.

[0032] In addition, embodiments of the present invention can solve the problem that the focal length for each channel is not constant due to warping that occurs in chips with multiple channels, which makes it time-consuming to obtain the focal value for each channel and acquire an image.

[0033] In addition, the embodiment of the present invention can prevent the problem that the amount of sample injected into the channel is small, so if the image is not captured and counted quickly, the sample will dry out, resulting in measurement errors. [Brief explanation of the drawings]

[0034] [Figure 1] 1 is a plan view showing a sample chip of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 2] 1 is a plan view illustrating an example of a focal mark provided on a sample tip of a microparticle counter using a multi-channel sample tip according to an embodiment of the present invention; [Figure 3] 10 is an image showing an example of determining a warpage correction value based on the difference between the focus value of a specific focus mark and the focus value of an adjacent channel in a sample chip of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 4] This is a comparison image showing an image taken with a focus value in which a warp correction value is substituted for the focus value of an adjacent focus mark when observing the F01 channel of a sample chip of a microparticle counting device using a multi-channel sample chip according to one embodiment of the present invention, and an image taken with automatic focus adjustment. [Figure 5] This is a comparison image showing an image taken with a focus value in which a warp correction value is substituted for the focus value of an adjacent focus mark when observing the F08 channel of a sample chip of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention, and an image taken with automatic focus adjustment. [Figure 6]This is a constructed image showing an example of how to obtain an area by capturing an image of a certain area of ​​a grid chip with a lattice pattern composed of 200 μm intervals, then calculating the average distance in pixels to calculate the μm value per pixel, and substituting it for the total image resolution. [Figure 7] 7 is an image showing an example of finding an area according to image resolution using a focal mark near the A01 channel by applying the method for finding an area described in FIG. 6. [Figure 8] 7 is an image showing an example of finding an area according to image resolution using a focal mark near the F01 channel by applying the method for finding an area described in FIG. 6. [Figure 9] 1 is a plan view showing an example of a focal mark of a sample chip of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention, the focal mark being made of a grid. [Figure 10] 1 is a plan view illustrating an example of a procedure for acquiring channel images of a multi-channel sample chip of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 11] 1 is a partially enlarged perspective view of a microparticle counting device using a multi-channel sample chip according to an embodiment of the present invention, viewed from one side. [Figure 12] 1 is a perspective view of a microparticle counting device using a multi-channel sample chip according to an embodiment of the present invention, viewed from one side. [Figure 13] 1 is a perspective view of a fine particle counting device using a multi-channel sample chip according to an embodiment of the present invention, seen from another side. [Figure 14] FIG. 2 is a partially enlarged perspective view showing a Z-axis moving unit and a Z-axis driving unit of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 15] 1 is a partially enlarged perspective view showing the upper configuration of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 16] FIG. 2 is a partially enlarged perspective view showing the optical system of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 17]1 is a partially enlarged perspective view showing the lower structure of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 18] 1 is a conceptual diagram showing the principle of topology mapping using coordinate values ​​and focal values ​​of some focal marks in a sample chip of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 19] 10 is a table showing an example of focal values ​​of focal marks corresponding to each channel position determined by topology mapping in a sample chip of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention. [Figure 20] 10 is a plan view showing a sample chip of a fine particle counter using a multi-channel sample chip according to another embodiment of the present invention. FIG. [Figure 21] 21 is a layout diagram and an actual photographed image showing a focus mark applied to the lower plate of the sample chip of FIG. 20. [Figure 22] 1 is a plan view showing a case where the sample chips of a fine particle counter using a multi-channel sample chip according to an embodiment of the present invention are divided into groups. DETAILED DESCRIPTION OF THE INVENTION

[0035] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosure will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art. Like reference numerals refer to like elements throughout the specification.

[0036] FIG. 1 is a plan view showing a sample chip of a fine particle counter using a multi-channel sample chip according to one embodiment of the present invention; FIG. 2 is a plan view showing an example of a sample chip of a fine particle counter using a multi-channel sample chip according to one embodiment of the present invention, in which a focus mark is provided; FIG. 3 is an image showing an example of determining a warp correction value based on the difference between the focus value of a specific focus mark on the sample chip of a fine particle counter using a multi-channel sample chip according to one embodiment of the present invention and the focus value of an adjacent channel; FIG. 4 is a comparison image showing a comparison between an image taken at a focus value in which a warp correction value is substituted for the focus value of an adjacent focus mark when observing the F01 channel of the sample chip of a fine particle counter using a multi-channel sample chip according to one embodiment of the present invention, and an image taken by automatic focus adjustment; and FIG. 5 is a comparison image showing an image taken at a focus value in which a warp correction value is substituted for the focus value of an adjacent focus mark when observing the F08 channel of the sample chip of a fine particle counter using a multi-channel sample chip according to one embodiment of the present invention. 6 is a comparison image comparing an image taken by automatic focus adjustment with an image taken by automatic focus adjustment. FIG. 6 is a structural image showing an example of obtaining an image of a certain area of ​​a grid chip with a lattice pattern arranged at intervals of 200 μm, then calculating the average distance in pixels to calculate the μm value per pixel and substituting it for the total image resolution to obtain the area. FIG. 7 is an image showing an example of obtaining an area based on image resolution using a focal mark near the A01 channel by applying the area obtaining method described in FIG. 6. FIG. 8 is an image showing an example of obtaining an area based on image resolution using a focal mark near the F01 channel by applying the area obtaining method described in FIG. 6. FIG. 9 is a plan view structural diagram showing an example of the focal mark of a sample chip of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention consisting of a grid. FIG. 10 is a plan view structural diagram showing an example of the procedure for obtaining channel images of a multi-channel sample chip of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention.

[0037] 1 to 10, a method for counting fine particles using a multi-channel sample chip according to one embodiment of the present invention is a method for counting fine particles in a sample contained in each channel 110 on a sample chip 100 having a plurality of channels 110 and focal marks 120, and can include the steps of: calculating the difference between the focal value for a specific focal mark 120 among the plurality of focal marks 120 and the focal value of a channel 110 adjacent to the specific focal mark 120 to obtain a warp correction value; measuring the focal values ​​for each of the plurality of focal marks 120; acquiring an image through the focus value obtained by substituting the warp correction value for the focus value of the adjacent focal mark 120 while sequentially moving between each of the channels 110; and counting fine particles in the sample contained in each channel 110 through the acquired image.

[0038] 1, the sample chip 100 may be configured with multiple channels 110. In this embodiment, the number of channels 110 is 48, but this is not limited thereto and may be modified to various numbers as needed.

[0039] The sample chip 100 may be configured by joining an upper plate and a lower plate, and the channel 110 may be configured to form a space of a certain height between the upper plate and the lower plate. The height of the channel 110 is preferably set to 10 to 100 μm, so that fine particles contained in the sample can be observed in a stationary state rather than floating.

[0040] A sample inlet 112 for introducing a sample may be provided on one side of the channel 110, and a sample outlet 114 may be provided on the other side of the channel to allow air and excess sample inside the channel 110 to be discharged.

[0041] Meanwhile, the sample chip 100 may have a plurality of focal marks 120 for obtaining focusing information for correcting the warpage height deviation of each channel 110. As shown in FIG. 2, a plurality of the focal marks 120 may be provided at positions close to each channel 110.

[0042] The focal mark 120 may have a shape of a cluster of many protrusions and may have a circular shape as a whole, as shown in the upper image of Fig. 3. Alternatively, the focal mark 120 may have a grid shape, as shown in the middle image of Fig. 3. In addition, the focal mark 120 may be modified into various shapes.

[0043] In this embodiment, the focal mark 120 is located on the lower plate of the upper and lower plates. The focal mark 120 is used to check the degree of warpage of the sample chip 100. By pre-profiling focusing information for correcting the warpage height deviation of each channel 110 through the focal mark 120, a topology regarding the height deviation of the sample chip 100 can be created. In other words, the warpage information of the entire sample chip 100 is pre-profiled and stored.

[0044] Specifically, in one embodiment of the present invention, a method for counting fine particles using a multi-channel sample chip first calculates the difference between the focus value for a specific focus mark 120 among the multiple focus marks 120 and the focus value of the channel 110 adjacent to the specific focus mark 120 to obtain a warp correction value.

[0045] 3, the focus value of the specific focus mark 120 is 175 μm (images are taken assuming both the case where the focus mark is a protrusion shape and the case where the focus mark is a grid shape), and the focus value of the channel 110 adjacent to it is 240 μm, so the warpage correction value is calculated as the difference, 65 μm. The reference positions of the specific focus mark 120 and the adjacent channel 110 for determining this warpage correction value may be specified in advance.

[0046] In the step of determining the warp correction value, the focus value for the specific focus mark 120 is determined by automatic focus adjustment, and the focus value of the channel 110 adjacent to the specific focus mark 120 is determined by manual focus adjustment or automatic focus adjustment, but is not limited to this.

[0047] Next, a focus value is measured for each of the plurality of focus marks 120. At this time, the measurement of the focus value for each of the plurality of focus marks 120 may be performed by automatic focus adjustment.

[0048] Since the number of focus marks 120 is less than the number of channels 110, even if focus value measurement for the focus marks 120 is performed by autofocusing, it takes less time than if autofocusing were performed for all of the channels 110. As shown in Figure 2, it can be seen that there are 42 focus marks 120, which is less than the number of 48 channels 110. Furthermore, as shown in another embodiment in Figure 9, it can be seen that there are 36 focus marks, which is less than the number of 48 channels 110.

[0049] Ultimately, the focus value of the focus mark 120 is assumed to represent the focus value of the adjacent channel 110, and the number and position of the focus mark 120 can be modified as necessary.

[0050] This configuration allows for image acquisition and counting of fine particles for multiple channels 110 in a short time, thereby realizing a high-throughput fine particle counting method.

[0051] In this embodiment, after the warpage correction value is obtained, the focus value of each focus mark 120 is measured, but it is also possible to change the order of steps, i.e., after the focus value of each focus mark 120 is obtained, the warpage correction value can be obtained in that order.

[0052] Thereafter, the image is acquired for each channel 110 while moving sequentially between the channels 110. The order of moving between the channels 110 is, for example, as shown in Fig. 10, but is not limited thereto and can be modified in any way as long as the order allows efficient movement to each adjacent channel 110.

[0053] At this time, the focal length adjustment in each channel 110 can be performed by substituting the warp correction value into the focal value of the adjacent focal mark 120 previously obtained in the previous step, thereby enabling quick image acquisition.

[0054] 4 and 5 show examples of cases where images of the F01 and F08 channels 110 shown in Fig. 1 are acquired using focus values ​​obtained by substituting the warp correction value determined in Fig. 3 for the focus values ​​of each adjacent focus mark 120. That is, the image in the middle is an image obtained when the warp correction value is applied, and the image on the far right is an image acquired by autofocus for the same channel 110.

[0055] In the case of Figure 4, the focus value of the focus mark 120 when the warp correction value is substituted is 325 μm, and the focus value taken through autofocus is 315 μm, and it can be seen that there is only a very slight difference between the two focus values, and both images show similar clarity so that there is no difference in counting fine particles. Similarly, from Figure 5, it can be seen that the difference in focus value is only around 10 μm, and there is no difference in clarity either.

[0056] In this way, the fine particle counting method according to the present invention can obtain images with almost the same clarity as when all of the channels 110 are obtained by automatic focus adjustment, but by quickly adapting the focal length, images can be obtained quickly.

[0057] Then, the microparticles in the sample contained in each channel are counted through the acquired images, and ultimately, the concentration of the microparticles can be determined by calculating the number of counted microparticles and the volume of the observed sample.

[0058] When applying the grid-shaped focal mark 120, the volume for the acquired image can be calculated in the following manner.

[0059] First, as shown in Fig. 6, in the grid-shaped focal mark 120, each grid pattern can be configured at intervals of, for example, 200 µm. A certain area of ​​such a focal mark 120 is acquired as an image, and the pixel-unit distance of one grid in the acquired image is calculated, and then this is used to determine how many µm there are per pixel.

[0060] In Figure 6, 108 pixels are 200 μm, so we can see that the distance per unit pixel is 1.85 μm / pixel. By substituting this into the image resolution of 1280 x 1024, we can calculate the total area.

[0061] That is, the width is 1280 × 1.85 = 2368 μm, and the length is 1024 × 1.85 = 1894 μm. The area is then calculated as 2368 μm × 1894 μm = 4.484 mm 2 Multiplying this by the height of the designed channel 110 gives the volume of the observed image of the channel 110. Of course, such calculation of area and volume and counting of fine particles are performed automatically on the system.

[0062] The above-mentioned warping of the sample chip 100 causes the focal length to differ even for images with the same resolution, resulting in different areas due to different distances per unit pixel. However, by correcting the area value using the grid-shaped focal mark 120 as described above and applying this to the adjacent channel 110, errors in volume calculation can be minimized.

[0063] 7 and 8 show examples of calculating the corrected area when calculating the focus values ​​for each grid-shaped focus mark 120. For example, in FIG. 7, the distance per unit pixel calculated via the focus mark 120 adjacent to the A01 channel 110 was 1.8466 μm / pixel. When this was substituted for the resolution (1280 × 1024), the area was found to be 4.4695 mm 2 This area value is applied to the A01 channel 110 etc. adjacent to the focal mark 120 to calculate the volume and concentration.

[0064] FIG. 8 shows the area value obtained by the same correction through the focus mark 120 adjacent to the F01 channel 110. The distance per pixel is 1.8472 μm / pixel, and the area value is 4.4722 mm. 2 Again, this area value is applied to the F01 channel 110 etc. adjacent to the focal mark 120 to calculate the volume and concentration.

[0065] When the grid-shaped focus mark 120 is used in this way, it is possible to quickly capture an image by applying the focus value, and it is also advantageous in that it is possible to correct even area errors.

[0066] In conclusion, the above-described counting method enables rapid image acquisition for the multi-channel sample chip 100, counting of fine particles, and accurate volume calculation for concentration calculation. Below, a detailed description will be given of a fine particle counting device that realizes the above-described fine particle counting method.

[0067] Figure 11 is a partially enlarged oblique view of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention, viewed from one side; Figure 12 is a partially enlarged oblique view of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention, viewed from one side; Figure 13 is a partially enlarged oblique view of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention, viewed from another side; Figure 14 is a partially enlarged oblique view showing the Z-axis moving section and Z-axis driving section of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention; Figure 15 is a partially enlarged oblique view showing the upper structure of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention; Figure 16 is a partially enlarged oblique view showing the optical system of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention; and Figure 17 is a partially enlarged oblique view showing the lower structure of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention.

[0068] 1 to 17, a fine particle counting device 200 using a multi-channel sample chip according to one embodiment of the present invention may broadly comprise a sample chip 100 having a plurality of channels 110 for accommodating a sample containing fine particles, a stage 210 on which the sample chip 100 is placed, a light source unit 220 for irradiating light onto the channels on the sample chip 100 placed on the stage 210, an objective lens unit 260 for magnifying an image of the sample contained in the channel through which the light has passed, an image sensor unit 270 for acquiring an image related to the image magnified by the objective lens unit 260, a fine particle counting unit for counting fine particles in the sample contained in each channel 110 via the acquired image, and a stage moving unit for moving the stage.

[0069] The multi-channel sample chip 100 having the above-described structure is applied to the fine particle counting device 200. As described above, the correction of the warpage height deviation of each channel 110 is performed by calculating the difference between the focus value for a specific focus mark 120 among the plurality of focus marks 120 and the focus value of the channel 110 adjacent to the specific focus mark 120, and substituting the obtained warpage correction value for the focus value of each focus mark 120, as before.

[0070] The sample chip 100 may be placed on a stage 210 that is formed to correspond to the shape of the sample chip 100. The stage 210 may include a stage cover 212 that can cover the sample chip 100 after the sample chip 100 is placed on it. The stage cover 212 may have a hollow shape that allows the channel 110 and the focal mark 120 to be seen.

[0071] A light source unit 220 may be provided on the upper part of the stage 210. The light source unit 220 is positioned on the upper part of the stage 210 while being supported by a light source support unit 222, and serves to irradiate light onto a channel on the sample chip 100 placed on the stage 210.

[0072] Depending on the characteristics of the particles to be counted, various light sources such as an LED, laser, halogen lamp, xenon lamp, and mercury lamp can be selected and used as the light source unit 220. For example, when counting red blood cells, it is preferable to use a lamp or LED that emits ultraviolet-visible light as the light source unit 220. When counting white blood cells or somatic cells that contain cell nuclei, it is preferable to use a laser as the light source unit 220.

[0073] Although not shown, an incident light adjusting lens may be further included in front of the light source unit 220 to adjust the amount and focal length of light emitted from the light source unit 220 and irradiate it onto the sample chip 100.

[0074] Meanwhile, an objective lens unit 260 may be provided below the stage 210. The objective lens unit 260 may serve to magnify the image of the sample accommodated in the channel through which the light passes. The objective lens unit 260 may be configured to use a lens of any magnification as needed. In order to grasp the overall distribution of particles in the channel 110 on the sample chip 100, it is preferable to observe at a low magnification, and in this embodiment, a 4x objective lens is used.

[0075] An image sensor unit 270 is provided below the objective lens unit 260. The image magnified by the objective lens unit 260 can be acquired as an image through the image sensor unit 270. The image sensor unit 270 can be of various types, such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor).

[0076] The light source unit 220, the objective lens unit 260, and the image sensor unit 270 are arranged in a line on the Z axis to form an optical system.

[0077] The fine particle counting unit (not shown) counts fine particles in the sample accommodated in each channel 110 through the image acquired by the image sensor unit 270. The fine particle counting unit can be realized as software, and can be installed as a program or application on a PC, server, or mobile terminal connected to the fine particle counting device 200 wirelessly or via a wire, or can be incorporated on-chip into a microcomputer in the fine particle counting device 200.

[0078] Meanwhile, a stage moving unit may be provided to move the stage 210. The stage moving unit is realized to be able to move the stage 210 accurately in three axial directions.

[0079] First, the stage moving unit includes an X-axis moving unit 232 and an X-axis driving unit 230. The X-axis driving unit 230 includes a motor and an encoder, and provides a driving force that enables the X-axis moving unit 232 connected to the stage 210 to precisely adjust and move the stage 210 in the X-axis direction.

[0080] Similarly, the stage moving unit includes a Y-axis moving unit 242 and a Y-axis driving unit 240. The Y-axis driving unit 240 includes a motor and an encoder, and provides a driving force that enables the Y-axis moving unit 242 connected to the stage 210 to precisely adjust and move the stage 210 in the Y-axis direction.

[0081] The stage 210 can be moved in the XY plane via the X-axis moving unit 232 and the Y-axis moving unit 242, thereby allowing each channel 110 and focal mark 120 on the sample chip 100 to move to correspond to the above-mentioned optical system. This also makes it possible to move between each channel 110 sequentially or to each focal mark 120 sequentially.

[0082] The stage moving unit includes a Z-axis moving unit 252 and a Z-axis driving unit 250. The Z-axis driving unit 250 also includes a motor and an encoder, and provides a driving force that enables the Z-axis moving unit 252 connected to the stage 210 to precisely adjust and move the stage 210 in the Z-axis direction. The Z-axis moving unit 252 moves the stage in the Z-axis direction, thereby enabling adjustment of the focal length.

[0083] A power supply unit 282, a communication unit 284, and a motor control unit 286 may be provided at the bottom of the fine particle counting device 200. The power supply unit 282 serves to supply power to the entire device, the communication unit 284 serves to transmit and receive data or control commands to and from an external PC, server, or mobile terminal via a wireless or wired method, and the motor control unit 286 serves to control the motor installed in the stage moving unit.

[0084] Figure 18 is a conceptual diagram showing the principle of topology mapping using the coordinate values ​​and focus values ​​of some focus marks in a sample chip of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention, Figure 19 is a table showing an example of the focus values ​​of focus marks corresponding to each channel position in a sample chip of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention obtained by topology mapping, Figure 20 is a plan view showing a sample chip of a fine particle counting device using a multi-channel sample chip according to another embodiment of the present invention, Figure 21 is a layout diagram and actual photographed image showing the focus marks applied to the lower plate of the sample chip of Figure 20, and Figure 22 is a plan view showing the sample chip of a fine particle counting device using a multi-channel sample chip according to one embodiment of the present invention when divided into groups.

[0085] Referring to Figures 18 to 22, the focus values ​​of the multiple focus marks 120 can be configured by measuring the focus values ​​of some of the focus marks 120 out of all the focus marks 120, and using this to perform topology mapping of the degree of warping of the entire sample chip 100 to determine the focus values ​​of the remaining focus marks 120.

[0086] For example, as shown in Fig. 18, when the focus values ​​of A and F are measured and known, the focus value at any point D between A and F can be calculated and found. Then, in a similar manner, the focus values ​​at B, C, and E can also be found. In this manner, the coordinate values ​​of some of the focus marks 120 can be used to perform topology mapping of the degree of warpage of the entire sample chip 100, thereby finding the focus values ​​of the remaining focus marks 120.

[0087] 19 shows a specific example of a topology map obtained by calculating the focus values ​​of the focus mark 120. First, the reference focus mark focus values ​​A01, A08, F01, and F08 are obtained, and then the focus values ​​of the remaining focus marks 120 are obtained.

[0088] Example 1: Calculate B01 to E01 using the height difference between A01 and F01. B01=A01+((F01-A01) / F(100000)×B(20000)) =1000+((2000-1000) / 100000×20000) =1200 Example 2: A02 to A07 are calculated using the height difference between A01 and A08. A02=A01+((A08-A01) / 08(126000)×02(18000)) =1000+((2000-1000) / 126000×18000) =1143

[0089] By using the coordinates and focus values ​​of some of the focus marks 120 in the above-described manner, the focus values ​​of the remaining focus marks 120 can be obtained through topology mapping.

[0090] 18 and 19 show only linear warpage patterns in the above-described topology mapping, but the present invention is not limited to this. For example, by appropriately adjusting the position and number of focal marks to be measured, various types of topology mapping are possible, and therefore various warpage patterns can be predicted and applied.

[0091] This allows the topology mapping function to be applied to capture images of each channel more quickly and accurately without measuring the overall focus mark 120 .

[0092] Meanwhile, as described above, the pattern of the focus mark 120 can be modified into various forms.

[0093] As shown in Figures 20 and 21, the grid-shaped focus mark 120 may be embossed. If the grid pattern presented in the previous embodiment is formed by intaglio, it may be perceived as opaque and difficult to measure the focus value. To overcome this, a embossed grid shape can be applied. More preferably, as can be seen from the actual photographed image in Figure 21, the visibility can be further improved by forming it as an embossed black square.

[0094] Meanwhile, as shown in FIG. 22, the steps of determining the warp correction value, measuring the focus value for the focus mark 120, and acquiring the image can be configured to be repeated for each group formed by dividing the sample chip 100.

[0095] Next, the above-mentioned method will be explained in order based on FIG.

[0096] (1) Measure the focus value of A01 channel 110 by manual focus adjustment. (2) Measure the focus value of the focus mark 120 in group 1 by autofocus adjustment. (3) The warpage correction value is calculated from the values ​​of (1) and (2). (4) Apply the warp correction value of group 1 and sequentially acquire the A01 and A02 images through the corrected focus value. (5) The focus value of the focus mark of group 2 is calculated, and the warp correction value of (3) is applied to sequentially acquire images of A03 and A04. (6) The focus value of the focus mark of group 3 is calculated, and the warp correction value of (3) is applied to sequentially acquire images of A05 and A06.

[0097] In this case, groups to which the same warpage compensation value is applied can be designated in advance as in (4), (5), and (6). When measuring different samples within the specimen chip 100, the warpage compensation value is measured anew in the group to which the measurement position belongs, and an image is acquired by adjusting the focus to the corrected value, and in this case, the group to which the same warpage compensation value is applied is designated in advance.

[0098] The fine particle counting method using a multi-channel sample chip according to the embodiment of the present invention described above, and the fine particle counting device that realizes this method, make it possible to observe a large number of samples in a short period of time, and even if warping occurs during the manufacturing process due to the large size of the sample chip, this can be corrected to enable accurate counting of fine particles.

[0099] In addition, this solves the problem that the focal length for each channel is not constant due to warping that occurs in chips with many channels, and therefore it takes a long time to obtain the focal value for each channel and acquire an image.

[0100] Furthermore, since the amount of sample injected into the channel is small, if the image is not captured and counted quickly, the sample will dry out, which can prevent measurement errors.

[0101] Although the present invention has been described above with reference to one embodiment thereof, those skilled in the art will be able to implement the present invention in various modifications and variations without departing from the spirit and scope of the present invention as set forth in the claims below. Therefore, it should be understood that any modified implementations that essentially include the elements of the claims of the present invention are included within the technical scope of the present invention.

Claims

1. A method for counting microscopic particles in a sample contained in each channel on a sample chip having a plurality of channels and a focal mark, comprising: calculating a difference between a focus value for a specific focus mark among the plurality of focus marks and a focus value of a channel adjacent to the specific focus mark to obtain a warpage correction value; measuring respective focus values ​​for said plurality of focus marks; a step of acquiring an image of each channel through a focus value obtained by substituting the warp correction value for a focus value of a focus mark adjacent to each channel while moving between the channels in sequence; and counting the number of microparticles in the sample contained in each channel via the acquired images.

2. 2. The method of claim 1, wherein the number of the focus marks is less than the number of the channels.

3. 2. The method of claim 1, wherein the focal mark has a shape in which a number of protrusions are clustered together.

4. 2. The method of claim 1, wherein the focus mark has a grid shape.

5. 5. A method for counting fine particles using a multi-channel sample chip as described in claim 4, characterized in that the lattice pattern of the grid-shaped focus marks is used to determine a distance per unit pixel, the distance is substituted into the image resolution to determine an area, and the area is applied to the area of ​​the adjacent channel image.

6. The step of obtaining the warpage correction value includes: determining a focus value for said particular focus mark by automatic focus adjustment; 2. The method for counting fine particles using a multi-channel sample chip according to claim 1, wherein the focus value of the channel adjacent to the specific focus mark is determined by manual focus adjustment or automatic focus adjustment.

7. 2. The method for counting fine particles using a multi-channel sample chip according to claim 1, wherein the step of measuring the focus values ​​for each of the plurality of focus marks comprises obtaining the focus values ​​by automatic focus adjustment.

8. A method for counting microscopic particles in a sample contained in each channel on a sample chip having a plurality of channels and a focal mark, comprising: measuring respective focus values ​​for said plurality of focus marks; calculating a difference between a focus value for a specific focus mark among the plurality of focus marks and a focus value of a channel adjacent to the specific focus mark to obtain a warpage correction value; a step of acquiring an image of each channel through a focus value obtained by substituting the warp correction value for a focus value of a focus mark adjacent to each channel while moving between the channels in sequence; and counting the number of microparticles in the sample contained in each channel via the acquired images.

9. a sample tip having a plurality of channels for accommodating a sample including microparticles; a stage on which the sample chip is placed; a light source unit that irradiates light onto a channel on the sample chip placed on the stage; an objective lens section for magnifying an image of a sample contained in the channel through which the light passes; an image sensor unit for acquiring an image related to the image magnified by the objective lens unit; a fine particle counting unit that counts fine particles in the sample accommodated in each channel based on the acquired image; A stage moving unit that moves the stage, The sample chip further includes a plurality of focal marks for obtaining focusing information for correcting the warp height deviation of each of the channels, and each of the channels and the focal marks are adjacent to each other.

10. The correction of the warp height deviation of each channel includes:

10. A fine particle counting device using a multi-channel sample chip as described in claim 9, characterized in that the warp correction is performed by calculating the difference between the focus value for a specific focus mark among the multiple focus marks and the focus value of a channel adjacent to the specific focus mark, and substituting the warp correction value obtained for the focus value of each focus mark.

11. 10. The fine particle counter using a multi-channel sample chip according to claim 9, wherein the number of the focus marks is less than the number of the channels.

12. 10. The fine particle counting device using a multi-channel sample chip according to claim 9, wherein the focal mark has a shape in which a number of protrusions are clustered together.

13. 10. The fine particle counting device using a multi-channel sample chip according to claim 9, wherein the focus mark has a grid shape.

14. 10. The method for counting fine particles using a multi-channel sample chip as claimed in claim 1 or 8, wherein the focus values ​​of the plurality of focus marks are determined by measuring the focus values ​​of some of the focus marks among all the focus marks, and using the measured values, the degree of warping of the entire sample chip is subjected to topology mapping to determine the focus values ​​of the remaining focus marks.

15. 5. The method for counting fine particles using a multi-channel sample chip according to claim 4, wherein the grid-shaped focus mark is embossed.

16. 10. A method for counting fine particles using a multi-channel sample chip as claimed in claim 1 or 8, wherein the steps of determining the warp correction value, measuring the focus value for the focus mark, and acquiring the image are repeated for each group formed by dividing the sample chip.

17. 17. The method for counting fine particles using a multi-channel sample chip according to claim 16, wherein groups to which the same warpage compensation value is applied are designated in advance.

Images