JP5122930B2 - Evanescent wave generator and observation apparatus using the same - Google Patents

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, a total reflection microscope (observation apparatus) using an evanescent wave can observe only a part of cells (about 100 nm above the total reflection surface) from the total reflection surface, and a clear observation diagram with reduced background. Therefore, the state of cells that grow in contact with the culture vessel is attracting attention as being able to observe more clearly.

  Specifically, for example, a prism or the like is used on the bottom surface of the sample container on which the cell to be observed is mounted (which is a refractive index boundary surface and serves as a total reflection surface), and excitation light is irradiated at a predetermined angle to thereby sample the sample container. Measurement (observation) sample (cell) between 100 nm above the total reflection surface due to the evanescent wave generated on the total reflection surface at the bottom surface (refractive index boundary surface and total reflection surface) Is excited.

  In this way, by limiting the area where light (evanescent wave) reaches to about 100 nm and detecting scattered light and fluorescence generated from the measurement sample (cell) in that area, the situation of the measurement sample becomes clearer. Thus, background noise can be suppressed, and clearer fluorescence observation is possible (for example, see Patent Document 1).

As an observation apparatus using such an evanescent wave, there is also an observation apparatus that uses the principle of the surface plasmon resonance phenomenon. In this apparatus, when a measurement sample (cell) is placed on the surface of the metal thin film formed on the upper surface of the prism, and light (laser) is incident on the prism side surface of the metal thin film on which the measurement sample is placed via the prism. Then, resonance with the electron density wave SP (surface plasmon) occurs at the interface between the metal thin film and the measurement sample. At this time, if the incident angle of the light (laser) is changed and totally reflected by the prism-side surface of the metal thin film, a phenomenon that the reflected light attenuates at a certain incident angle, that is, surface plasmon resonance occurs. The incident angle (resonance angle) of light that causes this surface plasmon resonance depends on the dielectric constant of the measurement sample, and by measuring this resonance angle, the measurement sample on the surface of the metal thin film can be observed. is there. That is, by adjusting the incident angle of light and measuring the change in resonance angle, the measurement sample process (for example, interaction between antibody and antigen) can be quantitatively analyzed in real time on the surface of the metal thin film ( For example, see Patent Document 2).
Japanese Patent Laid-Open No. 9-61346 Japanese Patent No. 2758904

  By the way, in the conventional total reflection microscope, in order to enable illumination and observation with a single lens, a high-magnification objective lens having a large aperture is used, so it is difficult to process many observation objects in a short time. In addition, the cost of the apparatus has been increased. In recent regenerative medicine, cells are frequently cultured, and there is a demand for evaluating measurement samples (cells) cultured in as short a time as possible. And as regenerative medicine becomes widespread and the application in the medical field further advances, it becomes necessary to judge the cultured measurement sample as a whole. Therefore, there is a demand to observe or judge samples cultured on a microplate that can process many samples at once. However, conventional devices can observe multiple observation objects instantly or simultaneously. There wasn't.

  Moreover, in such an apparatus, it is necessary to make the excitation light irradiated to the observation object enter the point of total reflection at an incident angle that is an angle at which total reflection occurs, which is an angle greater than the critical angle. The critical angle for causing this total reflection (the angle formed between the line perpendicular to the plane of the point of total reflection and the excitation light) differs depending on the measurement sample, so that the incident angle is appropriate according to the measurement sample. Had to adjust to. Also, the closer to the critical angle, the farther the region where the evanescent wave arrives, the farther the distance from the total reflection surface becomes, and the farther away from the critical angle, the region where the evanescent wave reaches from the total reflection surface. Since the distance becomes shorter, changing the incident angle will change the illumination area (the evanescent wave arrival area can be changed from 100 nm), and adjustment of the incident angle is necessary to widen the observation range. Met. Conventionally, the incident angle is adjusted by changing the angle of the excitation light. However, since the point of total reflection changes when the angle of the excitation light is changed, the position of the excitation light irradiation means must also be moved so that the point of total reflection matches the target measurement sample. When each of the measurement samples is illuminated and intended to be observed at the same time, there is a problem that the workability is remarkably complicated.

  The present invention has been made to solve the conventional technical problems, and provides an evanescent wave generator capable of simultaneously observing a plurality of measurement samples and an observation apparatus using the generator. Objective.

The evanescent wave generator according to the first aspect of the present invention introduces a plurality of light beams into a prism, totally reflects the light on a plurality of evanescent wave generation surfaces, and generates an evanescent wave, which is parallel to the optical axis. A lens array in which a plurality of introduction lenses having a refraction function for concentrating the light at a single point regardless of the incident position when the light is incident, and a light incident for generating a plurality of parallel light beams A plurality of light beams that are incident on the respective introduction lenses of the lens array from the light incident means and pass through the respective introduction lenses, and the light incident means is provided for each introduction. It has a moving means for making it incident simultaneously and parallel to the optical axis of the lens, and is characterized in that light having a width narrower than the diameter of each introduction lens is incident on each introduction lens .

An evanescent wave generator according to a second aspect of the present invention is characterized in that, in the above invention, the introduction lens is a lens fragment formed by cutting away only a necessary portion of one lens.

According to a third aspect of the present invention, there is provided the evanescent wave generator according to the first aspect, wherein the light incident means has a predetermined width with respect to the direction of change of the incident position and emits light parallel to the optical axis of each introduction lens. A parallel light generating means for generating and a shielding member having a plurality of window holes through which a part of the light from the parallel light generating means passes, and the shielding member is moved to transmit light to each introduction lens. The incident position is changed simultaneously.

According to a fourth aspect of the present invention, there is provided the evanescent wave generator according to the third aspect , wherein the parallel light generating means converts the point light source and diffused light from the point light source into light parallel to the optical axis of each introduction lens. And a light source lens to be refracted.

An evanescent wave generator according to a fifth aspect of the invention is characterized in that in the invention according to the third or fourth aspect , a filter is provided that selectively allows light from the parallel light generating means to pass through according to the wavelength. To do.

According to a sixth aspect of the present invention, there is provided an evanescent wave generator according to the first or second aspect of the invention, wherein the light incident means has a plurality of laser light sources for generating laser light, and the laser light from each laser light source is received. The laser beam is incident on each introduction lens, and the laser light sources are simultaneously moved to simultaneously change the incident position of the laser light on each introduction lens.

The observation device according to a seventh aspect of the present invention is directed to a scattered light of a plurality of measurement samples by the evanescent wave generated on each evanescent wave generation surface of the evanescent wave generation device according to any one of the first to sixth aspects, and / or Alternatively, a means for detecting fluorescence is provided.

An observation device according to an eighth aspect of the invention is a means for detecting a plurality of reflected light intensity changes due to surface plasmon resonance using the evanescent wave generated in the evanescent wave generation device according to any one of the first to sixth aspects. It is provided with.

  According to the present invention, in the evanescent wave generating device that generates the evanescent wave by the light introduced into the prism, a plurality of light beams are introduced into the prism and totally reflected on each of the plurality of evanescent wave generating surfaces. An evanescent wave can be generated at each measurement point.

  Thereby, for example, a plurality of measurement samples respectively accommodated in each well of the microplate can be illuminated simultaneously and observed simultaneously or instantaneously, and the observation efficiency can be remarkably improved.

In particular, when light parallel to the optical axis is incident, regardless of the incident position, a lens array in which a plurality of introduction lenses having a refraction function for concentrating the light at one point are arranged in parallel. A light incident means for generating parallel light, and enters each of the introduction lenses of the lens array from the light incidence means, and introduces a plurality of light beams respectively passing through the introduction lenses into the prism, The incident means has a moving means for simultaneously making incident light parallel to the optical axis of each introduction lens, and allows light having a width narrower than the diameter of each introduction lens to enter each introduction lens. Only the incident angles of a plurality of light beams can be changed at the same time without changing the reflection point.

  Here, it is necessary to make the excitation light irradiated to the observation target incident on the point of total reflection at an incident angle that is an angle at which total reflection occurs or more than a critical angle. Since the critical angle for causing this total reflection differs depending on the measurement sample, it must be adjusted so as to have an appropriate incident angle according to the measurement sample. Also, the closer to the critical angle, the farther the region where the evanescent wave arrives, the farther the distance from the total reflection surface becomes, and the farther away from the critical angle, the region where the evanescent wave reaches from the total reflection surface. Since the distance becomes shorter, changing the incident angle will change the illumination area (the evanescent wave arrival area can be changed from 100 nm), and adjustment of the incident angle is necessary to widen the observation range. It is. Conventionally, the incident angle is adjusted by changing the angle of the excitation light. In this case, changing the angle of the excitation light also changes the point of total reflection, so the point of total reflection is the target. The position of the excitation light irradiating means must be moved so as to match the measurement sample of this, which has caused complication of workability, but according to the present invention, the work involved in adjusting the incident angle with respect to a plurality of measurement points Can be performed remarkably simply.

  In particular, when a plurality of light beams are introduced into a single introduction lens having a large diameter, a plurality of light beams are aggregated at the focal point of the introduction lens and cannot be observed at multiple points. If a lens array is configured by arranging the lenses for introduction and light is incident on each introduction lens, the incident position of the light on each introduction lens is changed, so that the light for each measurement point is changed. The incident angle can be changed significantly.

Further, if the introduction lens is a lens piece formed by cutting away only a necessary portion of one lens as in the invention of claim 2 , the introduction lens can be made to the minimum necessary size. Therefore, it is possible to reduce the size of the lens array.

According to the invention of claim 3, in each of the above inventions, the light incident means has a predetermined width with respect to the direction of change of the incident position, and generates parallel light that is parallel to the optical axis of each introduction lens. A light generating means and a shielding member having a plurality of window holes through which a part of the light from the parallel light generating means passes, and the incident position of the light to each introduction lens is moved by moving the shielding member Since the change is made at the same time, only the necessary portion of light can be made incident on each introduction lens by the shielding member. Further, by moving the shielding member, only the incident angle can be changed without changing the position of each total reflection. Accordingly, any light incident means capable of generating light parallel to the optical axis of each introduction lens can be used as the light source.

In the invention of claim 4, in the above invention , the parallel light generating means includes a point light source and a light source lens that refracts diffused light from the point light source into light parallel to the optical axis of each introduction lens. A point light source makes it possible to obtain light having a wide range of wavelengths.

In the invention of claim 5, since the filter according to claim 3 or 4 is provided with a filter that selectively allows the light from the parallel light generating means to pass through according to the wavelength, the light of an arbitrary wavelength is provided by the filter. Can be extracted freely.

According to the invention of claim 6, in the invention of claim 1 or 2 , the light incident means has a plurality of laser light sources that generate laser light, and each laser light from each laser light source is introduced. The total reflection angle can be easily changed by allowing the laser light sources to be simultaneously moved and simultaneously changing the incident positions of the laser light to the introduction lenses. Further, by using a plurality of laser light sources having a high output per unit area, a plurality of stronger evanescent waves can be obtained.

According to the observation device of the invention of claim 7 , scattered light of a plurality of measurement samples by the evanescent wave generated on each evanescent wave generation surface of the evanescent wave generation device according to any one of claims 1 to 6 , In addition, since a means for detecting fluorescence is provided, scattered light and fluorescence of a plurality of measurement samples to be observed such as cells can be detected.

According to the observation device of the eighth aspect of the invention, a plurality of reflected light intensity changes due to surface plasmon resonance using the evanescent wave generated in the evanescent wave generation device according to any one of the first to sixth aspects. Therefore, the measurement sample process can be quantitatively analyzed in real time.

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

  FIG. 1 is a configuration diagram of an observation apparatus S provided with an evanescent wave generator 1 according to an embodiment of the present invention, and FIG. 2 is a partially enlarged view of FIG. This observation device S includes an evanescent wave generation device 1 and a light detection means 20 described later. Specifically, the observation device S of the present embodiment uses an evanescent wave generated by the evanescent wave generator 1 as a measurement sample that becomes an observation target substance such as a cell (in this embodiment, the measurement target is made to fluoresce by being irradiated with excitation light). The evanescent wave is generated on the upper surface 40C of the bottom wall 40B of the sample container 40 by irradiating from the lower surface of the sample container 40 containing the cells to be emitted), and the sample container 40 which is the generation surface of the evanescent wave is generated. The cells located within a predetermined range from the upper surface 40 of the bottom wall 40B of the bottom are excited to generate scattered light and fluorescence, and the scattered light and fluorescence are arranged on one side of the sample container 40 (upward in FIG. 1). The detection means 20 is configured to detect.

  The sample container 40 of the embodiment is a glass microplate, and includes a plurality of wells 40A as shown in FIG. 2, and a measurement sample is accommodated in each well 40A. Then, an evanescent wave is generated on the upper surface 40C of the bottom wall 40B of each well 40A (bottom wall of the microplate corresponding to the well 40A) to excite cells within a predetermined range, thereby generating scattered light and fluorescence. To observe.

  The light detection means 20 is means for simultaneously or instantaneously detecting scattered light and fluorescence emitted from the cells in each well 40A by the excitation light from the evanescent wave generator 1. The light detection means 20 of the present embodiment includes a lens or an objective lens 45 for collecting scattered light and fluorescence from a cell, an optical filter (fluorescence filter) 46 for removing a specific wavelength from the scattered light and fluorescence, An imaging lens (not shown) that forms an image of scattered light and fluorescence that has passed through the optical filter 46, and a CCD camera 48 that detects and images a light image from the imaging lens two-dimensionally. The means for detecting scattered light and fluorescence is not limited to the light detection means of this embodiment, and any detection means can be used as long as it can detect scattered light and fluorescence. For example, although a CCD camera is used in the embodiment, a light receiving element such as a photodiode may be used.

  Further, it is effective to use a means for detecting a change in reflected light intensity due to surface plasmon resonance using an evanescent wave as the light detecting means. Specifically, the observation apparatus in that case is an observation apparatus using the principle of the surface plasmon resonance phenomenon. The apparatus arranges a measurement sample (cell) on the surface of a metal (for example, gold) thin film formed on the upper surface of the prism, and transmits light (through the prism 5 to the surface of the metal thin film on which the measurement sample is arranged via the prism 5. When a laser is incident, resonance with an electron density wave SP (surface plasmon) occurs at the interface between the metal thin film and the measurement sample.

  At this time, if the incident angle of the light (laser) is changed and totally reflected by the surface of the metal thin film on the prism 5 side, a phenomenon that the reflected light attenuates at a certain incident angle, that is, surface plasmon resonance occurs. The incident angle (resonance angle) of light causing this surface plasmon resonance depends on the dielectric constant of the measurement sample, so measure this resonance angle, that is, detect the change in reflected light intensity due to surface plasmon resonance using an evanescent wave. As a result, the measurement sample on the surface of the metal thin film can be measured. Therefore, by adjusting the incident angle of light and measuring the change in resonance angle, the process of the measurement sample (for example, interaction between antibody and antigen) can be qualitatively and quantitatively analyzed in real time on the surface of the metal thin film. .

  Next, the above-described evanescent wave generator of the present invention will be described. The evanescent wave generator of the present invention introduces a plurality of light beams into a prism and totally reflects the light on a plurality of evanescent wave generation surfaces. Specifically, the evanescent wave generator 1 of this embodiment includes a prism 5, a lens array 30, and a light incident means 10, introduces a plurality of light beams into the prism 5, and generates a plurality of evanescent wave generation surfaces. (In the present embodiment, the evanescent wave generating surface is totally reflected at the upper surface 40C of the bottom surface 40B of the sample container 40).

  The prism 5 of the embodiment is made of BK7 (manufactured by SCHOTT GLASS), and its refractive index is 1.52. One surface (left side surface in FIG. 1) of the prism 5 is a light incident portion for allowing excitation light from a light incident means 10 described later to enter the prism 5.

  On the other hand, the lens array 30 described above is formed by arranging a plurality of circular introduction lenses 6 in parallel as shown in FIG. Each introduction lens 6 is a lens having a refraction function for concentrating light at one point regardless of the incident position when light parallel to the optical axis L of the introduction lens 6 is incident. The introduction lenses 6 are all arranged with the optical axis L in parallel. In the present embodiment, an achromatic lens is used as the introduction lens 6. This achromatic lens is a lens that corrects chromatic aberration caused by the wavelength of light. Specifically, two lenses (convex lens and concave lens) having different refractive indexes and chromatic dispersion are bonded together to correct the focal position shift between the two colors (usually red and blue). In the embodiment, an achromat lens is used as the introduction lens 6. However, an apochromat lens in which spherical aberration and coma are corrected by combining three or more lenses and chromatic aberration is smaller than that of the achromat lens is introduced. The present invention is also effective when used as the lens 6 for an automobile.

  On the other hand, the light incident means 10 described above is a means for generating a plurality of parallel light beams and causing the parallel light beams to enter the introduction lenses 6 of the lens array 30, and introduces the light into the prism 5. That is, in this embodiment, an excitation light source for introducing a plurality of light beams respectively incident on the respective introduction lenses 6 of the lens array 30 and passed through the respective introduction lenses 6 into the prism 5, and the introduction lenses. And a moving means for simultaneously moving the light incident means 10 in the direction in which the incident angle of each light changes with respect to the surface where the evanescent wave is generated, while making the light incident parallel to the optical axis L of 6. The light incident means 10 is configured so that light having a narrower width than the diameter of each introduction lens 6 (D shown in FIG. 1 in this embodiment) is incident on each introduction lens 6 simultaneously.

  Specifically, the light incident means 10 of this embodiment has a predetermined width that can correspond to the dimensions of the lens array 30 in the incident position changing direction (diameter direction of each introduction lens 6 constituting the lens array 30). And 10 A of parallel light generation means which generate | occur | produces the light parallel to the optical axis L of each introduction lens 6, and the shielding member 55 are provided. The shielding member 55 is a member for causing a part of the light from the parallel light generating means 10A to enter each of the introduction lenses 6, and is introduced to allow a part of the light from the parallel light generating means 10A to pass. The same number (plural) of window holes 56 (slits) as the number of lenses 6 are provided. Each window hole 56 has a narrower width than the diameter of each introduction lens 6, and the shielding member 55 is disposed between the parallel light generating means 10 </ b> A and the introduction lens 6.

  In this case, the shielding member 55 is moved in a direction orthogonal to the optical axis L of each introduction lens 6 by the moving means constituted by a motor, a rack and pinion gear, and the like. The incident position of the light to 6 is changed. In addition, you may move the shielding member 55 to the direction shown with the broken line of FIG. As a result, only a part of the light from the light incident means 10 (parallel light generating means 10 </ b> A) that has passed through each window hole 56 of the shielding member 55 is incident on each introduction lens 6. Therefore, in this embodiment, it is possible to change the incident angle by moving the shielding member 55 without moving the parallel light generating means 10A which is a light source. Specifically, the point of total reflection in each well 40 </ b> A is changed by moving the shielding member 55 and adjusting the shielding member 55 so that only a necessary portion of light is incident on each introduction lens 6. Therefore, the incident angle of light can be easily changed simultaneously in each well 40A.

  The lens array 30 is disposed between the light incident means 10 and the prism 5, and is incident on each introduction lens 6 of the lens array 30 and passes through each introduction lens 6 as shown in FIG. The strip light is configured to be introduced into the prism 5.

  As described above, by providing the shielding member 55 having the window hole 56 through which a part of the light can pass and moving the shielding member 55, the incident position of the light to each introduction lens 6 is changed at the same time. Therefore, the incident angle can be changed simultaneously without changing the position of total reflection in each well 40A. Accordingly, any parallel light generating means capable of generating light parallel to the optical axis L of each introduction lens 6 can be used as a light source.

  When a plurality of excitation light beams from the light incident means 10 are incident on any part of each introduction lens 6 in parallel to the optical axis L of each introduction lens 6, each introduction lens 6 is turned on. The light that has passed through is refracted and concentrated at a certain point regardless of the incident position and wavelength on each introduction lens 6. Therefore, a measurement sample in each well 40A is arranged at one point where the light is concentrated, and each excitation light irradiated to the measurement sample is incident at an incident angle equal to or greater than a critical angle at which total reflection occurs. Each of the measurement samples is totally reflected by the critical surface of each measurement sample, and at that time, each measurement sample within a predetermined range above the total reflection surface is excited by an evanescent wave generated above the total reflection surface.

  As described above, the lens array 30 is provided which includes a plurality of introduction lenses 6 having a refraction function for concentrating a plurality of light beams from the light incident means 10 at one point regardless of the incident position and wavelength. Thus, each light having a width narrower than the diameter D of the introduction lens 6 is incident on each introduction lens 6 from the light incident means 10, and the light that has passed through each introduction lens 6 is introduced into the prism 5. Each light that has passed through each introduction lens 6 is concentrated at one point regardless of the incident position or wavelength on each introduction lens 6. That is, no matter what position the light from the light incident means 10 is incident on each introduction lens 6, the light that has passed through each introduction lens 6 is always concentrated at one point. Even when the position of 10 is changed, the respective points of total reflection are not changed, and only the incident angles of the respective lights incident on the respective points are changed simultaneously.

  In this way, in the present invention, a plurality of light beams from the light incident means 10 are introduced into the prism 5 and totally reflected on the plurality of evanescent generation surfaces, so that a plurality of points (the upper surface of the bottom wall 4B of each well 40A) 40C), an evanescent wave can be generated. As a result, if each measurement sample is arranged on the evanescent wave generation surface of each well 40A, it becomes possible to simultaneously illuminate each measurement sample and observe it simultaneously or instantaneously, thereby significantly improving the observation efficiency. become able to.

  In particular, when light parallel to the optical axis L is incident, a lens array 30 in which a plurality of introduction lenses 6 having a refractive function for concentrating the light to one point regardless of the incident position; A light incident means 10 for generating a plurality of parallel light beams. The light incident means 10 is incident on each introduction lens 6 of the lens array 30 from the light incidence means 10, and the plurality of light beams that have passed through each introduction lens 6. An upper surface of the bottom wall 40B of the sample container 40, which is a surface on which the evanescent wave is generated in the embodiment of FIG. 1 while being introduced into the prism 5 and simultaneously incident in parallel with the optical axis L of each introduction lens 6. Since there is a moving means for moving the incident angle of the light with respect to 40C in a direction to change, light having a width smaller than the diameter of each introduction lens 6 is made incident on each introduction lens 6. Reflection point without change, it is possible to change only the angle of incidence of the plural rows of light simultaneously.

  Thereby, it becomes possible to perform the operation | work accompanying adjustment of the incident angle with respect to several measurement points remarkably simply. Here, when a plurality of light beams are introduced into a single introduction lens having a large diameter, a plurality of light beams are aggregated at the focal point of the introduction lens, and multiple points cannot be observed. Since the introduction lens 6 is arranged side by side to form the lens array 30 and light is incident on each introduction lens 6, by changing the incident position of the light on each introduction lens 6, The incident angle of light with respect to each measurement point (the upper surface 40C of the bottom wall 40B of each well 40A) can be changed significantly.

  Next, another embodiment of the present invention will be described with reference to FIG. 3 that have the same reference numerals as those in FIGS. 1 and 2 have the same or similar effects and will not be described. 3 includes a point light source 67 (for example, a xenon lamp, a mercury lamp, etc.) and a light source lens 68. The light source lens 68 is a lens for bending the diffused light from the point light source 67 into light parallel to the optical axis L of each introduction lens 6, and is disposed between the point light source 67 and the shielding member 55. Has been. The shielding member 55 is the same as that of the first embodiment shown in FIGS.

  In this case, the light of the point light source 67 is light including many wavelength components due to the performance of the point light source (for example, a lamp), and is diffused light. The diffused light can be refracted into light parallel to the optical axis L of each introduction lens 6. That is, the diffused light from the point light source 67 is converted into light parallel to the optical axis L of each introduction lens 6 in the process of passing through the light source lens 68, and then only necessary portions of light are transmitted by the shielding member 55. Adjustment is made so that the light enters the introduction lens 6. As a result, it is possible to make each of the introduction lenses 6 enter a necessary portion of light parallel to the optical axis L of the introduction lens 6. Further, the light passing through each introduction lens 6 is concentrated at one point as described in detail in the above embodiment. Thereby, even when a point light source is used, the light incident angle can be easily changed without changing the point of total reflection in each well 40A by adopting the configuration as in the present embodiment described in detail above. Will be able to change.

  In the present embodiment or the first embodiment, for example, in the configuration of the present embodiment, the parallel light generating means 65 is arranged between the parallel light generating means 65 and each introduction lens 6 as shown in FIG. If the filter 70 (for example, a band pass filter etc.) which selectively passes the light according to a wavelength is installed, even if it is a case where the light containing a several wavelength is used as a light source, the said filter 70 Therefore, it is possible to cut light other than the required specific wavelength and pass only the light having the specific wavelength (light having a preset wavelength). By providing the filter 70 as described above, light having an arbitrary wavelength can be freely selected from light from a light source including light having a plurality of wavelengths.

  In each of the above embodiments, the light incident means 10 and 60 are constituted by the parallel light generating means 10A and 65 and the shielding member 55. However, the light incident means described in claim 1 of the present invention includes It is not limited. FIG. 5 is a diagram for explaining an embodiment in this case. 5 that have the same reference numerals as those in FIGS. 1 to 4 have the same or similar effects or functions, and the description thereof will be omitted.

  The light incident means 50 of the present embodiment uses a laser light source (for example, a solid laser) that generates laser light as the excitation light source described above. That is, the light incident means 50 of this embodiment has a plurality of laser light sources 52 that generate parallel laser beams (laser beams having a narrower width than the diameter of the introduction lens 6), The laser light is incident on the introduction lens 6 at the same time, and the laser light incident positions on the introduction lenses 6 can be changed simultaneously by moving the laser light sources 52 at the same time.

  Specifically, the moving means is composed of, for example, a motor and a rack and pinion gear, and an evanescent wave is generated while light is incident parallel to the optical axis L of each introduction lens 6 of the lens array 30. The laser light source 52 is simultaneously moved in a direction to change the incident angle of a plurality of light beams with respect to the upper surface 40C of the bottom wall 40B of each well 40A of the sample container 40, which is a surface to be processed. Each laser light source 52 is configured to be movable in a direction perpendicular to the optical axis L of each introduction lens 6 as indicated by solid line arrows. The moving means of this embodiment may be any means that can move a plurality of light beams simultaneously in a direction orthogonal to the optical axis L of each introduction lens 6, and is shown by, for example, a broken line arrow in FIG. 1. Even moving in such a direction is effective.

  Then, a plurality of excitation light (laser light) from each laser light source 52 of the light incident means 50 is incident on any part of each introduction lens 6 in parallel to the optical axis L of each introduction lens 6. The light that has passed through each introduction lens 6 is refracted and concentrated at a certain point regardless of the incident position and wavelength on each introduction lens 6. Therefore, a measurement sample in each well 40A is arranged at one point where the light is concentrated, and each excitation light irradiated to the measurement sample is incident at an incident angle equal to or greater than a critical angle at which total reflection occurs. Each of the measurement samples is totally reflected by the critical surface of each measurement sample, and at that time, each measurement sample within a predetermined range above the total reflection surface is excited by an evanescent wave generated above the total reflection surface.

  Thus, in this case as well, a lens formed by juxtaposing a plurality of introduction lenses 6 having a refractive function for concentrating a plurality of laser beams from the light incident means 50 at one point regardless of the incident position and wavelength. An array 30 is provided so that light having a width narrower than the diameter D of the introduction lens 6 is incident on each introduction lens 6 from the light incident means 50, and the light that has passed through each introduction lens 6 is entered into the prism 5. By introducing, each laser beam that has passed through each introduction lens 6 is concentrated at one point regardless of the incident position and wavelength on each introduction lens 6. That is, no matter what position the multiple laser beams from the light incident means 50 are incident on each introduction lens 6, the laser light that has passed through each introduction lens 6 is always concentrated on one point. Even when the position of the incident means 50 is changed, the respective points of total reflection are not changed, and only the incident angles of the respective laser beams incident on the respective points are changed at the same time.

  In particular, in this case, since laser light is used as excitation light, a plurality of strong evanescent waves can be obtained by a plurality of laser lights having a high output per single area.

  Next, FIG. 6 shows another embodiment of each introduction lens 6 constituting the lens array 30 of each embodiment. In this case, each introduction lens 6 does not use a single circular lens, but uses a piece cut in half in the embodiment in a plane parallel to the optical axis L of the introduction lens 6. That is, in this case, each introduction lens 6 is a lens that is excised leaving only a portion (half from the center in the embodiment) necessary for changing the incident angle of the excitation light incident on the evanescent wave generation point. ing.

  In this case, the excitation light is narrower than the radius of the half-sized introduction lens 6 cut in this way.

  In this way, if each introduction lens 6 is made into a lens fragment formed by cutting away only a necessary portion of one lens, each introduction lens 6 can be made to the minimum necessary size. When a plurality of introduction lenses 6 are arranged in parallel, the lens array 30 can be reduced in size.

  In each of the above embodiments, a glass microplate was used as the sample container. However, the material is not limited to this, and the measurement samples may be arranged at a plurality of locations on the undivided container. The present invention is also effective when each of the containers is used and a measurement sample is accommodated in each container, or when a plurality of measurement samples are arranged directly on the prism without using the container.

It is a block diagram of the observation apparatus provided with the evanescent wave generator of one Example of this invention. Example 1 FIG. 2 is a partially enlarged view of the evanescent wave generator of FIG. 1. It is explanatory drawing of the evanescent wave generator of the other Example of the observation apparatus of FIG. 1 (Example 2). It is a figure explaining the other evanescent wave generator of the observation apparatus of FIG. FIG. 10 is a diagram for explaining still another evanescent wave generator of the observation apparatus of FIG. 1 (Example 3). FIG. 10 is a diagram for explaining still another evanescent wave generator of the observation apparatus of FIG. 1 (Example 4).

DESCRIPTION OF SYMBOLS S Observation apparatus 1 Evanescent wave generator 5 Prism 6 Introduction lens 10, 50, 60 Light incident means 20 Photodetection means 40 Sample container 40A Well 40B Bottom wall 45 Objective lens 48 CCD camera

Claims (8)

In an evanescent wave generator that introduces a plurality of light beams into a prism, totally reflects each of a plurality of evanescent wave generation surfaces, and generates an evanescent wave,
When light parallel to the optical axis is incident, a lens array in which a plurality of introduction lenses having a refraction function for concentrating the light to one point regardless of the incident position;
A light incident means for generating the plurality of parallel light beams,
The light incident means is incident on each introduction lens of the lens array, and a plurality of light beams respectively passing through the introduction lenses are introduced into the prism.
The light incident means has a moving means for simultaneously entering parallel to the optical axis of each introduction lens, and makes light having a width narrower than the diameter of each introduction lens enter each introduction lens . An evanescent wave generator characterized by that. 2. The evanescent wave generator according to claim 1, wherein the introduction lens is a lens fragment formed by cutting away only a necessary portion of one lens . The light incident means has a predetermined width with respect to the changing direction of the incident position, and generates parallel light generating means for generating light parallel to the optical axis of each introduction lens, and the parallel light generating means And a shielding member having a plurality of window holes through which a part of light from the light passes, and the incident position of the light to each introduction lens is changed simultaneously by moving the shielding member. The evanescent wave generator according to claim 1 or 2. The said parallel light generation means has a point light source and the lens for light sources which refracts the diffused light from this point light source to the light parallel to the optical axis of each said introduction lens. Evanescent wave generator. 5. The evanescent wave generation device according to claim 3, further comprising a filter that selectively allows light from the parallel light generation means to pass through according to a wavelength . The light incident means has a plurality of laser light sources for generating laser light, and makes the laser light from each laser light source enter each introduction lens, and simultaneously moves each laser light source to introduce each of the laser light sources. The evanescent wave generator according to claim 1 or 2 , wherein the incident position of the laser beam on the lens is simultaneously changed . 7. The apparatus according to claim 1, further comprising means for detecting scattered light and / or fluorescence of a plurality of measurement samples by evanescent waves generated on the respective evanescent wave generation surfaces. Observation device using the evanescent wave generator. 7. The evanescent wave generator according to claim 1, further comprising means for detecting a plurality of reflected light intensity changes due to surface plasmon resonance using each of the evanescent waves . Observation device.

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