JP2008268063A - Evanescent wave generating device, and observation apparatus using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an evanescent wave generating device for providing evanescent waves by making the wave total reflect over a wide range at high density, and provide an observation apparatus capable of observing a measurement sample over a wide range with high sensitivity, by using the evanescent wave generating device. <P>SOLUTION: The evanescent wave generation device comprises a prism 5, having a pair of two faces 5A, 5B which face with each other in parallel, and at least a pair of two faces 5C, 5D which face each other in parallel, and are orthogonal to the two faces 5A, 5B, and a light incidence means 10 by which light enters in the prism 5 at a predetermined angle from the one face 5A out of any of the two faces. A part, except for a light incidence part 8 of the one face 5A and the face 5B facing to the one face 5A in parallel are formed of mirror surfaces 7. By the light incidence means 10, the light is incident in the prism 5 at an angle by which total reflection on a pair of two faces orthogonal to faces with the light incidence parts 8 and reflection on the mirror surfaces 7 are repeated in the prism 5. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to an evanescent wave generator for generating an evanescent wave and an observation apparatus using the same.

  In recent years, in tissue engineering, research on tissue regeneration based on cell culture has attracted attention and is said to change the quality of medicine. Among them, by applying cultured cells to a medical device, techniques for improving the compatibility with the living body at the time of transplantation (for example, techniques for placing cultured cells on a bone matrix) and culturing into cells having various functions Techniques (for example, techniques for differentiating mesenchymal stem cells into cells such as cardiac muscle, blood vessels, and skin) have been rapidly advanced due to the development of regenerative medicine. In such a cell culture technique, it is necessary to evaluate the state of the cultured cell and use it for medical purposes, and devices and techniques therefor are being developed along with the progress of tissue engineering.

  In particular, an observation device using evanescent waves can observe only a part of cells (about 100 nm above the total reflection surface) from the total reflection surface, and can obtain a clear observation map with reduced background. Therefore, the state of cells that are in contact with the culture vessel and proliferate is attracting attention as being able to observe more clearly. Specifically, as shown in FIG. 39, this apparatus uses a prism 105 or the like on a bottom surface (a refractive index boundary surface and a total reflection surface) of a sample container 140 on which a cell to be observed is placed at a predetermined angle. The excitation light is irradiated and reflected on the bottom surface (refractive index boundary surface and total reflection surface) of the sample container 140, and at that time, about 100 nm above the total reflection surface by the evanescent wave generated on the total reflection surface. This excites the observation sample (cell).

  By limiting the region where light (evanescent wave) reaches in this way to about 100 nm and detecting scattered light and fluorescence generated from the observation sample (cell) in that region, the state of the observation sample can be made clearer. Detection is possible, background noise can be suppressed, and clearer fluorescence observation is possible. However, even in such an apparatus, the range that can be observed in the planar direction is only the place where light hits the total reflection surface. That is, the observable range depends on the width (thickness) of the light beam (FIG. 40). In order to solve this problem, as shown in FIG. 41, increasing the total reflection point to widen the generation range of the evanescent wave is one means of widening the observation range in the plane direction. In particular, in the field of regenerative medicine, there has been a strong demand for observing the state of the entire cell sample used for treatment in a short time rather than local observation of cells.

  In order to widen the observation range in this way, an apparatus that causes total reflection multiple times on the total reflection surface has been developed (see, for example, Patent Document 1).

The apparatus described in Patent Document 1 has an observation sample placed on a slide glass, and excitation light is incident from the side surface of the slide glass, and the slide glass is used as an optical path through which the excitation light propagates. That is, the excitation light that has entered the slide glass from the side surface of the slide glass travels through the slide glass while being totally reflected by one surface and the other surface, and exits from the opposite side surface. At this time, since the evanescent wave is generated at the point (point) where the excitation light is totally reflected, scattered light and fluorescence can be generated from the cells in the arrival area of the evanescent wave. With such an apparatus, the observation range can be expanded to the surface.
Japanese Patent Laid-Open No. 9-61346

  By the way, in such an apparatus using an evanescent wave, in order to obtain a wide range and a highly sensitive observation surface, the number of total reflections is increased and the points of total reflection on each surface become denser. That is, it is necessary to increase the density of total reflection points on the total reflection surface. However, when the above device is used, in order to increase the number of total reflections, it is necessary to make the thickness of the slide glass thinner. However, there is a limit to reducing the thickness of the slide glass. Sensitive observation could not be realized.

  In view of such a problem, the present invention has been made to solve the conventional technical problem, and an evanescent wave can be obtained by causing total reflection in a wide range and at a high density. It is an object of the present invention to provide an evanescent wave generator and an observation device that can realize observation of an observation target substance such as a cell with high sensitivity and in a wide range using the evanescent wave generator.

  The evanescent wave generator according to the first aspect of the present invention includes a set of two parallelly facing surfaces, a prism having at least one pair of parallelly facing two surfaces orthogonal to the two surfaces, and any two surfaces. A light incident means for entering light into the prism at a predetermined angle from any one of the surfaces, and a portion excluding the light incident portion on one surface and a surface facing the one surface in parallel are configured as mirror surfaces The light incident means is characterized in that light is incident on the prism at an angle at which total reflection on a pair of two surfaces orthogonal to the surface having the light incident portion and reflection on the mirror surface are repeated in the prism. And

  An evanescent wave generator according to a second aspect of the invention includes a set of two parallelly facing surfaces, a prism having at least one pair of parallelly facing two surfaces orthogonal to the two surfaces, and any one of the two surfaces. A light incident means for entering light into the prism at a predetermined angle from any one of the surfaces, and a portion excluding the light incident portion on one surface and a surface facing the one surface in parallel are configured as mirror surfaces The light incident means has any one of a pair of two surfaces orthogonal to the surface having the light incident portion and a container surface disposed directly on one surface facing the one surface in parallel, or the light incident portion. The total reflection on the container surface and the reflection on the mirror surface are repeated in the prism, with one of the two surfaces orthogonal to the surface and one surface facing the surface parallel to the surface being interposed via the medium. The light is incident on this prism at a certain angle. .

  An evanescent wave generator according to a third aspect of the present invention is characterized in that any one of a pair of two surfaces orthogonal to a surface having a light incident portion is configured as a mirror surface in each of the above inventions.

  The observation device according to the fourth aspect of the present invention is the observation device according to claim 4, wherein one of the two surfaces orthogonal to the surface having the light incident portion or one of the two surfaces orthogonal to the surface having the light incident portion and the one surface. The container surface arranged directly on one surface parallel to the surface, or one of the two surfaces orthogonal to the surface having the light incident portion and one surface facing the surface parallel to the one surface via the medium 4. The evanescent wave generator according to claim 1, further comprising means for detecting scattered light and / or fluorescence of the measurement sample by an evanescent wave generated on the arranged container surface. Is used.

  An observation apparatus according to a fifth aspect of the invention is characterized in that, in the invention according to the fourth aspect of the invention, a plurality of types of filters that selectively pass scattered light and / or fluorescence in accordance with the wavelength are provided.

  According to the evanescent wave generator of the first aspect of the present invention, a set of two parallelly facing surfaces and a prism having at least one pair of parallelly facing two surfaces orthogonal to the two surfaces, A light incident means for entering light into the prism at a predetermined angle from any one of the surfaces, and the portion excluding the light incident portion on one surface and the surface facing the one surface in parallel are configured as mirror surfaces. In addition, the light incident means makes light enter the prism at an angle at which total reflection on a pair of two surfaces orthogonal to the surface having the light incident portion and reflection on the mirror surface are repeated in the prism. Thus, more total reflection is generated at a high density on a pair of two surfaces orthogonal to the surface having the light incident portion, and an evanescent wave can be easily generated in a wide area.

  This makes it possible to observe cells and the like with a wide range and with high sensitivity using the evanescent wave generator.

  According to the evanescent wave generator of the invention of claim 2, any one of the two sets of parallel opposing surfaces and at least one set of the parallel opposing prisms orthogonal to the two surfaces, A light incident means for entering light into the prism at a predetermined angle from any one of the surfaces, and the portion excluding the light incident portion on one surface and the surface facing the one surface in parallel are configured as mirror surfaces. In addition, the light incident means is either a container surface disposed directly on one surface of the pair of two surfaces orthogonal to the surface having the light incident portion and one surface parallel to the one surface, or the light incident portion. The total reflection at the container surface and the reflection at the mirror surface are arranged in the prism, with one of the two surfaces orthogonal to the surface having a surface and one surface parallel to the one surface facing the surface. Since light is incident on this prism at an angle repeated at A container surface arranged directly on one of the two surfaces orthogonal to the surface having the projecting portion and one surface parallel to the one surface, or a set orthogonal to the surface having the light incident portion A total surface of the container disposed between the surface of one of the two surfaces and the surface that is parallel to the one surface via the medium causes more total reflection to occur at a high density, so that evanescent waves can be easily generated in a wide area. Be able to occur.

  This makes it possible to observe cells and the like with a wide range and with high sensitivity using the evanescent wave generator.

  According to the invention of claim 3, since any one of the two surfaces orthogonal to the surface having the light incident part in each of the above inventions is constituted by a mirror surface, it is orthogonal to the surface having the light incident part. The light can be totally reflected on any one surface of the set of two surfaces, the container surface arranged directly on the one surface, or only the container surface arranged on the one surface via the medium. In particular, if impurities such as dust adhere to the surface to be totally reflected, light scattering is difficult to occur there, and total reflection cannot be obtained, so the surface to be totally reflected, or the surface of the container directly disposed on the one surface, or By using only the surface of the container disposed through the medium, it is possible to suppress as much as possible the inconvenience that a light transmission defect occurs.

  According to the observation device of a fourth aspect of the present invention, a set of two surfaces orthogonal to the surface having the light incident portion of the evanescent wave generator according to any one of the first to third aspects, or the light incident portion. The container surface disposed directly on one surface of the pair of two surfaces orthogonal to the surface having a surface and the surface facing the one surface in parallel, or the pair of two surfaces orthogonal to the surface having the light incident portion Since it has means for detecting scattered light and / or fluorescence of the measurement sample by an evanescent wave generated on the surface of the container disposed through the medium on one surface of the surface and parallel to the one surface, It becomes possible to detect scattered light and fluorescence of a measurement sample which is an observation target substance such as a cell.

  As a result, it becomes possible to detect the scattered light and fluorescence of the measurement sample using the evanescent wave generator of each of the inventions described above, and not a local observation range as in the conventional apparatus, but a wide range and high An observation apparatus that enables sensitive observation can be constructed. In particular, it is effective when observing a measurement sample that cannot be directly arranged on the prism surface, for example, a cell in a culture container, and if the medium is to be inserted, the prism and the container are brought into close contact with each other. The refractive index can be adjusted, and total reflection at the interface between the container and the measurement sample can be caused.

  Further, in the invention of claim 4, if a plurality of types of filters that selectively pass scattered light and / or fluorescence according to wavelength are provided as in claim 5, detection is performed according to the purpose of observation and the like. The wavelength can be freely selected.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 shows a configuration diagram of an observation apparatus S1 including an evanescent wave generation apparatus 1 according to an embodiment of the present invention. This observation device S1 includes an evanescent wave generation device 1 described later and a light detection means 20. Specifically, the observation device S1 of the present embodiment uses a measurement sample that becomes an observation target substance such as a cell from the evanescent wave generated by the evanescent wave generation device 1 (in this embodiment, the measurement target is made to fluoresce when irradiated with excitation light). By irradiating from the lower surface of the sample container 40 containing the cells to be emitted), scattered light and fluorescence are detected from the cells by the light detection means 20 arranged on one side (upper in FIG. 1) of the sample container 40. It is configured.

  The light detection means 20 is means for detecting scattered light and fluorescence from the cell by the excitation light from the evanescent wave generator 1. The light detection means 20 of this embodiment passes through an objective lens 45 for condensing scattered light and fluorescence from the cell, an optical filter 46 for removing a specific wavelength from the scattered light and fluorescence, and the optical filter 46. An imaging lens 47 that forms an image of the scattered light and fluorescence that has been formed, and a CCD camera 48 that two-dimensionally detects and images a light image from the imaging lens 47. The means for detecting scattered light and fluorescence is not limited to the light detection means 20 of the embodiment, and any detection means can be used as long as it can detect scattered light and fluorescence. For example, in the embodiment, the CCD camera 48 is used, but a light receiving element such as a photodiode may be used. In FIG. 1, reference numeral 50 denotes a lens, and 52 denotes an optical fiber, which efficiently collects scattered light and fluorescence and guides them to a light receiving element.

  Next, the evanescent wave generator 1 of the present invention described above will be described. The evanescent wave generator 1 according to the present invention includes a prism 5 having a set of two oppositely facing surfaces and at least one set of two oppositely facing surfaces orthogonal to the two surfaces, and one of the two surfaces. Light incident means 10 is provided for injecting light into the prism 5 at a predetermined angle from any one surface.

  The prism 5 of the embodiment is made of BK7 (manufactured by SCHOTT GLASS), and its refractive index is 1.52. The prism 5 has a set of two oppositely facing surfaces 5A and 5B and a set of two oppositely facing surfaces 5C and 5D perpendicular to the two surfaces. Specifically, as shown in FIG. 2, the prism 5 of the present embodiment is configured by a rectangular parallelepiped having a longitudinal dimension (length) L of 82 mm and a height dimension (thickness) T of 36 mm. A light incident portion 8 for excitation light from the light incident means 10 described later is formed on the one surface 5A. The light incident portion 8 is an excitation light incident point for allowing the excitation light from the light incident means 10 to enter the prism 5. Further, a portion of the one surface 5A excluding the light incident portion 8 and a surface 5B that faces the one surface 5A in parallel are configured as a mirror surface 7.

  The mirror surface 7 is formed on the surface 5A (front surface in FIG. 2) of the prism 5 and the portion excluding the upper end portion and the lower end portion of the surface 5B (back surface in FIG. 2) facing it. The mirror surface 7 of the present embodiment has a height dimension (thickness) of 19 mm, and the center in the height direction of each surface 5A, 5B of the prism 5 and the center in the height direction of the mirror surface 7 are substantially the same. Are provided on the surfaces 5A and 5B.

  In this embodiment, the light incident portion 8 as the excitation light incident point is set to the lower side of the mirror surface, and the excitation light is incident on the prism 5 from here. However, the present invention is not limited to this. It does not matter even if it is on the upper side. Further, in this embodiment, the mirror surface 7 is formed on the surface 5A (front surface in FIG. 2) and the surface 5B (back surface in FIG. 2) opposite to the upper surface portion and the lower end portion, respectively. The configuration is not limited to this. For example, various configurations as shown in FIGS. 25 to 30 can be applied. That is, the one surface 5A has any configuration as long as the mirror surface 7 is formed on a surface other than the light incident portion 8 that causes at least the excitation light from the light incident means 10 to enter the prism 5, as shown in FIGS. This is not limited to the configuration of 30 to 28. In particular, the entire surface 5B may be a mirror surface 7 as shown in FIG.

  On the other hand, the light incident means 10 described above is an excitation light source that makes light incident on the prism 5 at a predetermined angle. The light incident means 10 of the present embodiment includes a lamp (for example, a mercury lamp), an optical filter, and a lens. The light of this lamp contains wavelengths of various components, but by passing through the optical filter, only light other than the specific wavelength is cut by the optical filter, and light of a specific wavelength (light of a preset wavelength) ) Pass through the optical filter, and the light passing therethrough is emitted as excitation light through the lens and the wave plate 11.

  The excitation light emitted from the light incident means 10 may have any wavelength, but the wavelength may be selected according to the characteristics of the cell to be detected.

  By the way, in a conventional observation apparatus using an evanescent wave, as shown in FIG. 40, an excitation light is irradiated to a point on the lower surface of a cell or the like and totally reflected there to generate an evanescent wave. Generated scattered light and fluorescence. That is, such a conventional device can observe only a local observation range, that is, a cell in the vicinity of the focus in a portion where the excitation light is totally reflected, and a wide range of observation can be performed. could not. In particular, in the field of regenerative medicine, there has been a strong desire to observe the entire state in a shorter time than such local observation.

  In this way, in order to enable observation over a wider range, more total reflection is caused on the lower surface of the target cell or the like (observation target substance) to obtain an evanescent wave due to total reflection in a wide area. There must be. For the purpose of observation in such a wide range, an evanescent wave generator is also developed in which cells are placed on the slide glass 60 shown in FIG. 38 and light is incident on the slide glass 60 to propagate the light. Have been. In this case, the excitation light that has entered the slide glass 60 from one side surface proceeds through the slide glass 60 while being totally reflected by the one surface 60A and the other surface 60B of the slide glass 60, and exits from the other side surface. At this time, an evanescent wave is generated at a location where the light is totally reflected, and scattered light and fluorescence can be generated from a cell in the arrival area of the evanescent wave.

  By arranging cells on the slide glass 60 as described above and propagating light to the slide glass 60 to cause a plurality of total reflections, it becomes possible to generate more evanescent waves on the lower surface of the cell, and the observation range Can be expanded to the surface.

  By the way, in such an apparatus using an evanescent wave, in order to realize a wide and highly sensitive observation surface, the number of total reflections is increased and the points of total reflection on each surface become denser. That is, it is necessary to cause total reflection more densely on the total reflection surface. In this case, in the above apparatus, the thickness of the slide glass 60 must be made thinner. However, since there is a limit to reducing the thickness of the slide glass, it is difficult to make the point of total reflection dense. Therefore, high-sensitivity observation could not be realized.

  Therefore, in the present invention, as described above, the mirror 5 is formed on the prism 5 and the pair of parallelly opposed two surfaces 5A and 5B as described above, and orthogonal to the two surfaces 5A and 5B on which the mirror surface 7 is formed. A pair of parallel opposing two surfaces 5C, 5D, or the surface 5D and the surface of the sample container 40 directly disposed on the one surface 5C opposing the surface 5D, or the surface 5D and the surface 5D. Thereafter, total reflection on the surface of the sample container 40 disposed on the opposite surface 5C through a medium (imulsion oil, oil, aqueous solution of sucrose or salt, gel, silicon, etc.) for adjusting the refractive index. It is assumed that the excitation light from the light incident means 10 enters the prism 5 at such an angle that the reflection on the mirror surface 7 is repeated.

  In this case, as described above, the excitation light from the light incident means 10 is applied to the prism surfaces 5C and 5D, the surface of the sample container 40 directly disposed on the prism surfaces 5C and 5D, or the prism surfaces 5C and 5D. The light is incident on the prism 5 at an angle such that total reflection and reflection on the mirror surface 7 are repeated on the surface of the sample container 40 disposed through the medium for adjusting the refractive index, and the light is incident on each of the prism surfaces 5C and 5D. It is necessary to find an optimum angle that closes the point of total reflection.

  The optimum angle is determined in consideration of the material of the prism (particularly the refractive index), the length dimension L and the thickness dimension T, and the refractive index of the target substance when the target substance is arranged directly on the surface 5C of the prism 5. Need to be set. Further, when the sample container 40 is arranged on the prism surface 5C as in the apparatus S1 of FIG. 1 and the measurement sample to be observed is stored in the sample container 40, in addition to the above, the material of the sample container 40 (particularly, , Refractive index), thickness dimension, and the like. Further, when the sample container 40 is disposed on the prism surface 5C via a medium for adjusting the refractive index, the refractive index of the medium for adjusting the refractive index must be taken into consideration.

  In the present embodiment, an optimal incident angle determination method when a measurement sample (for example, a cultured cell) is placed directly on the surface 5C of the prism 5 described above will be described. In this case, the total reflection surface is a boundary surface between the prism surface 5C of the prism 5 and the detection target.

  First, when excitation light is incident on the prism 5 from the light incident means 10 at a certain angle, for example, 37 degrees as shown in FIG. 3, the angle of total reflection (total reflection angle) occurring on the prism surface 5C is set to snell. (Formula (1)).

n1 × sin θ1 = n2 × sin θ2 (1)
In this case, in Equation (1), n1 is the refractive index of air (that is, 1), n2 is the refractive index of the prism 5 (1.52 described above), and θ1 is the incidence of excitation light from the light incident means 10. Since the angle (here 37 degrees) and θ2 is the angle of the excitation light immediately after entering the prism 5 (see FIG. 3),
1sin37 = 1.52sinX
X = 23.32.
It becomes. X is the angle of the excitation light immediately after entering the prism 5 as described above, and the sum of the angle of the excitation light immediately after entering the prism 5 and the reflection angle (total reflection angle) generated at the prism surface 5C is Because it is 90 degrees,
Total reflection angle + X = 90
That is,
Total reflection angle = 90-X
When the above calculated value is substituted for X,
Total reflection angle = 90-23.32 = 66.68
Therefore, when the excitation light is incident on the prism 5 from the light incident means 10 at an incident angle of 37 degrees, the total reflection angle at the prism is 66.68 degrees.

  From the above, the total reflection angle A on the prism surface 5C in the case of the incident angle θ1 of the excitation light entering the prism 5 from the light incident means 10 is n1 for the refractive index on the incident side of the excitation light and n2 for the refractive index of the prism 5. Then, it can be derived from the following formula (2).

A = 90−sin ⁻¹ (n1 × sin θ1 / n2) (2)
Next, when the target substance is arranged on the prism surface 5C of the prism 5 according to the mathematical formula (1), the angle at which total reflection occurs on the lower surface of the target substance (total reflection angle) is obtained. In this case, in Equation (1), n1 is the refractive index of the prism 5 (1.52 described above), n2 is the refractive index of the measurement sample, θ1 is the angle B at which total reflection occurs, and θ2 is 90 degrees.

Here, if the measurement sample placed on the prism surface 5C of the prism 5 is water as shown in FIG. 4, the refractive index of water is 1.33.
n1 × sin θ1 = n2 × sin θ2
1.52 × sinB = 1.33 × sin90
sinB = 1.33 / 1.52
B = 61.04498
It becomes. Therefore, when the target substance is water, total reflection occurs when the angle of reflection (total reflection angle) generated on the prism surface 5C is 61.05 degrees or more.

On the other hand, if the measurement sample placed on the prism surface 5C of the prism 5 is, for example, a cell having a refractive index of 1.38,
n1 × sin θ1 = n2 × sin θ2
1.52 × sinB = 1.38 × sin90
sinB = 1.38 / 1.52
B = 65.216
It becomes. Therefore, when the measurement sample is a cell having a refractive index of 1.38, total reflection occurs when the angle of reflection (total reflection angle) generated on the prism surface 5C is 65.22 degrees or more.

  Based on the above results, the incident angle of the total reflection generated on the prism surface 5C when the excitation light is incident from the light incident means 10 into the prism 5 within the range of the incident angle that is equal to or larger than the angle at which the total reflection occurs on the prism surface 5C. Dependency was investigated. The results are shown in FIGS. FIGS. 5 to 9 show optical paths of the excitation light that passes through the prism 5 when the excitation light is incident on the prism 5 at each incident angle. 6 to 9, the mirror surface 7 provided on the surfaces 5A and 5B of the prism 5 is omitted, but actually the mirror surface 7 exists as shown in FIG.

  As shown in FIG. 5, when the excitation light is incident on the prism 5 at an incident angle of 34 degrees, the excitation light incident on the prism 5 does not sufficiently repeat the prism 5, and the surface 5A, Leakage immediately leaked from the portion where the mirror surface 7 of 5B was not arranged, and the points of total reflection on the prism surface 5C (the same applies to the prism surface 5D) could not be made dense.

  FIG. 6 shows the result when the excitation light is incident on the prism 5 at an incident angle of 35 degrees. In this case as well, the excitation light that has entered the prism 5 leaks immediately from the portion of the surfaces 5A and 5B where the mirror surface 7 is not disposed (thick line arrows shown in FIG. 6). The point of total reflection on the surface 5C (same for the prism surface 5D) could not be made dense.

  FIG. 7 shows the result when the excitation light is incident on the prism 5 at an incident angle of 36 degrees. In this case, the excitation light is repeatedly reflected in the prism 5 without leaking immediately from the portion of the surfaces 5A and 5B where the mirror surface 7 is not disposed. Therefore, the point of total reflection on the prism surface 5C (also the prism surface 5D) can be made relatively dense.

  Further, FIG. 8 shows a result when the excitation light is incident on the prism 5 at an incident angle of 37 degrees. In this case, the excitation light is reflected repeatedly inside the prism 5 without leaking immediately from the prism 5. Therefore, the point of total reflection on the prism surface 5C (also the prism surface 5D) can be made denser.

  FIG. 9 shows the result when the excitation light is incident on the prism 5 at an incident angle of 38 degrees. In this case, the excitation light incident on the prism 5 is not reflected by the mirror surfaces 7 of the surfaces 5A and 5B, but leaks from the portion where the mirror surfaces 7 of the surfaces 5A and 5B are not arranged, as indicated by thick arrows. Oops.

  From the above results, when the prism 5 is used, when the incident angle is 37 degrees as shown in FIG. 8, the points of total reflection on the prism surface 5C (same for the prism surface 5D) are in the most dense state. I found out that I could do it.

  On the other hand, FIG. 10 shows the result that the most preferable high-density total reflection can be generated on the prism surface when a prism other than the prism 5 is used. In this case, a prism 6 having the same thickness dimension as that of the prism 5 (36 mm) and a different length dimension (77 mm) was used. The material of the prism 6 is BK7 (manufactured by SCHOTT GLASS), which is the same as that of the prism 5, and the refractive index is 1.52.

  When this prism 6 is used, when the incident angle is set to 40 degrees as shown in FIG. 10, the points of total reflection on the prism surface 6C (the same applies to the prism surface 6D) can be brought into the most dense state. .

  Thus, even when prisms of the same material are used, the optimum angle of incidence differs because the point of total reflection changes in the prism by changing the ratio between the thickness dimension and the length dimension. I understood it. Therefore, the angle of incidence and the density of the points of total reflection on the prism surface were measured using several prisms having different thickness and length ratios. The result is shown in FIG. FIG. 11 shows the density of total reflection points generated on the prism surface when the incident angle is changed using prisms of various ratios (length dimension / thickness dimension), that is, total reflection per 1 mm unit of the prism surface. Shows the number of times it occurred. In FIG. 11, the result of FIG. 7 using the prism 5 is the result of using a prism with a ratio of 2.28 and setting the incident angle to 36 degrees (that is, the total reflection point density of 0.1433 (times / mm)). The result of setting the incident angle to 37 degrees using the prism with the same ratio (2.28) corresponds to the result of FIG.

  In FIG. 11, when the number of total reflections per 1 mm unit of the prism surface is 0.09 (times / mm) to 0.14 (times / mm), total reflection is obtained over the entire prism surface. Although it was possible, the point of total reflection could not be made dense. Therefore, it is considered that sufficient sensitivity cannot be obtained. On the other hand, in the case of 0.14 (times / mm) to 0.35 (times / mm), total reflection can be obtained over the entire prism surface, and the points of total reflection are relatively dense. It was. Therefore, it is considered that good sensitivity can be obtained. Furthermore, when the number of total reflections in the unit of 1 mm of the prism surface was 0.35 (times / mm) or more, the points of total reflection became more dense.

  In FIG. 12, the number of total reflections per 1 mm unit of the prism surface in FIG. 11 is 0.09 (times / mm) to 0.14 (times / mm), 0.14 (times / mm) to The density of the points of total reflection on the prism surface was evaluated from 1 to 3, with 0.35 (times / mm) being 2, and 0.35 (times / mm) being 3 or more. In this way, according to each prism, by selecting an incident angle such that the density evaluation value shown in FIG. 12 is 2 or more, it becomes possible to obtain an observation surface with good sensitivity over a wide range, Preferably, by selecting an incident angle such that the density evaluation value is 3, it is possible to obtain a wider and more sensitive observation surface.

  Next, the deviation of the reflection point of light for each reflection pattern and the efficiency of the total reflection will be described. FIG. 23 shows a reflection pattern when excitation light is incident from below the mirror surface 7 configured on the surface 5A, and FIG. 24 shows an example of a reflection pattern when excitation light is incident from above the mirror surface 7 of the surface 5A. Show. In the case of FIG. 23, when excitation light is incident on the prism 5 from the surface 5A by the light incident means 10, the incident excitation light is totally reflected by the prism surface 5D and then reflected by the mirror surface 7 of the surface 5B. After total reflection at the surface 5C, the surface returns to the surface 5A. Then, the light is reflected by the mirror surface of the surface 5A, and again undergoes total reflection by the prism surface 5D, and a state of total reflection is formed while repeating these processes. After passing through the first incident point on the surface 5A, the prism surface 5D, the surface 5B, and the prism surface 5C, the surface 5A is returned to the surface 5A again. The relationship between the dimension T, the length dimension L of the prism 5, and the incident angle θ of light incident on the prism from the light incident means 10 can be expressed by the following mathematical formula (3).

k = 2 × [TL × tan {sin−1 (n1 / n2 × sin θ)}] (3)
Here, assuming that the light beam density of the excitation light emitted from the light incident means 10 is constant within the light beam, the following relationship exists between the width (light beam width) of the excitation light and k. That is, when excitation light is incident from the lower side of the mirror surface 7 formed on the surface 5A as shown in FIG. 23, when k calculated by Equation (3) is k <0 or k = 0, the prism All the light leaks from the excitation light incident on 5 after one total reflection. If 0 <k <excitation light width, a dense total reflection point can be obtained, but some light leaks. Considering only the density of total reflection points, the closer the value of k is to 0, the more total reflection points increase, but more light leaks. If k = the width of the excitation light, dense total reflection can be obtained without light leakage, and the most efficient total reflection surface without light leakage can be obtained. Furthermore, if k> the width of the excitation light, a point of total reflection can be obtained in the prism 5, but the total reflection density decreases as the difference between the width of k and the excitation light increases.

  On the other hand, when the excitation light is incident from above the mirror surface 7 of the surface 5A as shown in FIG. 24, if k calculated by the equation (3) is set to −k <0 or −k = 0, the light enters the prism 5. All the light leaks from the excitation light after one total reflection. If 0 <−k <excitation light width, a dense total reflection point can be obtained, but some light leaks. Considering only the density of total reflection points, the closer the value of −k is to 0, the more total reflection points increase, but more light leaks. When −k = the width of the excitation light, dense total reflection can be obtained without light leakage, and the most efficient total reflection surface with no light leakage can be obtained. Furthermore, if -k> excitation light width, a point of total reflection can be obtained in the prism 5, but the total reflection density decreases as the difference between -k and the width of excitation light increases.

  In this way, by making the width of the excitation light incident on the prism 5 from the light incident means 10 closer to k, the total reflection point is made dense to obtain an efficient total reflection surface with no light leakage. Can do. Therefore, a highly sensitive observation surface can be obtained over a wide range.

  As described in detail above, from the light incident means 10 to the excitation light prism so that total reflection on the prism surfaces 5C and 5D intersecting the surfaces 5A and 5B on which the surface 7 is formed and reflection on the mirror surface 7 are repeated in the prism 5. By determining the incident angle of the excitation light incident on the prism surface, more total reflection is generated at the prism surfaces 5C and 5D at a high density, and an evanescent wave by total reflection can be easily generated in a wide area of the prism surfaces 5C and 5D. Be able to get.

  As a result, the observation of the measurement sample using the evanescent wave generator 1 can be realized in a wide range and at a high density. In other words, the evanescent wave generator 1 is applied to an observation device having means for detecting scattered light and fluorescence obtained by the evanescent wave generated on the prism surface of the evanescent wave generator, thereby providing the evanescent wave generator 1. It is possible to generate scattered light and fluorescence using and to detect this. Therefore, it is possible to construct an observation apparatus capable of observing a measurement sample with high sensitivity over a wide range rather than a local observation range as in the conventional apparatus.

  The above results shown in FIGS. 5 to 12 are results when water and cells are directly placed on the prism surface 5C. Therefore, in order to observe a measurement sample that cannot be directly arranged on the prism surface 5C, for example, a cell in the culture container, the sample container 40 is placed on the upper surface of the prism 5 like the observation device S1 in FIG. It is preferable to store the measurement sample in the container 40.

  However, in the above case, it becomes difficult to bring the prism surface 5C and the bottom surface of the sample container 40 into close contact with each other, the total reflection surface becomes the prism surface 5C, and evanescent waves generated on the prism surface 5C are generated in the sample container 40. It was difficult to reach the measurement sample.

  Therefore, when the sample container 40 is used as described above, a medium for adjusting the refractive index is inserted between the sample container 40 and the surface 5C in order to bring the surface 5C of the prism 5 into close contact with the sample container 40. It is desirable. Thus, by inserting the medium between the sample container 40 and the surface 5C on which the container 40 is installed, the surface 5C and the sample container 40 can be brought into close contact with each other to adjust the refractive index, and the sample container 40 can be adjusted. And total reflection can be caused at the interface of the measurement sample.

  When the medium is inserted between the sample container 40 and the surface 5C as described above, it is necessary to determine the optimum incident angle in consideration of the refractive index of the sample container 40 and the refractive index of the medium. is there. At this time, since the angle of total reflection changes depending on the difference between the refractive index of the medium and the refractive index of the measurement sample (when the measurement sample and the medium are in direct contact), it is necessary to pay attention to this point. In this case, it is an essential condition that the refractive index of the medium is larger than the refractive index of the measurement sample. In particular, it is preferable to use a medium having substantially the same refractive index as that of the prism 5, but it is necessary to select the medium with care so as to totally reflect at the interface with the measurement sample. As described above, oil (such as immersion oil), an aqueous solution such as sucrose or sodium chloride, gel, silicon or the like can be considered as the medium.

  The prism of the present invention is not limited to the shape and size shown in the embodiment, and for example, the prism shown in FIGS. 32 to 36 can be used. That is, in the present invention, a prism having a pair of parallel opposing surfaces and at least one pair of parallel opposing surfaces orthogonal to the two surfaces, any one of the two surfaces. Any shape is effective as long as a light incident portion is formed on the surface 7 and a mirror surface 7 is formed on a surface that is parallel to the surface excluding the light incident portion.

  Furthermore, in the present embodiment, the mirror surface 7 is formed on a pair of parallelly facing two surfaces 5A and 5B of the prism 5, but in addition to the surfaces 5A and 5B, the two surfaces 5A are shown in FIG. Any one of a pair of two surfaces 5C and 5D orthogonal to 5B (surface 5D in FIG. 37) can also be a mirror surface 7. In this case, light is totally reflected only on one surface 5C, the surface of the sample container 40 directly disposed on the one surface 5C, or only the surface of the sample container 40 disposed on the one surface 5C via the medium. In particular, if impurities such as dust adhere to the surface to be totally reflected, light scattering is difficult to occur there, resulting in a problem that total reflection cannot be obtained. Therefore, as shown in FIG. 37, the surface 5D facing in parallel with the surface 5C used for observation is also a mirror surface, so that the total reflection is performed on the surface 5C, or the surface of the sample container 40 disposed directly on the surface 5C, or the medium. Only the surface of the sample container 40 disposed via the surface of the sample container 40 can be used, and the inconvenience that the light transmission defect occurs can be suppressed as much as possible.

  The observation device S1 shown in the above embodiment is provided with one optical filter 46. However, the present invention is not limited to this, and may be provided with an optical filter switching means 30 having a plurality of optical filters as shown in FIG. There is no problem. Specifically, the observation device S2 shown in FIG. 13 irradiates the evanescent wave emitted from the evanescent wave generator 1 from the lower surface of the sample container 40 in which the measurement sample (for example, cultured cells) is accommodated, thereby scattering light. Or fluorescent light, and sequentially passes through an objective lens 45 disposed on one side (upper in FIG. 13) of the sample container 40 and any one of the optical filters provided in the optical filter switching means 30, so that scattered light and fluorescent light are emitted. A configuration in which light having a specific wavelength is removed is detected and picked up by a light receiving element 49 such as a CCD camera or a photodiode. In FIG. 13, the same reference numerals as those in FIGS. 1 to 12 have the same or similar effects or actions, and thus the description thereof is omitted here.

  The optical filter switching means 30 is for selecting scattered light and fluorescence detected by the light receiving element 49. The optical filter switching means 30 of the present embodiment is configured by a turret type optical filter turret including a plurality of types of optical filters that selectively allow scattered light and fluorescence to pass through according to the wavelength.

  Specifically, the optical filter turret of the present embodiment is arranged on a disk-shaped substrate 32 configured to be rotatable by a rotary motor 31 as shown in FIG. Four optical filters 35 to 38 are arranged, and are rotatably provided by a rotary motor 31 via a rotary shaft 33 having one end attached to the center of the substrate 32. In FIG. 14, the right side of the rotation shaft 33 (the position of the optical filter 35 in FIG. 14) is the use position in the observation apparatus S2 of the present embodiment. That is, the substrate 32 is rotated by the rotary motor 31 and moved to a predetermined use position (the position of the optical filter 35 in FIG. 14), so that each of the optical filters 35 to 38 is caused to emit scattered light and fluorescence according to the wavelength. Can be used selectively. As described above, the observation apparatus S2 according to the present embodiment includes a plurality of types of optical filters 35 to 38 that selectively pass scattered light and fluorescence according to the wavelength. Therefore, the wavelength to be detected according to the purpose of observation and the like is determined. You can choose freely.

  In the embodiment, the optical filter turret is used as the optical filter switching means 30 as described above, but the filter according to claim 4 is not limited to this. That is, the invention of claim 4 only needs to include a plurality of types of filters that selectively allow scattered light and fluorescence to pass through according to the wavelength. For example, as shown in FIG. A plurality of optical filters (three optical filters 35 to 37 in FIG. 15) are arranged at equal intervals in the longitudinal direction, and a rack and pinion type switching device configured to be movable in the longitudinal direction may be used. In addition, a slide type that manually switches a plurality of types of filters may be used.

  In the evanescent wave generator 1 shown in each of the above embodiments, the incident position of the excitation light can be configured to be movable. As directions for moving the incident position of the excitation light, two directions of the X-axis direction (that is, the length L direction) and the Y-axis direction shown in FIGS. 16 and 17 are conceivable. 16 is a perspective view of the prism 5 and the sample container 40 of FIG. 1 or FIG. 13, and FIG. 17 is a plan view of the sample container 40 of FIG.

  In this case, the prism 5 itself may be configured to be movable in the X-axis direction or the Y-axis direction, or the sample container 40 may be configured to be movable. The light incident means 10 may include a means for moving the incident position of the excitation light.

  For example, when the incident position of excitation light can be moved in the Y-axis direction, the light incident means 10 includes a scanning device 70 (that is, means for moving the incident position of excitation light) as shown in FIG. The incident position of the excitation light from the excitation light source 72 can be moved in the Y-axis direction by a beam scanning method in which the scanning device 70 is moved in the direction indicated by the arrow in FIG.

  Further, as shown in FIG. 19, a galvano mirror 75 may be provided so that the excitation light from the excitation light source 72 of the light incident means 10 enters the prism 5 via the galvano mirror 75 (galvano mirror). Scanning method). In this case, the incident position of the excitation light from the excitation light source 72 can be moved in the Y-axis direction by moving the galvanometer mirror 75 in the direction indicated by the arrow in FIG.

  On the other hand, in the evanescent wave generator 1 shown in each of the above embodiments, the light incident means 10 may include a means for widening the excitation light, and this means may be configured to change the width of the excitation light. . For example, if a beam expander 78 is provided as shown in FIG. 20, the excitation light from the excitation light source 72 can be widened by the beam expander 78 (a widening method using a beam expander optical system). .

  Furthermore, the wavelength of the excitation light from the light incident means 10 of the evanescent wave generator 1 shown in the first embodiment may be configured to be changeable. Specifically, for example, as shown in FIG. 21, two or more excitation light sources having different wavelengths are provided (in FIG. 21, two excitation light sources 1 and 2), and these are moved so that they can be used selectively. The pumping light from the pumping light source 1 may be guided to the collimating lens 82 via the optical fiber 80 and the pumping light from the pumping light source 2 may be similarly transmitted through the optical fiber 81 as shown in FIG. In this way, the position of the contact lens 82 is moved, and two or more excitation light sources (two excitation light sources 1 and 2 in FIG. 22) are selectively used. can do.

  In each embodiment, the measurement sample is placed on the prism surface 5C. However, actually, the excitation light is totally reflected by the pair of facing surfaces 5C and 5D intersecting the surfaces 5A and 5B constituting the mirror surface 7. It is also possible to use this prism surface 5D. However, in the evanescent wave generator 1 of each embodiment, the prism surface 5D is the lower surface, so it is actually difficult to use the surface 5D. Therefore, in the evanescent wave generator 1 of each embodiment, it is preferable to use the prism surface 5C as the upper surface.

It is a block diagram of the observation apparatus provided with the evanescent wave generator of one Example of this invention. Example 1 It is a figure which shows the prism and mirror surface of the evanescent wave generator of FIG. It is explanatory drawing at the time of irradiating excitation light with the incident angle of 37 degree | times to the prism of FIG. It is explanatory drawing at the time of arrange | positioning water or a fluorescent material to the prism surface of the prism of FIG. It is a figure which shows the optical path of the excitation light in a prism at the time of making excitation light enter into the prism of FIG. 2 with the incident angle of 34 degree | times. It is a figure which shows the optical path of the excitation light in a prism at the time of making excitation light enter into the prism of FIG. 2 with the incident angle of 35 degree | times. It is a figure which shows the optical path of the excitation light in a prism at the time of making excitation light enter into the prism of FIG. 2 with the incident angle of 36 degree | times. It is a figure which shows the optical path of the excitation light in a prism at the time of making excitation light inject into the prism of FIG. 2 with the incident angle of 37 degree | times. It is a figure which shows the optical path of the excitation light in a prism at the time of making excitation light enter into the prism of FIG. 2 with the incident angle of 38 degree | times. It is a figure which shows the optical path of the excitation light in a prism at the time of making excitation light enter at an incident angle of 40 degree | times using another prism. It is a figure which shows the density of the point of the total reflection which arises on a prism surface at the time of changing an incident angle using the prism of each ratio (length dimension / thickness dimension). It is the figure which evaluated the density of the point of total reflection of FIG. It is a block diagram of the observation apparatus of the other Example of this invention. (Example 2) It is a figure which shows the optical filter switching means of FIG. It is a figure which shows another optical filter switching means. It is a perspective view of the prism and sample container of FIG. 1 or FIG. (Example 3) It is a top view of the sample container of FIG. It is a figure which shows an example of the means to move the incident position of the excitation light which injects into a prism. It is a figure which shows the other example of the means to move the incident position of the excitation light which injects into a prism. It is a figure which shows an example of the means to widen the excitation light which injects into a prism. It is a figure which shows an example which changes the wavelength of the excitation light which injects into a prism. It is a figure which shows the other example which changes the wavelength of the excitation light which injects into a prism. It is a figure which shows the pattern of the reflection in a prism. It is another figure which shows the pattern of reflection within a prism. It is a figure which shows an example (2nd example) of a mirror surface. It is a figure which shows an example (3rd example) of a mirror surface. It is a figure which shows an example (4th example) of a mirror surface. It is a figure which shows an example (5th example) of a mirror surface. It is a figure which shows an example (6th example) of a mirror surface. It is a figure which shows an example (7th example) of a mirror surface. It is a figure which shows an example (8th example) of a mirror surface. It is a figure which shows an example (2nd example) of a prism. It is a figure which shows an example (3rd example) of a prism. It is a figure which shows an example (4th example) of a prism. It is a figure which shows an example (5th example) of a prism. It is a figure which shows an example (6th example) of a prism. It is another figure which shows another prism and a mirror surface. It is a figure which shows the optical path of the conventional evanescent wave generator. It is a figure explaining the conventional evanescent wave generator. It is another explanatory drawing of the evanescent wave generator of FIG. It is a figure explaining one means to expand the observation range of a sample.

Explanation of symbols

S1, S2 Observation device 1 Evanescent wave generator 5, 6 Prism 5A, 5B surface 5C, 5D Prism surface 7 Mirror surface 8 Incident part 10 Light incident means 20 Light detection means 30 Optical filter switching means 31 Rotation motor 32, 39 Substrate 33 Rotation Axis 35-38, 46 Optical filter (filter)
40 Sample container 45 Objective lens 47 Imaging lens 48 CCD camera

Claims (5)

A pair of parallel opposing surfaces;
A prism having at least one pair of parallel opposing two surfaces orthogonal to the two surfaces;
A light incident means for entering light into the prism at a predetermined angle from any one of the two surfaces;
The portion excluding the light incident part on one surface and the surface facing in parallel with the one surface are configured as mirror surfaces,
The light incident means makes light incident on the prism at an angle at which total reflection on the set of two surfaces orthogonal to the surface having the light incident portion and reflection on the mirror surface are repeated in the prism. The evanescent wave generator characterized by performing. A pair of parallel opposing surfaces;
A prism having at least one pair of parallel opposing two surfaces orthogonal to the two surfaces;
A light incident means for entering light into the prism at a predetermined angle from any one of the two surfaces;
The portion excluding the light incident part on one surface and the surface facing in parallel with the one surface are configured as mirror surfaces,
The light incident means is either a container surface disposed directly on one surface of the pair of two surfaces orthogonal to the surface having the light incident portion and facing the one surface in parallel, or the light incident Total reflection on the container surface disposed via a medium on one surface of the pair of two surfaces orthogonal to the surface having the portion and one surface facing the one surface in parallel with the reflection on the mirror surface An evanescent wave generator, wherein light is incident on the prism at an angle repeated in the prism.   3. The evanescent wave generator according to claim 1, wherein one of the two surfaces perpendicular to the surface having the light incident portion is a mirror surface. 4.   One surface of the set of two surfaces orthogonal to the surface having the light incident portion or one surface of the set of two surfaces orthogonal to the surface having the light incident portion and one surface facing the one surface in parallel The container surface disposed directly on the surface, or the one surface of the pair of two surfaces orthogonal to the surface having the light incident portion, and the surface disposed in parallel with the one surface through the medium The observation using the evanescent wave generator according to any one of claims 1 to 3, further comprising means for detecting scattered light and / or fluorescence of the measurement sample by an evanescent wave generated on the surface. apparatus.   The observation apparatus according to claim 4, comprising a plurality of types of filters that selectively allow the scattered light and / or fluorescence to pass through according to wavelength.

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