JP5641170B2 - Capillary array sheet inspection apparatus and inspection method - Google Patents

Description

  The present invention generally relates to a capillary array sheet inspection apparatus and an inspection method, and more particularly to a capillary array used for detecting biopolymers such as nucleic acids, proteins, polypeptides, and polysaccharides. The present invention relates to an inspection apparatus and an inspection method for an array sheet.

  The capillary array sheet is a biochip on which a sample such as a biopolymer probe such as a nucleic acid is fixed. This capillary array sheet has a plurality of through holes two-dimensionally arranged in the sheet, and a gel in which a sample is fixed is filled in these through holes.

  The capillary array sheet having such a configuration is, for example, a fiber assembly (three-dimensional array) formed by regularly arranging hollow fibers in which a gel on which a sample is fixed is held in an internal space. It is manufactured by slicing the aggregate in a direction orthogonal to the direction in which the fibers extend.

  In the capillary array sheet manufactured in this way, when the gel is injected into the internal space of the hollow fiber, bubbles may be involved, and a through-hole that is not completely filled with the gel may occur. .

  As a capillary array sheet inspection device for detecting a through-hole that lacks or lacks such a gel, the gel filled in the through-hole is used as a lens to fill the through-hole. The MTF of each through hole is calculated from the image of the MTF measurement lattice imaged by the gel (MTF lattice image), and whether or not the gel is properly filled from the calculated MTF value (that is, whether there is a gel defect) ) Is known (see Patent Document 1).

JP 2008-107331 A

  However, in the apparatus and method disclosed in Patent Document 1, an MTF lattice image is acquired using one fixed MTF measurement lattice. For this reason, if the direction in which the MTF measurement grating used (lattice direction) extends coincides with or is close to the “blur” direction of the image caused by the gel defect, the “blur” is MTF. Since it does not overlap with the light and dark portions of the lattice image, it becomes a clear lattice image similar to a defect-free through hole. As a result, there was a problem that a high MTF value similar to that of a through-hole having no defect was calculated, and a gel defect could not be detected correctly.

  The present invention has been made to solve such problems, and provides an inspection apparatus and inspection method for a capillary array sheet that can reliably detect gel defects in the through holes of the capillary array sheet. The purpose is to provide.

According to the present invention,
A capillary array sheet inspection apparatus,
A light source;
Capillary array sheet holding means for fixing the capillary array sheet;
A grating for MTF measurement disposed at a predetermined position between the light source and the capillary array sheet held by the capillary array sheet holding means, wherein the grating direction attached to the movable part of the uniaxial stage is MTF measurement grating means including a plurality of different MTF measurement gratings;
Imaging means for capturing an image of each of the plurality of MTF measurement gratings imaged by the capillary array sheet to obtain a plurality of MTF grating images;
An MTF value of each through hole of the capillary array sheet is calculated using the plurality of MTF lattice images, and the lowest MTF value among them is used as the MTF value of each through hole. Detect the presence or absence of gel defects in each through-hole,
The plurality of MTF measurement gratings include four kinds of gratings having different grating directions,
The plurality of MTF measurement gratings have different grating directions by 45 °,
An imaging range limiting means for limiting the imaging range, which is disposed between the light source and the imaging means;
A capillary array sheet inspection apparatus is provided.

  According to the present invention having such a configuration, it is possible to acquire a plurality of MTF lattice images by the MTF measurement lattices in different lattice directions, so that accurate defect detection can be performed regardless of the direction of the gel defects. Is possible.

According to another aspect of the invention,
A capillary array sheet inspection method comprising:
The MTF measurement grid images of four MTF measurement grids mounted on the movable part of the uniaxial stage and having different grid directions by 45 ° are arranged between the light source and the imaging means to limit the imaging range. Obtaining via an image range limiting means ;
Calculating an MTF value of each through hole of the capillary array sheet from each of the two or more MTF measurement lattice images, and determining the lowest MTF value among them as an MTF value to be inspected;
Detecting the presence or absence of a gel defect in each through hole of the capillary array sheet using the determined MTF value.
Inspection method characterized by that.

  According to the present invention, there is provided a capillary array sheet inspection apparatus and inspection method capable of reliably detecting the presence or absence of a gel defect regardless of the direction of the gel defect in the through hole of the capillary array sheet. .

  Hereinafter, a capillary array sheet inspection apparatus and an inspection method according to a preferred embodiment of the present invention will be described with reference to the accompanying drawings.

First, a capillary array sheet for inspecting a defect with the capillary array sheet inspection apparatus and the inspection method of the embodiment of the present invention will be described.
FIG. 1 is a schematic plan view showing the configuration of the capillary array sheet 1. Similar to the conventional capillary array sheet, the capillary array sheet 1 includes a sheet-like base material portion 2 and a plurality of through holes 4 extending through the base material portion 2 in the thickness direction. . The through holes 4 are arranged so as to form a two-dimensional pattern on the surface of the base material portion 2, and the through holes 4 are filled with a gel 6 on which a biopolymer probe such as a nucleic acid is immobilized.

  The capillary array sheet 1 of the present embodiment is a fiber assembly (three-dimensional array) in which a plurality of biopolymer-immobilized gel-holding hollow fibers in which biopolymers such as different nucleic acids are immobilized are regularly arranged. Is sliced in a direction perpendicular to the fiber axis.

  However, in the present invention, a capillary array sheet manufactured by another method, for example, a plurality of stacked sheet-like members or block-like members are formed with a plurality of holes with a laser beam or the like, and these holes are different. Produced by injecting a biopolymer probe such as nucleic acid and gel precursor solution, polymerizing the gel precursor solution in the hole, and then cutting it at a desired thickness in the direction perpendicular to the direction in which the hole extends Capillary array sheets can also be used.

  FIG. 2 is a schematic diagram showing the configuration of the capillary array sheet inspection apparatus 8 according to a preferred embodiment of the present invention. The capillary array sheet inspection apparatus 8 includes a light source 10 including a halogen lamp or an LED element capable of adjusting the amount of light, an imaging range limiting member 12 for limiting irradiation light from the light source 10, and a single axis. Immersion in which four MTF (Modulation Transfer Function) measurement gratings 16a, 16b, 16c, and 16d attached to the stage 14 and the capillary array sheet 1 are immersed in a liquid and fixed inside. And a tank 18.

The capillary array sheet inspection apparatus 8 further generates an image (MTF lattice image) of the MTF measurement lattice 16 formed by the gel 6 filled in the through holes 4 of the capillary array sheet 1 with the light from the light source 10. And an imaging device 22 for imaging.
The imaging device 22 includes an objective lens 24 directed to the light source 10 and a CCD sensor 26 as imaging means. Image data obtained by the CCD sensor 26 is sent to an image processing device (not shown) for predetermined image analysis. Thus, the MTF is evaluated.

  The imaging range limiting member 12 is configured so that the MTF lattice images formed by the gels 6 in the adjacent through holes 4 of the capillary array sheet 1 do not overlap on the CCD sensor 26 of the imaging device 22. 22 has a function of limiting the range of the MTF lattice image by the through hole. As the imaging range limiting member 12, for example, a metal plate having a hole, a field stop capable of adjusting the size of the hole, or the like is used. The position where the imaging range limiting member 12 is provided, the size of the hole of the imaging range limiting member 12 and the like are appropriately selected according to the measurement conditions.

  The immersion tank 18 is a water tank-shaped container made of a light transmissive material such as glass or quartz. The immersion tank 18 contains a light transmissive liquid 20 such as water, in which the capillary array sheet 1 has a surface orthogonal to the optical axis of the objective lens 24 of the imaging device 22. That is, it is immersed (so as to be orthogonal to the straight line connecting the light emitting part of the light source 10 and the light receiving part of the imaging device 22), and is appropriately fixed by a mechanism not shown.

  By dipping the capillary array sheet 1 in the liquid 20, it is possible to prevent the gel 6 held in the through holes 4 of the capillary array sheet 1 from being dried. The liquid 20 is preferably highly transparent, and any liquid may be used as long as it prevents the gel 6 from drying and does not contaminate or deteriorate the gel 6. Examples of liquids other than water include salt solutions and glycerol solutions. Moreover, you may add antiseptic | preservative etc. to these solutions.

  FIG. 3 is a schematic plan view showing the configuration of the MTF measurement gratings 16a, 16b, 16c, and 16d used in the capillary array sheet inspection apparatus 1 of the present embodiment. The MTF measurement gratings 16a, 16b, 16c, and 16d are known MTF measurement gratings, and have a portion with low light transmittance (black portion) shown in black and a light transmittance shown with white. It is a lattice in which high portions (white portions) are alternately arranged. The width of each white part and the black part is the same, the width of a pair of white part and black part is called a line pitch (mm), and the reciprocal of the line pitch is the spatial frequency (lp / mm).

  The spatial frequency of the grating depends on various conditions such as the size of the through hole of the capillary array sheet to be inspected, the thickness of the capillary array sheet, the magnification of the objective lens 24 of the imaging device 22, and the numerical aperture. Therefore, an appropriate value is selected. The MTF measurement grids 16a, 16b, 16c, and 16d are formed by a known method such as partial vapor deposition of a metal so as to form a grid on a substrate such as a glass plate.

  In the present embodiment, as shown in FIG. 4, a plurality of MTF measurement gratings 16 a, 16 b, 16 c, and 16 d having different grating extending directions (lattice directions) are movable parts 14 a of the uniaxial stage 14. Installed on. The movable portion 14 a is disposed so as to be orthogonal to the optical path of the irradiation light from the light source 10 toward the objective lens 24. Therefore, by moving the uniaxial stage 14 in the direction of arrow A, any one of the plurality of MTF measurement gratings 16a, 16b, 16c, and 16d having different grating extending directions (lattice directions) is supplied from the light source 10. It is selectively arranged at a position (predetermined position) crossing the optical path of the irradiation light. Such a uniaxial stage 14 serves as a lattice direction changing means.

  The plurality of MTF measurement gratings 16a, 16b, 16c, and 16d are arranged so that the grating direction of each grating is 45 ° with respect to the adjacent grating. Specifically, for example, the first MTF measurement grid 16a is arranged in the slicing direction (moving direction of the cutting blade) when the capillary array sheet 1 manufactured in the immersion tank 18 is fixed. The second MTF measurement grating 16b is arranged so as to be perpendicular to each other, and the second MTF measurement grating 16b is arranged so that the grating direction is rotated by 45 ° from the grating direction of the first MTF measurement grating 16a. 3 MTF measurement grating 16c is arranged so that the grating direction is rotated by 45 ° from the grating direction of the second MTF measurement grating 16b, and the fourth MTF measurement grating 16d is The direction is arranged so as to be a direction rotated by 45 ° from the lattice direction of the third MTF measurement lattice 16c.

  As a result, by moving the uniaxial stage 14 in the direction of arrow A, it passes through any one of a plurality of four MTF measurement gratings 16a, 16b, 16c, and 16d whose grating extending directions (lattice directions) differ by 45 °. The MTF lattice image by the obtained light can be acquired sequentially or selectively.

  In this embodiment, the four MTF measurement gratings are arranged so that the grating directions are different by 45 ° in the rotation direction. However, the number of MTF measurement gratings and the angle difference between the grating directions are limited to these values. Is not to be done.

  Furthermore, in this embodiment, four MTF measurement gratings 16a, 16b, 16c, and 16d having different grating directions are selectively and sequentially arranged on the optical path, thereby transmitting the MTF gratings having different grating directions. A plurality of MTF lattice images transmitted through MTF lattices having different lattice directions are obtained by rotating one MTF measurement lattice in a direction parallel to the lattice plane. The structure which acquires may be sufficient.

Next, a capillary array sheet inspection method using the capillary array sheet inspection apparatus 8 will be described.
First, the capillary array sheet 1 for defect detection is fixed in the immersion bath 18, the first MTF measurement grid 16 a is arranged at a predetermined position on the optical path from the light source 10, and the irradiation light is emitted from the light source 10. Let it emit. The MTF lattice image of the first MTF measurement grating 16a is connected to the imaging position by the lens action of the gel 6 in each through-hole 4 of the capillary array sheet 1, and this is an imaging device 22 arranged at the imaging position. The image is taken by the CCD sensor 26.

  Next, the uniaxial stage 14 is operated to change the first MTF measurement grating 16a to the second MTF measurement grating 16b, and similarly, the imaging device 22 captures an image of the second MTF measurement grating 16b. . Further, the same operation is repeated for the third and fourth MTF measurement gratings 16c and 16d.

The MTF grating images of the four types of MTF measurement gratings 16a, 16b, 16c, and 16d obtained by the CCD sensor 26 are processed by an image processing apparatus, and the following formula (1 ) To calculate the MTF value for each through hole. The four types of MTF values obtained from the same through hole are respectively compared with the determination reference value (threshold value) of the defect state of the gel to determine the gel defect for each through hole.
MTF = {(I _max −I _min ) / (I _max + I _min )} × 100 (%) (1)
Here, I _max and I _min are the maximum light amount value (I _max ) and the minimum light amount value (I _min ) of the light amount line profile of the image of the imaged MTF measurement grating.

Hereinafter, a method for calculating the MTF value of each through hole from the MTF values of the four types of images of the MTF measurement grating will be described.
FIG. 5 is a schematic plan view of the capillary array sheet 1 to be inspected by the inspection apparatus of the present embodiment. The capillary array sheet 1 is a fiber assembly (three-dimensional array) formed by regularly arranging a number of biopolymer-immobilized gel-carrying hollow fibers on which biopolymers such as different nucleic acids are immobilized. Since the capillary array sheet is manufactured by slicing in a direction perpendicular to the fiber axis, gel defects that extend parallel to the slicing direction, which is the moving direction of the blade during slicing, are likely to occur.

  If the angle (slice angle) formed by the center line 28 in the X direction (that is, the lateral width direction) of the capillary array sheet 1 and the slice direction 30 is θ, the gel 6 in the through hole 4 has Therefore, the defect 32 extending at an angle θ is likely to occur.

FIG. 6 is an enlarged schematic plan view of the through hole 4 having the gel defect shown in FIG.
Here, for example, with respect to the through-hole 4 having the gel 6 having the defect 32 extending in parallel with the slice direction 30, a lattice in a direction parallel to the slice direction as shown in FIG. 7 (that is, the inclination angle of the lattice is θ When the MTF lattice image is acquired by using the MTF measurement lattice 16, the MTF lattice image is parallel to the slice direction 30 due to the defect 32 extending in parallel with the slice direction 30, that is, a white portion or a black color. It extends in a direction along the direction in which the portion extends and is “blurred”. On the other hand, the image is less likely to blur in the horizontal direction of the lattice, that is, in the direction crossing the white portion or the black portion.

  As a result, the image change (blurring) due to the defect 32 hardly appears in the MTF lattice image (that is, it is difficult to read from the MTF lattice image), and the MTF value is high even though the gel is missing. There is a high possibility that an erroneous determination that there is no defect will be made.

In the inspection apparatus of the present embodiment described above, a total of four types of MTF measurement gratings 16a and 16b in which the grating direction is rotated by 45 ° from the reference direction with the direction orthogonal to the slice direction (slice angle + 90 °) as the reference direction. 16c, 16d are used. Then, a total of four MTF lattice images are obtained from each of the four types of MTF measurement lattices 16a, 16b, 16c, and 16d having different lattice directions by 45 °. Then, the MTF values for each through hole are calculated using these four MTF lattice images, and the lowest value among them is adopted as the MTF value of the through hole.
The MTF value thus obtained is compared with a preset threshold value, and when the through hole MTF value is lower than the threshold value, it is determined that there is a defect. Thus, by adopting the lowest MTF value, the gel defect can be accurately determined.

  Depending on the surface state of the gel 6 filled in the through-hole 4 in one capillary array sheet, the imaging position of one through-hole may be different from the imaging position of other through-holes. . In such a case, if the position of the objective lens is fixed at a position where an image is obtained at an imaging position through a certain through-hole, the image of the MTF measurement grating that has passed through the through-hole having a different imaging position is Or it becomes a back pin. As a result, the MTF value calculated based on the image is lower than the MTF value calculated in the focused state, and the gel is not properly filled even though the gel is properly filled. May be determined.

  In the present embodiment, the objective lens 24 is moved at a predetermined pitch over a predetermined distance in the optical axis direction, for example, about 3 mm at a pitch of 100 μm, and the objective lens 24 is stopped every 100 μm for MTF measurement. An image of the lattice is taken, and the MTF value is calculated using the lattice image at the optimum focus position.

  The present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the invention described in the claims, and these are also included in the scope of the present invention. Needless to say.

Examples of the present invention will be described below.
Prepare two capillary plates with a thickness of 0.1 mm in which 19 × 12 = 228 holes with a diameter of 0.32 mm are arranged at intervals of 0.42 mm in the center of the capillary array sheet fabrication plate at intervals of 0.42 mm. The two perforated plates were overlapped so that the holes of each perforated plate were aligned. After passing through the hollow holes made of polycarbonate colored by carbon black (outer diameter 0.28mm, inner diameter 0.18mm, length 500mm) through all the aligned holes of the perforated plate, these two sheets were passed through the fibers 50 mm apart, filled with polyurethane resin colored with carbon black between the separated perforated plates, a hollow column having a length of 50 mm and a 7 × 12 mm square shape that is not fixed with resin at both ends A fiber array was obtained.

  The hollow fiber in the obtained hollow fiber array is filled with the gel precursor solution and 40-type nucleic acid probes of different types while entraining bubbles, polymerizing the gel precursor solution, and then by microtome A capillary array sheet was obtained by cutting out a thin piece having a thickness of 0.25 mm in a direction perpendicular to the fiber axis.

Measurement of MTF The obtained capillary array sheet was immersed in a glass container filled with sterilized water. The immersed capillary array sheet was disposed so that the surface thereof was perpendicular to the optical axis of the objective lens attached to the microscope. Nikon CM-70L was used as a microscope, a lens having a magnification of 2 × and an aperture angle (NA) of 0.06 as an objective lens, and transmitted illumination (halogen lamp) as an illumination device.

  A metal plate with a 9 mm hole formed as an imaging range limiting member at a position moved 165 mm from the capillary array sheet toward the illumination device so that the center of the optical axis coincides with the optical axis of the objective lens. Attached to. Further, an MTF measurement grating (0.275 lp / mm, thickness 0.1 mm) made of copy paper was attached to a position moved 5 mm from the imaging range limiting member toward the illumination device.

The MTF measurement grid is rotated in the direction of 45 ° in the grid direction by 45 ° with the direction perpendicular to the slice direction of the capillary array sheet (slice angle + 90 °) as the reference direction of the grid direction. Each was attached to a movable stage so as to be displaced.
A lattice image of the MTF measurement lattice formed through each through hole of the capillary array sheet was imaged by a CCD sensor (BS-41L manufactured by Bitlan). Thereafter, the stage was moved to switch the MTF measurement grid and imaged in the same manner, and all four types of MTF measurement grid images were captured.

  FIG. 8 is an image of four types of captured MTF measurement gratings, and is an enlarged view of the same portion of the entire capillary array sheet image. In the figure, the image of the MTF measurement grating imaged through the through-hole surrounded by the square is the image blurry direction and the grating direction in the image using the MTF measurement grating of the reference angle + 90 ° (that is, the slice direction). Because of the coincidence, a clearer lattice is seen than in the lattice images in the other directions. However, in the other MTF measurement lattice images, the light and dark portions of the lattice are blurred and overlapped with each other, so that the lattice images are observed blurred. When the MTF values of the lattice images of the reference angle, the reference angle + 45 °, the reference angle + 90 °, and the reference angle + 135 ° are calculated, they are 7%, 15%, 16%, and 32%, respectively, which are the lowest values. 7% was adopted as the MTF value of the through hole, compared with a preset threshold value, and a defect was determined.

It is a typical top view which shows the structure of the capillary array sheet | seat which test | inspects a gel defect in preferable embodiment of this invention. 1 is a schematic drawing showing a configuration of a capillary array sheet inspection apparatus according to a preferred embodiment of the present invention. It is typical drawing which shows an example of the grating | lattice for MTF measurement used with the capillary array sheet | seat test | inspection apparatus of FIG. FIG. 2 is a schematic drawing for explaining a uniaxial stage and an MTF measurement grid used in the capillary array sheet inspection apparatus of FIG. 1. It is a typical top view for demonstrating the slice direction of a capillary array sheet | seat, and the direction of a defect. It is the typical top view to which one of the through-holes having a defect of Drawing 5 was expanded. It is a schematic drawing showing the relationship between the slice direction of the capillary array sheet and the lattice direction for MTF measurement. It is a figure which shows a part of image of four types of MTF measurement grating | lattices.

Explanation of symbols

1: Capillary array sheet 10: Light source 14: Uniaxial stage (lattice direction changing means)
16a, 16b, 16c, 16d: MTF measurement grating 22: Imaging device 32: Gel defect

Claims (2)

A capillary array sheet inspection apparatus,
A light source;
Capillary array sheet holding means for fixing the capillary array sheet;
A grating for MTF measurement disposed at a predetermined position between the light source and the capillary array sheet held by the capillary array sheet holding means, wherein the grating direction attached to the movable part of the uniaxial stage is MTF measurement grating means including a plurality of different MTF measurement gratings;
Imaging means for capturing an image of each of the plurality of MTF measurement gratings imaged by the capillary array sheet to obtain a plurality of MTF grating images;
An MTF value of each through hole of the capillary array sheet is calculated using the plurality of MTF lattice images, and the lowest MTF value among them is used as the MTF value of each through hole. Detect the presence or absence of gel defects in each through-hole,
The plurality of MTF measurement gratings include four kinds of gratings having different grating directions,
The plurality of MTF measurement gratings have different grating directions by 45 °,
An imaging range limiting means for limiting the imaging range, which is disposed between the light source and the imaging means;
Capillary array sheet inspection apparatus. A capillary array sheet inspection method comprising:
The MTF measurement grid images of four MTF measurement grids mounted on the movable part of the uniaxial stage and having different grid directions by 45 ° are arranged between the light source and the imaging means to limit the imaging range. Obtaining via an image range limiting means ;
The MTF value of each through hole of the capillary array sheet is calculated from each of the MTF measurement lattice images of the four MTF measurement lattices, and the lowest MTF value among them is determined as the MTF value to be inspected. And steps to
Detecting the presence or absence of a gel defect in each through hole of the capillary array sheet using the determined MTF value.
Inspection method characterized by that.