Slide glass

Abstract

This slide glass (1) is formed from at least one selected from the group consisting of quartz, rock crystal, silicon, sapphire, etc. Further, a plurality of first metal lines (21) extending in a first direction (x) and arranged at a prescribed spacing and a plurality of second metal lines (22) extending in a second direction (y) and arranged at a prescribed spacing are provided on a part of a surface (1a) on which a biological sample is to be placed or the like. A plurality of sub-regions (3) are defined by the metal lines (21, 22), and the metal lines (21, 22) are formed from at least one metal selected from the group consisting of Re, Ir, Ta, Ni, Cr, Pt, Au, Ti, and Si.

Description

Slide glass

[0001] The present disclosure relates to a slide glass that can be suitably used when performing LA-ICP-MS analysis of a biological sample.

[0002] There is a known technique in which a solid sample or a liquid sample is irradiated with a laser beam to evaporate and atomize the sample with its energy (laser ablation: LA), and the resulting sample is introduced into an inductively coupled plasma mass spectrometry (ICP-MS) apparatus to perform qualitative and quantitative analysis of the elements contained in the sample (Non-Patent Document 1, etc.: hereinafter sometimes abbreviated as "LA-ICP-MS analysis"). Since this LA-ICP-MS analysis enables atomization of a micro-region or an extremely surface layer region of about several μm in a sample by controlling the laser beam, it is a highly sensitive and quantitative method that is a characteristic of ICP-MS, and has been widely used for trace component analysis of electronic materials, biological samples, and the like.

[0003] On the other hand, recently, findings have been obtained that a specific cancer cell contains a significantly large amount of a specific metal element. Based on such a correlation, an inspection method for identifying cancer cells in a specimen tissue containing a lesion part based on the measurement result of the metal element distribution in the specimen tissue has attracted attention. In addition, there are times when trace component analysis at the cell unit is required instead of biological tissue. Therefore, many expectations are placed on a method of applying LA-ICP-MS analysis to such pathological examinations at the specimen tissue and cell unit levels.

[0004] By the way, in LA-ICP-MS analysis, generally, a sample is placed or supplied on a support member installed at a predetermined position, and laser ablation of the sample is performed while relatively moving the sample and the laser irradiation position. At present, as such a support member, a slide glass (for example, Patent Documents 1 and 2: sometimes called "slide glass" or "glass plate") that is widely used for observation and specimen preparation by an optical microscope is often used.

[0005] "Laser Ablation ICP Mass Spectrometry LA-ICP-MS", Internet <https: / / www.jfe-tec.co.jp / analysis / la-icp-ms.html>; Searched on September 12, 2024

[0006] Utility Model Registration No. 3141498, Publication No. 2004 / 044639, Brochure

[0007] However, even if predetermined alignment marks are provided to position the slide glass, if there are no such markers in the area where the sample is placed or supplied, it is difficult to accurately determine the actual location where the laser is irradiating the sample. As a result, when the sample is a specimen tissue, it may not be possible to sufficiently improve the positional accuracy of the obtained metal element distribution. Furthermore, depending on the material of the slide glass, background signals originating from the metals it contains may prevent the detection of trace metal components, especially in biological samples.

[0008] Therefore, this disclosure has been made in view of these circumstances, and aims to provide a slide glass that can improve the positional accuracy of the obtained metal element distribution by accurately determining the actual location where the laser irradiation is performed on the biological sample when performing LA-ICP-MS analysis of the biological sample, and can also reduce background signals caused by metal components contained in the material.

[0009] [1] To achieve the above objective, the slide glass according to this disclosure is used to perform qualitative and / or quantitative analysis of metal elements contained in a biological sample (such as human tissue samples or solutions containing cells) by performing ICP-MS analysis while irradiating the biological sample with a laser, and is formed of at least one selected from the group consisting of quartz, crystal, silicon (Si), sapphire, diamond, and alkali-free glass, and comprises a plurality of first metal wires extending in a first direction (e.g., x direction) and arranged at predetermined intervals on a part of the surface on which the biological sample is placed, and a plurality of second metal wires extending in a second direction (e.g., y direction) and arranged at predetermined intervals. Furthermore, a plurality of sub-regions are defined by these first and second metal wires, and furthermore, these first and second metal wires are formed of at least one metal selected from the group consisting of Re, Ir, Ta, Ni, Cr, Pt, Au, Ti, and Si.

[0010] In this configuration, a laser is irradiated onto a biological sample placed or supplied to multiple sub-regions, and microparticles formed from the biological sample are subjected to ICP-MS analysis to qualitatively and quantitatively determine the metal elements contained in the microparticles. During this process, the entire biological sample on the slide is measured by continuously performing ICP-MS analysis while relatively moving the biological sample and the laser irradiation position. When the laser irradiation passes over the positions of the first and second metal lines that define the multiple sub-regions on the surface of the slide, the metal at that location is micronized together with the biological sample and detected by ICP-MS. Therefore, based on the positions of these first and second metal lines, the position of the biological sample on each sub-region where the laser is actually irradiated can be accurately determined (identified).

[0011] Furthermore, since the first and second metal wires are formed from at least one of the metals described above, and these metal elements are not present in the human body, or are present in extremely small amounts, they do not interfere with the measurement of metal components in biological samples. In addition, since the slide glasses are formed from at least one of the materials described above, they contain very little metal component that could be problematic in the measurement of biological samples compared to other glass materials, etc., thus reducing background signals that could interfere with the measurement of trace metal components contained in biological samples.

[0012] [2] In the above configuration, multiple intersecting first grooves may be formed in each of the multiple sub-regions. In this case, in the case of a biological sample containing cells in a liquid, the biological sample solution diffuses along the first grooves, and the contained cells can be held at the intersections of the first grooves, thus enabling cell-level metal element analysis with excellent positional accuracy.

[0013] [3] In this case, the plurality of first metal wires and the plurality of second metal wires may be provided in a second groove formed on a part of the surface. In this configuration, the liquid and cells of the biological sample are easily diffused through the second groove into each of the plurality of sub-regions defined by the first metal wire and the plurality of second metal wires.

[0014] [4] Furthermore, when the biological sample is a liquid sample containing cells, the width of each of the multiple first grooves may be approximately equal to or smaller than the width of a cell, and the width of the intersection of the multiple first grooves may be approximately equal to or larger than the width of a cell. In this way, it becomes easier to retain cells at the intersection of the first grooves. In this disclosure, "approximately equal" means within ±10% of the reference value.

[0015] [5] The remaining portion of the surface may also include an unprocessed area. This allows, for example, a sticker or tape with the number or type of the biological sample to be attached to the unprocessed area, thereby improving user convenience.

[0016] [6] In addition, one or more alignment marks for positioning the slide glass may be provided at multiple corners on the surface, and matrix symbols (marks) for identifying each of the multiple sub-regions may be provided at the periphery of the surface. This has the advantage of making it easier to position the slide glass as a whole and to identify the location (address) of the sub-regions.

[0017] According to the slide glass provided in this disclosure, when performing LA-ICP-MS analysis of a biological sample, it is possible to improve the positional accuracy of the obtained metal element distribution by accurately determining the actual location where the laser irradiation is being performed on the biological sample, and to reduce background signals caused by metal components contained in the material.

[0018] (A) is a plan view (top view) showing the schematic configuration of a microscope slide according to one embodiment of the present disclosure, and (B) is a side view (front view) of the same. This is an enlarged plan view showing an example of the details of part A in Figure 1(A). This is an enlarged plan view showing an example of the details of part B in Figure 2. (A) is a plan view (top view) showing the schematic configuration of a microscope slide according to another embodiment of the present disclosure, and (B) is a side view (front view) of the same. This is an enlarged plan view showing an example of the details of part C in Figure 4(A). This is an enlarged plan view showing an example of the details of part D in Figure 5. This is an enlarged plan view showing an example of the details of part E in Figure 6. This is a cross-sectional view along the line VIII-VIII in Figure 7. This is a graph showing an example of signal intensity measurement when Mg contained in the microscope slide according to the present disclosure and conventional microscope slides is quantified by LA-ICP-MS analysis. (A) is a photograph showing the state of the surface of the slide glass according to this disclosure after laser irradiation in LA-ICP-MS analysis, and (B) is an image showing the result of imaging the metal wire grating by image processing based on the measurement results of the signal intensity for the constituent elements of the metal wire grating obtained by the LA-ICP-MS analysis.

[0019] This embodiment will be described below with reference to the attached drawings. To facilitate understanding of the description, the same reference numerals are used for identical components in each drawing whenever possible, and redundant descriptions are omitted. The following embodiments are illustrative examples for illustrating the present disclosure and are not intended to limit the present disclosure to these embodiments only. Furthermore, the present disclosure can be modified in various ways without departing from its essence. Moreover, those skilled in the art can adopt embodiments in which each of the elements described below is replaced with equivalent components, and such embodiments are also included within the scope of the present disclosure.

[0020] [First Embodiment] Figure 1(A) is a plan view (top view) showing the schematic configuration of a slide glass 1 according to one embodiment of the present disclosure, and Figure 1(B) is a side view (front view) of the same. Figure 2 is an enlarged plan view showing an example of details of part A in Figure 1(A), and Figure 3 is an enlarged plan view showing an example of details of part B in Figure 2. The slide glass 1 is suitably used to perform qualitative and quantitative analysis of metal elements contained in a biological sample by performing ICP-MS analysis (LA-ICP-MS analysis) while irradiating the biological sample with a laser.

[0021] As shown in Figure 1, the microscope slide 1 is a transparent substrate that is plate-shaped and substantially rectangular, with notches K formed at each corner, and is made of at least one material selected from the group consisting of quartz, crystal, silicon (Si), sapphire, diamond, and alkali-free glass. Furthermore, a portion of the surface 1a on the microscope slide 1 on which the biological sample is placed or supplied (approximately the left 3 / 4 of the area in the plane shown in Figure 1(A)) is provided with a plurality of metal wires 21 (corresponding to an example of the "first metal wire" in this disclosure) extending in the x direction (corresponding to an example of the "first direction" in this disclosure), and a plurality of metal wires 22 extending in the y direction perpendicular to the x direction (corresponding to an example of the "second direction" in this disclosure). A metal wire grid 2 is formed from these plurality of mutually orthogonal metal wires 21, 22 in a grid pattern (like a checkerboard) at equal intervals. In addition, a plurality of sub-regions 3 are defined by these metal wires 21, 22.

[0022] These metal wires 21 and 22 are each formed from at least one metal selected from the group consisting of Re, Ir, Ta, Ni, Cr, Pt, Au, Ti, and Si, and the constituent metals of the two may be the same or different. The metal wires 21 and 22 can be formed on the surface 1a of the slide glass 1, for example, by vapor deposition and appropriate patterning techniques. The remaining part of the surface 1a of the slide glass 1 (approximately the right quarter of the plane shown in Figure 1(A)) is left as an unprocessed area M for attaching, for example, a sticker or tape indicating the number or type of the biological sample. This contributes to improving user convenience.

[0023] Furthermore, alignment marks 4 are provided on the surface 1a of three of the four corners of the slide glass 1 to position it when it is placed on a support stand for laser irradiation. The shape, manufacturing method, and material of these alignment marks 4 are not particularly limited as long as their position can be recognized, for example, optically, magnetically, or physically (mechanically), and can be formed by printing, laser engraving, etc. Also, on the longer side of the periphery of the surface 1a, column code marks 51 are provided along the longitudinal direction (x direction) of the metal wire grid 2 to indicate the longitudinal positions of multiple sub-regions 3. Similarly, on the shorter side of the periphery of the surface 1a, row code marks 52 are provided along the shorter direction (y direction) of the metal wire grid 2 to indicate the short-direction positions of multiple sub-regions 3. This has the advantage of making it easier to identify the location (address) of the sub-regions 3. Furthermore, the shape, manufacturing method, material, etc., of the matrix code mark 5 (corresponding to an example of a "matrix code" in this disclosure), which is composed of these column code marks 51 and row code marks 52, are not particularly limited as long as their contents can be recognized, for example, optically, magnetically, or physically (mechanically), and can be formed by printing, laser engraving, etc.

[0024] Furthermore, as shown in Figures 2 and 3, each sub-region 3 defined by the metal wire grid 2 is a rectangular section on the surface 1a, and while there are no particular limitations on the side length D1 (i.e., the spacing between the metal wires 21 and 22), it is preferably 0.1 mm to 1 mm, more preferably 0.25 mm to 0.75 mm, and even more preferably about 0.5 mm. Also, the column code mark 51 corresponds to the column number of the sub-region 3 and can be represented by a number, for example, and the row code mark 52 corresponds to the row number of the sub-region 3 and can be represented by an alphabet, for example. Furthermore, while there are no particular limitations on the width D2 of the metal wires 21 and 22, it is preferably 0.01 mm to 0.1 mm (10 μm to 100 μm), more preferably 0.02 mm to 0.06 mm (20 μm to 60 μm), and even more preferably about 0.03 mm (30 μm).

[0025] With the slide glass 1 configured in this way, a biological sample is placed or supplied to multiple sub-regions 3, and the biological sample is irradiated with a laser. Microparticles formed from the biological sample are then introduced into ICP-MS analysis, and the metallic elements contained in the microparticles are qualitatively and quantitatively analyzed. At this time, by continuously performing ICP-MS analysis while relatively moving the position of the biological sample and the laser irradiation position, measurements are taken over the entire area of ​​the biological sample on the slide glass 1. When the laser irradiation passes over the positions of the metal lines 21 and 22 that define the multiple sub-regions 3 on the surface 1a of the slide glass 1, the metal at that location is atomized together with the biological sample and detected by ICP-MS. Therefore, based on the positions of the metal lines 21 and 22, the position of the biological sample on each sub-region 3 that is actually being irradiated with the laser can be accurately determined (identified).

[0026] Furthermore, the metal wires 21 and 22 are formed from at least one metal selected from the group consisting of Re, Ir, Ta, Ni, Cr, Pt, Au, Ti, and Si. Since these metal elements are not present in the human body, or are present only in extremely small amounts, they do not interfere with the measurement of metal components in biological samples. Moreover, since the slide glass 1 is formed from at least one material selected from the group consisting of quartz, crystal, silicon (Si), sapphire, diamond, and alkali-free glass, the content of metal components that could be problematic in the measurement of biological samples is very low compared to other glass materials. Therefore, background signals that could interfere with the measurement of trace metal components contained in biological samples can be reduced. Although some slide glass 1 materials contain Al, Al is an element that is frequently contaminated from the environment, so it does not pose a particular problem in analytical systems that do not measure Al in biological samples.

[0027] As a result, when the biological sample is a sample tissue, the positional accuracy of the metal element distribution (two-dimensional distribution) obtained as an analysis result can be significantly improved. Furthermore, if the biological sample is a solution containing cells, and the cells are evenly distributed on the slide glass 1, the cells can be more clearly distinguished based on the positions of the metal wires 21 and 22. This makes it easier to perform analysis at the cell level. In addition, since the detection peak of, for example, Ti in ICP-MS analysis is a nearly single, steep peak, it is useful for detecting the position of sub-region 3 on the slide glass 1. Moreover, if metals with excellent corrosion resistance are used as the metal wires 21 and 22, and the slide glass 1 is formed from the above-mentioned material, heat resistance and corrosion resistance can be improved, which is also preferable from the viewpoint of suppressing deterioration of the slide glass 1.

[0028] [Second Embodiment] Figure 4(A) is a plan view (top view) showing the schematic configuration of a slide glass 1' according to another embodiment of the present disclosure, and Figure 4(B) is a side view (front view) of the same. Figure 5 is an enlarged plan view showing an example of the details of part C in Figure 4(A), and Figure 6 is an enlarged plan view showing an example of the details of part D in Figure 5. The slide glass 1' is configured in the same way as the slide glass 1 in the first embodiment, except that it is equipped with metal wires 61 and 62 (metal wire grid 6) instead of metal wires 21 and 22 (metal wire grid 2), and that the metal wire grid 6 defines a plurality of sub-regions 7 instead of a plurality of sub-regions 3.

[0029] As shown in Figures 5 and 6, each sub-region 7 defined by the metal wire grid 6 is a rectangular section on the surface 1a, and its side length D3 (i.e., the spacing between the metal wires 61 and 62) is not particularly limited, but preferably 0.2 mm to 2 mm, more preferably 0.5 mm to 1.5 mm, and even more preferably about 1.0 mm. The width D4 of the metal wires 61 and 62 is not particularly limited, but preferably 0.05 mm to 0.3 mm, more preferably 0.075 mm to 0.2 mm, and even more preferably about 0.1 mm. Furthermore, each sub-region 7 has trenches 9, 9 (corresponding to an example of the "first trench" in this disclosure) that extend in the x and y directions, respectively, and intersect in the center of the sub-region 7. The metal wires 61 and 62 are provided on the bottom surface of a groove 10 (corresponding to an example of the "second trench" in this disclosure) formed in a grid pattern on a part of the surface 1a.

[0030] Here, Figure 7 is an enlarged plan view showing an example of the details of section E in Figure 6, and Figure 8 is a cross-sectional view along the line VIII-VIII in Figure 7. As shown in these figures, the intersection 9x of grooves 9, 9 is a rectangular section having a side length longer than the width of groove 9. More specifically, in the case of a biological sample being a liquid sample containing cells C, the width D5 of groove 9 is made to be approximately equal to or smaller than the width of the cells, for example, preferably 0.002 mm to 0.01 mm (2 μm to 10 μm), more preferably 0.003 mm to 0.008 mm (3 μm to 8 μm), and even more preferably about 0.005 mm (5 μm). Furthermore, the width D6 of the intersection 9x is approximately equal to or greater than the width of the cell. For example, it is preferably 0.004 mm to 0.02 mm (4 μm to 20 μm), more preferably 0.006 mm to 0.016 mm (6 μm to 16 μm), and even more preferably about 0.01 mm (10 μm).

[0031] Furthermore, the depth D7 of the grooves 9 and intersections 9x is not particularly limited and can be, for example, the same dimension as the width D5 of the groove 9. In addition, as shown in Figure 8, the groove side wall 9s of the intersection 9x has a counterbore (a slope such that the width D6 gradually increases towards the upper open end) formed during processing, and the width D8 of the upper end surface of the counterbore is exemplified to be about twice the width D6 of the bottom surface of the intersection 9x. In addition, the method of forming the grooves 9, 9 and intersections 9x is not particularly limited and can be appropriately selected and used from patterning methods such as dry etching, wet etching, and ashing, and as for dry etching, for example, it can be formed by laser processing.

[0032] In the slide glass 1' configured in this way, with the biological sample placed or supplied to multiple sub-regions 7, the biological sample is irradiated with a laser, and microparticles formed from the biological sample are introduced into ICP-MS analysis, where the qualitative and quantitative analysis of metal elements contained in the microparticles is performed. At that time, by continuously performing ICP-MS analysis while relatively moving the biological sample and the laser irradiation position, the entire biological sample on the slide glass 1 is measured. In particular, since grooves 9,9 with a width D5 as described above are formed in the sub-regions 7 of the slide glass 1', cells C contained in the biological sample can easily diffuse throughout the multiple sub-regions 7 by traveling along these grooves 9,9. Furthermore, since the grooves 9,9 are orthogonal to each other and their intersections 9x have a width D6 as described above, cells C can easily be deposited individually at these intersections 9x. Therefore, since cells C can be deposited individually within each sub-region 7, there is an advantage in that quantitative analysis at the cell level is easier to achieve compared to conventional methods. In addition, since the metal wires 61 and 62 are formed on the bottom surface of the groove 10 formed on a part of the surface 1a, there is an advantage in that the liquid of the biological sample, and consequently the cells C, can be further diffused through the groove 10 into each of the multiple sub-regions 7. Furthermore, since a recessed portion (slope) is provided on the groove side wall 9s of the groove 9, the liquid of the biological sample can be diffused even more easily.

[0033] Figure 9 is a graph showing examples of signal intensity measurements when Mg in a slide glass 1 according to this disclosure, formed from quartz, and a conventional slide glass formed from materials such as borosilicate glass was quantified by LA-ICP-MS analysis. In the figure, the solid line graph ("novel plate 24Mg") shows the results for slide glass 1 according to this disclosure, and the dashed line graph ("conv. plate 24Mg") shows the results for the conventional slide glass. The signal intensity was also measured for both slide glasses at laser outputs of 20 mW, 30 mW, and 40 mW. As shown in the figure, the signal intensity derived from Mg in slide glass 1 according to this disclosure was 10 times higher than that of the conventional slide glass. ５ ~10 ６ Level 10 ３ It was confirmed that the level had been reduced significantly by about two orders of magnitude, falling below the normal level.

[0034] Furthermore, Figure 10(A) is a photograph showing the state after laser irradiation in LA-ICP-MS analysis on the surface 1a of the slide glass 1 according to this disclosure, in which a sub-region 3 is defined by a metal wire grating 2 (example of Ti). Figure 10(B) is an image showing the result of color imaging of the metal wire grating 2 using known image processing based on the signal intensity measurement results for the constituent element (Ti) of the metal wire grating 2 obtained by the LA-ICP-MS analysis. Note that the transverse stripes appearing on the metal wire grating 2 in Figure 10(A) indicate the trajectory of the collimated laser light as it was scanned. Although Figure 10(B) is a monochrome image, the region on the metal wire grating 2 in the original color image is displayed in a color tone indicating high signal intensity. Thus, it has been confirmed that the shape and position of the metal wire grating 2 shown in Figure 10(A) are clearly reproduced in the image shown in Figure 10(B) using the slide glass 1 according to this disclosure.

[0035] The embodiments described above with reference to specific examples are provided to facilitate understanding of this disclosure and are not intended to limit its interpretation. In other words, this disclosure is not limited to these specific examples, and modifications made to these examples by those skilled in the art are also included within the technical scope of this disclosure, as long as they retain the features of this disclosure. Furthermore, the elements, arrangements, materials, conditions, shapes, dimensions, scales, etc., of each of the aforementioned specific examples are not limited to those exemplified unless otherwise specified and can be modified as appropriate. Moreover, the elements of each of the aforementioned specific examples can be combined in different ways as appropriate, as long as no technical inconsistencies arise.

[0036] For example, sub-regions 3 and 7 may be formed in shapes other than square, such as rectangles or other polygons, in which case the metal wire grids 2 and 6 may also be grid shapes other than simple grids. Furthermore, the planar shape of the slide glass 1 and 1' itself is not limited to rectangles. In addition, alignment marks 4 and matrix code marks 5 do not need to be provided. Also, the number of grooves 9 provided in each sub-region 7 may be three or more, and they may not be orthogonal to each other but intersect at sharper angles. Furthermore, counterbore (inclined) portions do not need to be provided on the groove side walls 9s of the grooves 9.

[0037] …1,1'…Slide glass, 1a…Surface, 2,6…Metal wire grid, 3,7…Subregions, 4…Alignment marks, 5…Matrix code marks, 51…Column code marks, 52…Row code marks, 9…Groove (first groove), 9x…Intersection, 10…Groove (second groove), 21,61…Metal wire (first metal wire), 22,62…Metal wire (second metal wire), C…Cell, D1,D3…Side length, D2,D4,D5,D6,D8…Width, D7…Depth, K…Notch, M…Unprocessed region, x…First direction, y…Second direction

Claims

1. A microscope slide used for qualitative and / or quantitative analysis of metal elements contained in a biological sample by performing ICP-MS analysis while irradiating the biological sample with a laser, wherein the microscope slide is formed of at least one selected from the group consisting of quartz, crystal, silicon, sapphire, diamond, and alkali-free glass, and comprises a plurality of first metal wires extending in a first direction and arranged at predetermined intervals on a part of the surface on which the biological sample is placed or supplied, and a plurality of second metal wires extending in a second direction and arranged at predetermined intervals, wherein a plurality of sub-regions are defined by the plurality of first metal wires and the plurality of second metal wires, and the plurality of first metal wires and the plurality of second metal wires are formed of at least one metal selected from the group consisting of Re, Ir, Ta, Ni, Cr, Pt, Au, Ti, and Si.

2. The microscope slide according to claim 1, wherein each of the plurality of sub-regions has a plurality of intersecting first grooves.

3. The microscope slide according to claim 2, wherein the plurality of first metal wires and the plurality of second metal wires are provided in a second groove formed on a part of the surface.

4. The microscope slide according to claim 2, wherein the biological sample is a liquid sample containing cells, the width of each of the plurality of first grooves is approximately equal to or smaller than the width of the cells, and the width of the intersection of the plurality of first grooves is approximately equal to or larger than the width of the cells.

5. The microscope slide according to claim 1 or 2, wherein the remainder of the surface includes an unprocessed area.

6. A microscope slide according to claim 1 or 2, wherein one or more alignment marks for positioning the microscope slide are provided at a plurality of corners on the surface, and matrix symbols for identifying each of the plurality of sub-regions are provided at the periphery of the surface.

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