JP2009122203A - Optical filter, filter set for fluorescence analysis and fluorescence measuring device using this optical filter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a required reflectance with a fewer number of layers by increasing the width of a transmission area of a filter having discontinuous two transmission areas and two reflection areas on the spectrum. <P>SOLUTION: An optical filter comprises: a substrate that is transparent in a wavelength area to be used; a transmission filter that is formed on one surface of the substrate and has a layered structure of successively and alternately deposited high refractive index layers and low refractive index layers, in which one of a shorter wavelength side and a longer wavelength side of a predetermined wavelength is rendered into a transmission area and the other area is rendered into a reflection area; and a notch filter that is formed on the other surface of the substrate, has a layered structure of successively and alternately deposited high refractive index layers and low refractive index layers and has a reflection area in a wavelength area distant from the above predetermined wavelength and within the transmission area of the transmission filter. Thus, the obtained filter has discontinuous two transmission areas and two reflection regions in the spectrum. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention uses an optical filter having a laminated structure in which high refractive index layers and low refractive index layers are alternately laminated on a transparent substrate, a filter set for fluorescence analysis combining a plurality of optical filters, and the optical filter. The present invention relates to a fluorescence measuring apparatus.

  An optical filter (hereinafter simply referred to as a filter) having a laminated structure in which a high refractive index layer and a low refractive index layer are alternately laminated on a transparent substrate is used.

  There are many products that transmit one wavelength band and cut other wavelength bands. Although there are many uses for filters, for example, in order to detect two or more types of fluorescent reagents using such a filter in fluorescence analysis, a mechanical switching mechanism of the filter is required as described in detail later. As a result, the optical system becomes complicated, and the fluorescence analysis may be hindered).

A filter having a plurality of reflection regions has also been proposed (see Patent Document 1). There are provided two repeating layers formed by alternately laminating a high refractive index layer and a low refractive index layer on a transparent substrate, and the average optical thickness of the high refractive index layer in each repeating layer is provided. The sum of the value and the average value of the optical film thickness of the low refractive index layer is set to be approximately equal to the respective reflection wavelengths with respect to the normal incident light. Specific examples include (0.55H 0.45L) ²⁷ 0.55H 0.476L (0.619H 0.501L) ²⁷ 0.619H 0.251L (assuming λ = 488 nm) or (0.489H 0.404L) ²⁷ 0.489H 0.427L (0.55H 0.45L L) ²⁷ 0.55H 0.225L (assuming λ = 543 nm) is given as a film design value. These two types of notation are optical thicknesses for each of the center wavelengths λ of the two reflection regions, and represent the design values of the same filter. Symbols H and L are a high-refractive index layer and a low-refractive index layer, respectively, and the preceding numbers indicate how many times the optical film thickness of the center wavelength of the design is, "27") indicates that the configuration in parentheses is repeated the number of times.
JP 2006-23471 A Japanese translation of PCT publication No. 2002-533096 JP 2001-299366 A Japanese Patent Laid-Open No. 2002-300894

  In the filter having a plurality of reflection regions introduced above, the widths of the transmission region and the reflection region are determined by the laminated film structure itself, and thus there is a great restriction on widening the bandwidth. Further, in the above example, a film structure of 100 layers or more is obtained in order to obtain a desired reflectance, and when the physical film thickness is about 15 μm, there is a problem in the productivity and reproducibility of the filter.

  One object of the present invention is a filter having two transmission regions and two reflection regions that are discontinuous in the spectrum, and the width of the transmission region is increased as compared with the filter having a plurality of reflection regions introduced above. It is an object of the present invention to obtain a filter that is easy to achieve and that can achieve the required reflectance with a smaller number of layers.

  Another object of the present invention is to provide a fluorescence measuring apparatus with improved measurement accuracy, although the configuration is simplified by using the filter of the present invention.

  The present invention provides a combination of a transmission filter and a notch filter by forming a short wavelength transmission filter or a long wavelength transmission filter on one surface of a transparent glass substrate and forming a notch filter on the other surface of the transparent glass substrate. As a result, a filter having two transmission regions and two reflection regions is to be formed.

  In other words, the filter of the present invention has a laminated structure in which a transparent substrate in a used wavelength region and one surface of the substrate are formed, and a high refractive index layer and a low refractive index layer are alternately laminated. A transmission filter in which one of the short wavelength side and the long wavelength side is a transmission region and the other is a reflection region, and a high refractive index layer and a low refractive index layer are alternately formed on the other surface of the substrate. And a notch filter having a reflection region in the transmission region of the transmission filter and in a wavelength region away from the predetermined wavelength, thereby providing two spectrally discontinuous two layers. It has a transmission area and two reflection areas.

  In the present invention, the transmission filter and the notch filter are formed as laminated structures independent of each other on the respective surfaces of the substrate. The notch filter is designed to reflect one wavelength band and transmit the other wavelength band. The short wavelength transmission filter or the long wavelength transmission filter is designed so as to be a reflection region from an arbitrary wavelength in the transmission region on the short wavelength side or the long wavelength side of the notch filter. By combining these designs, a desired reflectance can be achieved even if the film thickness of each filter is reduced, and productivity can be improved. Regarding the film thickness of the laminated film formed on one side of the substrate, the film thickness on one side is about 3 μm in the excitation light filter shown in the embodiment, and the film thickness on one side is about 5 μm in the fluorescent filter. In the filter having a plurality of reflection regions introduced above, the film thickness is considerably thinner than that of the film thickness of about 15 μm in order to obtain a desired reflectance. In the present invention, since the laminated film is formed on both surfaces of the substrate, the film thickness on both surfaces is the sum of the film thickness on one surface. Even if it is simply twice the film thickness on one side, it is considerably smaller than about 15 μm. Since the film forming process is performed on each side, productivity is greatly affected by the film thickness formed on one side. That is, when the film thickness formed on one surface of the substrate is increased, the time for continuous film formation increases, and there is a shortage of consumables such as vapor deposition materials. This is because a special device such as an automatic material supply device is required to cope with the shortage of consumables.

  The wavelength at the boundary between the transmission region and the reflection region in the transmission filter (referred to as “predetermined wavelength”) and the center wavelength of the reflection region of the notch filter can be set independently of each other. Depending on how far the center wavelength of the reflection region of the notch filter is set from the predetermined wavelength of the transmission filter, the width of the transmission region existing between the predetermined wavelength of the transmission filter and the reflection region of the notch filter is arbitrarily set. Can be set.

  The first form is a short wavelength transmission filter in which the transmission filter has a transmission region on the shorter wavelength side than the predetermined wavelength, and the transmission region and the reflection region are the first transmission region, the first transmission region in order from the short wavelength side. This is a reflection area, a second transmission area, and a second reflection area.

  The second form is a long wavelength transmission filter in which the transmission filter has a transmission region on the longer wavelength side than the predetermined wavelength, and the transmission region and the reflection region are in order from the short wavelength side, the first reflection region, This is a transmissive region, a second reflective region, and a second transmissive region.

  By changing the combination of the design of the notch filter and the short wavelength transmission filter or the long wavelength transmission filter, it is possible to design a two-wavelength band excitation light filter, a two-wavelength band fluorescent filter, and a two-wavelength band dichroic filter described later. Become. The filter set of the present invention is used in a fluorescence analyzer that analyzes a sample labeled with two types of fluorescent reagents. As a preferred first embodiment, two types of fluorescent reagents are excited. An excitation light filter that transmits two excitation wavelengths and a fluorescence filter that transmits two fluorescence wavelengths generated from a fluorescent reagent, wherein at least one of the excitation light filter and the fluorescence filter is composed of the optical filter of the present invention. Yes. When the optical filter of the present invention is used as a pumping light filter, the filter set of the present invention includes one two-wavelength band pumping light filter designed so that two transmission regions include two pumping wavelengths. When the filter of the present invention is used as a fluorescent filter, the filter set of the present invention includes one two-band fluorescent filter designed so that two transmission regions include two fluorescent wavelengths generated from the fluorescent reagent. . Here, the transmission region of the filter “transmits” or “includes” the excitation wavelength or the fluorescence wavelength does not mean that the transmission region of the filter must be wider than the excitation wavelength region or the fluorescence wavelength region. It means that at least a part of the excitation wavelength region or at least a part of the fluorescence wavelength region is transmitted.

  As a preferred second form of the filter set of the present invention, an excitation light filter that transmits two excitation wavelengths for exciting two types of fluorescent reagents, a fluorescent filter that transmits two fluorescence wavelengths generated from the fluorescent reagents, and A dichroic filter that reflects the two excitation wavelengths and transmits the fluorescence wavelengths, wherein at least one of the excitation light filter, the fluorescence filter, and the dichroic filter is composed of the optical filter of the present invention. To do. In particular, when the filter of the present invention is used for a dichroic filter, the filter set of the present invention includes two excitation wavelengths in two reflection regions and two fluorescence wavelengths in two transmission regions. Includes band dichroic filter. When the optical filter of the present invention is used as the excitation light filter or the fluorescence filter, it is as described above.

  In the filter set including the two-wavelength band excitation light filter or the two-wavelength band fluorescence filter, the reflection region of the two-wavelength band excitation light filter and the two-wavelength band fluorescence filter preferably has a reflectance of 90% or more on the spectrum. It is set to have a crossing characteristic. Since the present invention has a high degree of freedom in setting the transmission region or the reflection region, such setting is also easy. As a result, even if the excitation light component is reflected by the sample and is incident on the fluorescent filter, most of the reflected light is reflected, so that leakage light from the light source is less incident on the detector. Further, the reflection region of the two-wavelength band excitation light filter and the two-wavelength band fluorescence filter can be set so as to have a characteristic of intersecting with a reflectance of 99% or more on the spectrum. Light can be further prevented from entering the detector.

  In the filter set including the two-wavelength band dichroic filter, the reflection region of the excitation light filter and the two-wavelength band dichroic filter is preferably set to have a characteristic of intersecting with a reflectance of 90% or more on the spectrum. As a result, the sample can be reliably irradiated with the excitation light component that has passed through the excitation filter. Furthermore, it can be set so that the reflection region of the excitation light filter and the two-wavelength band dichroic filter intersect at 95% or more. In this case, the excitation light component that has passed through the excitation light filter can be more reliably irradiated onto the sample.

  In the filter set including the two-wavelength band fluorescent filter, the two-wavelength band fluorescent filter preferably has a wide transmission region so as to cover the emission spectrum of the fluorescent reagent. According to the present invention, since the transmission region width can be set by a combination of notch filter and short wavelength transmission filter or long wavelength transmission filter design, it is easy to widen the transmission region width. The transmissive area width can be set. As a result, an increase in fluorescence signal can be realized.

  When fluorescence measurement is performed using the filter sets of the preferable first and second embodiments, the S / N (signal to noise) ratio of fluorescence detection in the detector is improved.

  The fluorescence measuring apparatus of the present invention is a fluorescence measuring apparatus that measures a sample labeled with two kinds of first and second fluorescent reagents. The first form of the fluorescence measuring device has a first excitation light source that generates light including a first excitation wavelength that can excite the first fluorescence reagent, and a second excitation wavelength that can excite the second fluorescence reagent. An excitation optical system having a second excitation light source that generates light including the light and switching the light from each light source so that the light can be arranged on a common excitation light optical axis; An excitation light filter set to have two transmission regions each including a wavelength and a second excitation wavelength, and two reflection regions each including the first excitation wavelength and the second excitation wavelength, the optical filter of the present invention, And a single transmission region having two transmission regions that transmit two fluorescence wavelengths generated from the fluorescent reagent, reflecting excitation light from the excitation optical system toward the sample, and receiving and transmitting fluorescence from the sample. One die for two wavelength bands A fluorescent filter, a fluorescent filter that is set to have two transmission regions each including two fluorescent wavelengths generated from a fluorescent reagent, and is arranged to receive and transmit fluorescence transmitted through the dichroic filter, and a fluorescent filter And a photodetector arranged at a position for receiving the fluorescence that has passed through. Here, the first excitation light source that generates light that includes the first excitation wavelength capable of exciting the first fluorescent reagent is that the emission spectrum of the first excitation light source is wider than the first excitation wavelength region. This does not mean that the emission spectrum of the first excitation light source includes at least a part of the first excitation wavelength region. The same applies to the second excitation light source.

  In this case, one or both of the excitation light filter and the fluorescence filter is preferably a single two-wavelength band filter formed of the optical filter of the present invention set to have two transmission regions.

  The second form of the fluorescence measuring device has a first excitation light source that generates light including a first excitation wavelength that can excite the first fluorescence reagent, and a second excitation wavelength that can excite the second fluorescence reagent. A second excitation light source that generates light including the excitation optical system that irradiates the sample by switching the light from each light source and switching the light on the common excitation light optical axis; An excitation light filter set to have two transmission regions each including one excitation wavelength and a second excitation wavelength, a detector for detecting fluorescence generated from the sample, and the opposite side of the excitation optical system with respect to the sample Fluorescent light receiving optical system that is arranged at a position for receiving fluorescence from the sample and guides the received fluorescent light to the detector, and two transmission regions that are arranged in the fluorescent light receiving optical system and each include two fluorescent wavelengths generated from the fluorescent reagent Fireflies set to have And a filter, one or both of the excitation light filter and fluorescence filter is a single filter for two-wavelength band formed of the optical filter of the present invention that is configured with two transmission regions.

  An example of a sample to be measured by this fluorescence measuring apparatus is DNA, and the measurement item is, for example, a single nucleotide polymorphism (SNP).

  In the filter of the present invention, since the film thickness of the laminated structure for achieving a desired reflectance can be reduced, productivity can be improved. Furthermore, the width of the transmission region existing between the predetermined wavelength of the transmission filter and the reflection region of the notch filter can be arbitrarily set.

If the filter set of the present invention is used, a plurality of filters conventionally required in the fluorescence measuring apparatus can be integrated, and the number of parts can be reduced and the optical system can be simplified.
Furthermore, in the fluorescence measuring apparatus of the present invention, neither the filter nor the dichroic filter requires mechanical switching, so that fluorescence measurement can be performed with high accuracy.

  As a filter set used in a fluorescence measuring apparatus that performs analysis using two types of fluorescent reagents, a two-wavelength excitation light filter having two transmission regions that transmit two excitation wavelengths that excite the fluorescent reagent, fluorescence Two-band fluorescent filter with two transmission regions that transmit two fluorescence wavelengths generated from the reagent, two reflection regions that reflect two excitation wavelengths, and two transmission regions that transmit two fluorescence wavelengths A dichroic filter for two wavelength bands is required. Examples of the required specifications for each filter are shown in Tables 1 to 3.

A design example is shown below.
As for the film material constituting the laminated structure of the filter, the high refractive index film material is Ta ₂ O ₅ (the refractive index depends on the wavelength, but about 2.2), TiO ₂ (the refractive index depends on the wavelength, but about 2.4), Nb ₂ O ₅ , HfO ₂ or Al ₂ O ₃ , or a mixture containing these, and the low refractive index film material is SiO ₂ (the refractive index depends on the wavelength, but about 1.46) or a mixture thereof It is. Here, Ta ₂ O ₅ is used as the high refractive index film material, and SiO ₂ is used as the low refractive index film material. The substrate was optical glass BK7.

  Film formation was performed by a technique that does not cause a wavelength shift by vapor deposition, and the substrate temperature was 250 ° C. In a normal vapor deposition method, the density of the film is below the bulk. When the density is low, moisture or the like enters the gap, and the spectral characteristics change (shift) depending on the use environment. Therefore, in order to suppress the shift of the spectral characteristics, the film packing density is brought close to the bulk state by performing vapor deposition using an ion process such as ion-assisted vapor deposition, thereby preventing moisture from entering. “Method without wavelength shift” means such a film forming method.

(1) The excitation light filter was designed as follows.
The excitation light filter was manufactured by forming a short wavelength transmission filter on one surface of the substrate and forming a notch filter on the other surface of the substrate.

The design was performed with the base film configuration of the short wavelength transmission filter as (0.5 L H 0.5 L) ¹⁸ (with the center wavelength λ = 663.0 nm). Here, symbol H is a Ta ₂ O ₅ film, symbol L is a SiO ₂ film, and the number before each (the number is not described represents “1”) is the design center of the layer. This means how many times the optical film thickness is 1/4 of the wavelength. The numerical value on the right shoulder of the parenthesis (in this example, “18”) means that the configuration in the parenthesis is repeated as many times as the numerical value. The optical film thickness is the product of the physical film thickness and the refractive index of the film material. The meaning of the base film configuration shown below is also the same. The base film configuration means a laminated configuration based on a design (base design) that serves as a basis for optimization.

Therefore, the structure of the base film of this short wavelength transmission filter is that SiO ₂ films and Ta ₂ O ₅ films are alternately laminated in order from the bottom layer (0.5L H L H L... H 0.5L). A total of 37 layers is formed.

  A spectrum showing the optical characteristics of the filter is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). In the base film configuration of this design, the optical characteristics have ripples in the transmission region near the reflection region. In FIG. 1, “Illumination: WHITE” indicates that the irradiation light used for measurement is white light, “Medium: AIR” indicates measurement in air, and “Substrate: BK7” indicates that the substrate is BK7. “Exit: BK7” means that the exit medium is BK7 glass, “Detector: IDEAL” means that the detector is complete (no sensitivity wavelength dependence), “Angle: 0.0 ( deg) ”indicates that the measurement light is incident on the substrate from the vertical direction,“ Reference: 663.0 (nm) ”indicates that the laminated structure is designed with 663.0 nm as the central wavelength, and“ Polarization: Ave-”does not exist. Polarization, “First surface: Front” means that light is incident from the substrate surface.

  Next, optimization design was performed to reduce ripples based on this base film configuration. Optimization design is performed by adjusting the thickness of some layers at both ends of the laminated structure. If the ripple cannot be sufficiently reduced only by optimizing the base film configuration, some layers are added as adjustment layers at both ends. Optimization design methods for reducing ripple are known and were performed using commercially available software TF Calc® (product of Spectra. Inc.). The filter optimization design shown below was performed in the same manner.

  A spectrum showing the optical characteristics of the optimized filter is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). It can be seen that the ripple in the transmission region near the reflection region is reduced.

The design was made with the base film configuration of the notch filter as (L 3H) ²⁰ (with the center wavelength λ = 527.0 nm). A spectrum showing the optical characteristics of the filter is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). In the base film configuration of this design, the optical characteristics have ripples in the transmission region on both sides of the reflection region and on the side close to the reflection region.

  The width of the reflection band is determined by the difference in refractive index between the high refractive index material and the low refractive index material. The greater the difference in refractive index, the wider the width of the reflection band. However, the width of the reflection band can be widened by the optimized design, but depending on the film thickness error in manufacturing, a ripple may be generated in a part of the reflection band and the reflectance may be lowered.

  FIG. 4 shows a spectrum showing the optical characteristics of the filter after optimization design is performed to reduce ripples based on this base film configuration. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). It can be seen that the ripple in the transmission region near the reflection region is reduced.

  The optical filter of the excitation light filter in a state in which the short wavelength transmission filter having the optimized design is formed on one surface of the glass substrate and the notch filter having the optimized design formed on the other surface of the glass substrate. A spectrum showing the characteristics is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). The excitation light filter having this optical characteristic satisfies the required specifications shown in Table 1.

(2) The fluorescent filter was designed as follows.
The fluorescent filter was manufactured by forming a long wavelength transmission filter on one surface of the substrate and forming a notch filter on the other surface of the substrate.

The base film configuration of the long wavelength transmission filter was designed as (0.5H L 0.5H) ¹⁷ (with the center wavelength λ = 450.0 nm). A spectrum showing the optical characteristics of the filter is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). In the base film configuration of this design, the optical characteristics have ripples in the transmission region near the reflection region.

  FIG. 7 shows a spectrum showing the optical characteristics of the filter after the optimization design is performed to reduce the ripple based on this design. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). It can be seen that the ripple in the transmission region near the reflection region is reduced.

  The base film configuration of the notch filter was designed with (L 3 H) 17 (with the center wavelength λ = 585.0 nm). A spectrum showing the optical characteristics of the filter is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). In the base film configuration of this design, the optical characteristics have ripples in the transmission region on both sides of the reflection region and on the side close to the reflection region.

  FIG. 9 shows a spectrum showing the optical characteristics of the filter after optimization design is performed to reduce ripples based on this base film configuration. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). It can be seen that the ripple in the transmission region near the reflection region is reduced.

  The optical characteristics of a fluorescent filter in which a long-wavelength transmission filter with such an optimized design is formed on one surface of a glass substrate and a notch filter with an optimized design formed on the other surface of the glass substrate is shown. The spectrum is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). The fluorescent filter having this optical characteristic satisfies the required specifications shown in Table 2.

(3) The dichroic filter was designed as follows.
The dichroic filter was manufactured by forming a long wavelength transmission filter on one surface of the substrate and forming a notch filter on the other surface of the substrate.

The base film configuration of the long wavelength transmission filter was designed as (0.5H L 0.5H) ¹⁹ (center wavelength λ = 497.0 nm). A spectrum indicating the optical characteristics of the filter is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). In the base film configuration of this design, the optical characteristics have ripples in the transmission region near the reflection region.

  FIG. 12 shows a spectrum showing the optical characteristics of the filter after performing the optimization design in order to reduce the ripple based on this design. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). It can be seen that the ripple in the transmission region near the reflection region is reduced.

  The base film configuration of the notch filter was designed with (L 3 H) 17 (with the center wavelength λ = 625.0 nm). A spectrum showing the optical characteristics of the filter is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). In the base film configuration of this design, the optical characteristics have ripples in the transmission region on both sides of the reflection region and on the side close to the reflection region.

  FIG. 14 shows a spectrum showing the optical characteristics of the filter after optimization design has been performed to reduce ripples based on this base film configuration. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). It can be seen that the ripple in the transmission region near the reflection region is reduced.

  The optical characteristics of a dichroic filter in which a long-wavelength transmission filter with such an optimized design is formed on one surface of a glass substrate and a notch filter with an optimized design formed on the other surface of the glass substrate are shown. The spectrum is shown in FIG. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). The dichroic filter having this optical characteristic satisfies the required specifications shown in Table 3.

  FIG. 16 shows the optical characteristics of a filter set including the excitation light filter a, the fluorescence filter b, and the dichroic filter c manufactured based on the above design. It can be seen that the reflection regions of the excitation light filter a and the fluorescence filter b intersect with a reflectance of about 99%. Further, it can be seen that the reflection regions of the excitation light filter a and the dichroic filter c intersect each other at a reflectance of about 95%.

  FIG. 17 shows the optical characteristics of the existing bandpass filter and the two-wavelength excitation light filter and the two-wavelength fluorescent filter produced in the above-described embodiment. a is the fluorescence filter of an Example, b is the excitation light filter of an Example. c to f are existing bandpass filters, c is an excitation light filter (for yellow excitation light), d is a fluorescence filter (for yellow excitation light), e is an excitation light filter (for blue excitation light), and f is fluorescence. This is a filter (for blue excitation light). It can be seen that each filter of the example has a wider transmission region width and higher transmittance than the existing products. Even in the fluorescence measurement in which the filter of the example and the existing product were mounted and compared in the same fluorescence measuring device, the filter of the example could obtain a higher fluorescence signal than the existing product.

  FIG. 18 shows an example of the absorption and emission wavelength bands of LEDs and fluorescent reagents. a is the absorption spectrum of the fluorescent reagent FAM, b is the fluorescence spectrum emitted by the fluorescent reagent FAM, and c is the emission spectrum of the blue LED. From these spectra, the fluorescent reagent FAM can be excited by the blue LED to emit fluorescence. Further, d is an absorption spectrum of the fluorescent reagent Redmond Red, e is a fluorescent spectrum emitted by the fluorescent reagent Redmond Red, and f is an emission spectrum of the yellow LED. From these spectra, the fluorescent reagent Redmond Red can be excited by a yellow LED to emit fluorescence. As long as these LEDs and fluorescent reagents are used, it can be seen that fluorescence measurement can be performed with a good S / N ratio by using a filter set including each filter of the above-mentioned Examples.

  Next, an application example to a fluorescence measuring apparatus suitable for using the filter set of the present invention will be shown. A suitable example is a gene polymorphism detection device that detects a single nucleotide polymorphism of DNA.

  The followings have been proposed as methods or apparatuses for predicting the susceptibility of diseases using genetic polymorphism.

  In order to determine whether a patient is susceptible to sepsis and / or is likely to progress rapidly to sepsis, a nucleic acid sample is taken from the patient and the pattern 2 allele in the sample, or the pattern 2 allele When a marker gene that is linkage disequilibrium is detected, and a marker gene that is linkage disequilibrium with the pattern 2 allele or the pattern 2 allele is detected, it is determined that the patient is susceptible to sepsis (see Patent Document 2). ).

  For analysis of one or more single nucleotide polymorphisms in the human flt-1 gene, one or more positions of human nucleic acids: 1953, 3453, 3888 (each position in EMBL accession number X51602) ), 519, 786, 1422, 1429 (according to positions in EMBL accession number D64016, respectively), 454 (according to SEQ ID No. 3) and 696 (according to SEQ ID No. 5), refer to these polymorphisms By doing this, a flt-1 ligand-mediated disease is diagnosed (see Patent Document 3).

  Many methods have been reported for so-called typing for discriminating the base of an SNP site. A typical one is the following method.

  In order to perform typing on hundreds of thousands of SNP sites using a relatively small amount of genomic DNA, a plurality of base sequences including at least one single nucleotide polymorphism site are used using genomic DNA and a plurality of pairs of primers. At the same time, using a plurality of amplified base sequences, the bases of single nucleotide polymorphic sites contained in the base sequences are discriminated by a typing process. As the typing process, an invader method or a Tuckman PCR method is used (see Patent Document 4).

  In these methods, a fluorescent reagent is generally used to detect a single nucleotide polymorphism of DNA. In order to detect a fluorescent reagent, a light source for exciting the fluorescent reagent (excitation light source), a detector for detecting light emitted from the fluorescent reagent (fluorescence), and light from the excitation light source are directly detected. A filter is needed to prevent entry into the vessel.

  FIG. 19 shows a typical component configuration for conventional fluorescent reagent detection. The light from the light source 2 passes through the collimating lens 4 and then passes through the first filter (excitation light filter) 6 so that only the wavelength band necessary for excitation of the fluorescent reagent is selectively extracted. Next, the dichroic filter 8 selectively reflects only the wavelength band necessary for excitation of the fluorescent reagent, and irradiates the sample 12 through the lens 10. The fluorescence from the fluorescent reagent of the sample 12 is collected by the lens 10, and only the fluorescence wavelength band is selectively transmitted by the second filter (dichroic filter 8). The light transmitted through the dichroic filter 8 is collected and detected by the detector 16 through the lens 14 after unnecessary light other than fluorescence is removed by the third filter (fluorescence filter) 13.

  As the excitation light source 2, a laser, a lamp, a light emitting diode (LED), or the like is used. In recent years, a high-brightness LED is often used. This is because LEDs are smaller and cheaper than lasers, and have a longer life and higher efficiency than lamps.

  For the detector 16, a photomultiplier tube (photomultiplier: PMT), a photodiode (PD), a charge coupled device (CCD), a C-MOS image sensor, or the like is mainly used depending on the application.

  The filters 6, 8, and 13 are formed by forming a thin film on a transparent base material such as glass by a method such as vacuum vapor deposition so as to selectively transmit light.

  Among these optical components, filters 6, 8, and 13 are particularly important components that determine the signal-to-noise ratio (S / N ratio) during fluorescence measurement, and are suitable for the wavelength characteristics of the target fluorescent substance. It is necessary to select. Further, when a light source 2 having a relatively narrow spectrum width such as a laser or LED is used, a light source having a wavelength band that can efficiently excite a target fluorescent material must be selected.

  In contrast, in the detection of single nucleotide polymorphisms (SNPs) of DNA, in order to determine whether a target sample is a wild-type homozygote, a mutant-type homozygote or a heterozygote, usually two types are used. The above fluorescent reagents are used. That is, for example, the probe for detecting the wild type is labeled with the fluorescent reagent A, and the probe for detecting the mutant type is labeled with the fluorescent reagent B. After reacting the sample with the two types of probes, the signal from the fluorescent reagent A and the signal from the fluorescent reagent B are detected. As a result, when only the signal from the fluorescent reagent A is obtained, it can be seen that the sample is wild-type homo. Similarly, it can be seen that when only the signal from the fluorescent reagent B is obtained, the mutant is homozygous, and when the signals from both fluorescent reagents are obtained, it is heterogeneous.

  As described above, it is necessary to select optical components having different characteristics depending on individual fluorescent reagents, whereas it is necessary to use two or more types of fluorescent reagents in order to detect SNPs. Therefore, in order to detect a signal from each fluorescent reagent, a method of mounting a plurality of optical systems for each fluorescent reagent or a method of mechanically switching optical components and corresponding to a plurality of fluorescent reagents is employed.

  In order to detect two or more types of fluorescent reagents, the method of mounting a plurality of optical systems corresponding to the respective fluorescent reagents has a drawback that the entire optical system becomes large. For example, in order to construct an optical system corresponding to two types of fluorescent reagents, all the components shown in FIG. 19 such as a light source, a detector, a filter, and a lens must be individually arranged. The size is twice that of the fluorescent reagent optical system.

  In addition, since different optical systems are used for each fluorescent reagent, it is necessary to adjust optical components for each of the fluorescent reagents, which not only takes time but also causes large variations in adjustment for each optical system. Therefore, when calculating the ratio of fluorescence signals among a plurality of fluorescent reagents for SNP allele determination, the result largely reflects the variation in adjustment, making correct SNP allele determination difficult. ing.

  Furthermore, since each fluorescent reagent is composed of an optical system having a different optical axis, at least one of the sample and the optical system is moved by a motor or the like in order to measure two or more types of fluorescence for one sample. There is a need. Since a mechanism for driving by a motor or the like is required, the apparatus becomes complicated and large, and an error in driving can be an error in fluorescence measurement. As a result, when calculating the ratio of fluorescence signals among a plurality of fluorescent reagents for SNP allele determination, the calculation result greatly reflects the variation in driving, and it is difficult to determine the correct SNP allele. I'm making things.

  As described above, in order to detect two or more types of fluorescent reagents, the method of mounting a plurality of optical systems corresponding to the respective fluorescent reagents complicates not only the optical system but also the surrounding mechanical components. Cause variations in measurement. As a result, there is a problem that the SNP allele determination tends to be inaccurate.

  The method of mechanically switching optical components to handle multiple fluorescent reagents is mainly to switch the disk with multiple filters to a wavelength characteristic suitable for the target fluorescent reagent by moving it with a motor. Realized. In this method, parts such as lenses and detectors can be used in common for a plurality of fluorescent reagents, and it is not necessary to drive a sample or an optical system with a motor or the like in order to measure a plurality of fluorescent reagents. The feature is that more stable measurement is possible.

  However, since it is necessary to switch a plurality of filters according to the fluorescent reagent, a driving component such as a motor is required, and a disk on which a plurality of filters are mounted is also large. In addition, the measurement tends to be unstable because the optical component is moved by a motor. In particular, in the configuration using the dichroic filter, the optical axis of the excitation light applied to the sample is bent at 90 degrees by the dichroic filter. However, when the dichroic filter is switched by a motor, a slight error is caused by the optical axis. It will be a big gap of. As a result, when calculating the ratio of fluorescence signals among a plurality of fluorescent reagents for SNP allele determination, the calculation result greatly reflects the variation in driving, and it is difficult to determine the correct SNP allele. I'm making things.

  As described above, in order to detect two or more types of fluorescent reagents, in the method of mechanically switching optical components and supporting a plurality of fluorescent reagents, the mechanical parts for switching the filters are complicated, and the apparatus is large and complicated. There is a problem that it becomes something. In addition, there is a possibility that the fluorescence measurement becomes unstable depending on the configuration, and there is a problem that the allele determination of the SNP tends to be inaccurate.

  In order to solve the above-described problems, the filter of the embodiment of the present invention that can transmit two or more wavelength bands was used to detect two or more kinds of fluorescent reagents. As an example, FIG. 20 shows a configuration in which an excitation light filter, a dichroic filter and a fluorescence filter capable of transmitting two wavelength bands for detecting two types of fluorescent reagents are used. In this example, two types of LEDs are used as the light source and a photodiode is used as the detector, but the present invention is not limited to this.

  Two types of LEDs 20 and 22 having wavelengths with high excitation efficiency of the fluorescent reagent are used as light sources. As a fluorescent reagent for labeling a sample, fluorescent reagent A that emits fluorescence with a wavelength of 510 to 530 nm (green) when excited with a wavelength of 470 to 490 nm (blue) and excited with a wavelength of 580 to 600 nm (yellow to orange) Considering an example where a fluorescent reagent B that emits fluorescence having a wavelength of 620 to 640 nm (red) is used, in this case, it is suitable to use a blue LED and a yellow (or orange) LED. . The lights from the LEDs 20 and 22 are collimated by the lenses 4a and 4b, respectively, and then are made into one optical axis by the first dichroic filter 28. The dichroic filter 28 here may be a conventional dichroic filter that is divided into a transmission region and a reflection region with a predetermined wavelength as a boundary so as to transmit the yellow wavelength band and reflect the blue wavelength band.

  Next, the excitation light filter 30 of the embodiment that can transmit blue and yellow light and reflect green and red light is passed through. Further, the sample 12 is folded back to the portion of the sample 12 disposed below through the dichroic filter 32 of the embodiment capable of reflecting two wavelength bands of blue and yellow and transmitting green and red. Thereafter, the sample 12 collected by the lens 10 and irradiated with the fluorescent reagent is irradiated.

  The fluorescence from the two types of fluorescent reagents excited by the sample 12 and excited by two light beams, blue and yellow, has two different wavelengths such as green and red. These lights are again collected by the lens 10 disposed above the sample portion and pass through the dichroic filter 32. After that, it transmits the two wavelength bands of green and red and transmits the fluorescent filter 34 of the embodiment that can reflect blue and yellow. Finally, the light is collected by the lens 14 and converted into an electric signal by the photodiode 16.

  When detecting the signal of the fluorescent reagent A, only the blue LED 20 is turned on and the yellow LED 22 is turned off. Then, only the fluorescent reagent A is excited by the blue LED 20 to emit fluorescence, while the fluorescent reagent B is not excited and does not emit fluorescence. By doing so, it is possible to detect only the signal of the fluorescent reagent A. Next, when detecting the signal of the fluorescent reagent B, only the yellow LED 22 is turned on, and the blue LED 20 is turned off. Then, on the contrary, only the fluorescent reagent B is excited by the yellow LED 22 to emit fluorescence, and conversely, the fluorescent reagent A is not excited and does not emit fluorescence. In this way, the signals from the two fluorescent reagents can be read independently by the common photodiode 16 simply by turning on and off the two types of LEDs 20 and 22.

  In the apparatus of FIG. 20, two types of LEDs 20 and 22 are provided as light sources, and excitation light from the LEDs 20 and 22 is made into one optical axis by the dichroic filter 28 through the respective lenses 4a and 4b, but the dichroic filter 28 is used. It can also be set as the structure which does not. In a configuration in which the dichroic filter 28 is not used, the LED 20 and the lens 4a may be set as one set, and the LED 22 and the lens 4b may be set as another set so that these sets can be switched and arranged on the optical axis.

  Using the experimental apparatus shown in FIG. 20, it was verified that a filter having two transmission regions is effective for SNP allele determination. High-intensity light emitting diodes (LEDs) 20 and 22 of blue and yellow to orange were used as light sources. The emission spectrum of the LED used is shown in FIG. Light from the lighted LEDs 20 and 22 is collimated by a plano-convex lens 4a or 4b having a diameter of 20 mm and a focal length of 30 mm, and is reflected or transmitted by the dichroic filter 28 and is incident on the excitation light filter 30. As shown in FIG. 21B, the transmission characteristic of the excitation light filter 30 transmits light when the wavelength is 460 to 490 nm and 570 to 590 nm, and does not transmit light when the wavelength is 520 to 560 nm and 620 nm. Thus, by using the filter 30 having two different transmission regions, it is possible to realize a characteristic effective for both of the two types of LEDs 20 and 22 with one filter 30. The light transmitted through the excitation light filter 30 is then folded back by the dichroic filter 32 in the downward direction of the figure. The transmission characteristics of the dichroic filter 32 are shown in FIG. The dichroic filter 32 reflects light having wavelengths of 460 to 490 nm and 570 to 590 nm among light incident at an angle of 45 degrees, and transmits light having wavelengths of 520 to 560 nm and 620 nm. The light reflected by the dichroic filter 32 is collected by a plano-convex lens having a diameter of 12 mm and a focal length of 12 mm, and is applied to the sample 12.

  The fluorescence from the sample 12 is collected by the same plano-convex lens 10, passes through the dichroic filter 32, and further enters the fluorescence filter 34. The transmission characteristics of the fluorescent filter 34 are shown in FIG. The fluorescent filter 34 transmits light at 520 to 540 nm and 630 to 660 nm, and does not transmit light at ˜490 nm and 560 to 600 nm. The light transmitted through the fluorescent filter 34 is collected by the plano-convex lens 14 having a diameter of 12 mm and a focal length of 12 mm, and converted into an electric signal by the photodiode 16. The current from the photodiode 16 is converted into a voltage signal by a current-voltage conversion circuit (not shown) and then measured by a digital voltmeter (not shown).

  As a sample, purified DNA that had already been subjected to invader reaction in another experimental apparatus was used. FAM and Redmond Red were used as fluorescent reagents for labeling the probes arranged in the measurement part. The absorption spectra of these fluorescent reagents are shown in FIG. 22 (a), and the emission spectra are shown in FIG. 22 (b). Among the probes, the probe for detecting the wild type was labeled with FAM, and the probe for detecting the mutant type was labeled with Redmond Red.

  FIG. 23 shows the result of detecting the signal of the sample 12 using the blue LED 20 for the FAM measurement and the yellow to orange LED 22 for the Redmond Red measurement. Fluorescence from the fluorescent reagent is indicated by “fluorescence” in the figure, and reflection / scattering noise at the sample portion is indicated by “reflection / scattered light” in the figure. “Reflected / scattered light” is the result of measurement with water placed in place of the sample instead of the sample. Thus, a sufficiently large fluorescence signal with respect to noise could be obtained. Since the result of this measurement indicates that both fluorescence from FAM and fluorescence from Redmond Red were detected, it can be said that the sample used here has a hetero type.

  In order to detect two or more types of fluorescent reagents, the conventional technique uses a method of mounting a plurality of optical systems corresponding to each fluorescent reagent. However, not only the optical system but also the surrounding mechanical components are complicated. This was a cause of measurement variation among fluorescent reagents. As a result, there is a problem that the allele determination of the SNP tends to be inaccurate. Further, in the method of mechanically switching optical components and dealing with a plurality of fluorescent reagents, which has been used as another method of the prior art, the mechanical components for switching the filter become complicated, and the apparatus becomes large and complicated. There was a problem. In addition, there is a possibility that the fluorescence measurement becomes unstable depending on the configuration, and there is a problem that the allele determination of the SNP tends to be inaccurate.

  On the other hand, in the above method using the filter of the embodiment of the present invention, since most of the optical components such as a lens, a filter, and a detector are used in common with any fluorescent reagent, measurement between the fluorescent reagents is performed. Can be minimized. In addition, when detecting multiple fluorescent reagents, it is only necessary to turn on / off multiple LEDs, which eliminates the need for mechanical parts such as motors, so that measurements can be made stably and the device can be made compact. can do. As a result, SNP allele determination can be performed easily and accurately.

  As shown in the fluorescence detection apparatus shown in FIG. 24, among the excitation light filter, the fluorescence filter, and the dichroic filter used in the epi-illumination optical system, for example, the dichroic filter of the present invention may be used only for the dichroic filter 32. In that case, excitation light filters 30a and 30b each having a single transmission wavelength band are arranged in the optical path of the excitation light as the excitation light filters. On the fluorescent light receiving side, fluorescent filters 34a and 34b having a single transmission wavelength band for each fluorescent light are also used as fluorescent filters. For example, on the fluorescent light receiving side, the fluorescence transmitted through the dichroic filter 32 is separated into two fluorescent light paths according to the wavelength of the fluorescent light using a normal dichroic filter 28a having one transmission region and one reflection region. Is arranged with a fluorescent filter 34a having a transmission wavelength band for the fluorescence, and the fluorescence transmitted through the fluorescent filter 34a is condensed by the lens 14a and is incident on the photodiode 16a. In the other fluorescent light path separated by the dichroic filter 28a, a fluorescent filter 34b having a transmission wavelength band for the fluorescent light is disposed, and the fluorescence transmitted through the fluorescent filter 34b is collected by the lens 14b and incident on the photodiode 16b. Let In the photodiodes 16a and 16b, each fluorescence is converted into an electric signal.

  Even in the case of this embodiment, it is not necessary to arrange the dichroic filter corresponding to each excitation light on the optical axis, which can contribute to downsizing of the apparatus.

  In the embodiment of FIG. 24, an excitation optical system as shown in FIG. 20 may be combined by using the two-band excitation light filter 30 of the present invention instead of the excitation light filters 30a and 30b.

  In the embodiment of FIG. 24, a fluorescent light receiving optical system as shown in FIG. 20 may be combined using the two-wavelength band fluorescent filter 34 of the present invention instead of the fluorescent filters 34a and 34b.

  Although the embodiments so far use an epi-illumination optical system using a dichroic filter, a fluorescence measuring apparatus can be realized without using a dichroic filter. FIG. 25 shows an embodiment of a fluorescence measuring apparatus that does not use a dichroic filter.

  In the fluorescence measuring apparatus shown in FIG. 25, as the excitation light filter, excitation light filters 30a and 30b having a single transmission wavelength band are arranged in the optical path of the excitation light, respectively, as in the fluorescence measuring apparatus of FIG. On the side, a fluorescent filter 34 capable of transmitting two wavelength bands is used as in the embodiment of FIG. Excitation light that has been converted into one optical axis by the dichroic filter 28 of the excitation optical system is irradiated from an oblique direction onto the sample 12 that is collected by the lens 10a and labeled with a fluorescent reagent.

  The sample 12 is irradiated with excitation light, and the fluorescence from the two types of fluorescent reagents respectively excited by the excitation light of each wavelength is collected by the lens 10b of the fluorescence receiving optical system arranged on the opposite side to the excitation optical system. The light is transmitted through the fluorescent filter 34 of the embodiment having transmission bands in two wavelength bands. Finally, the light is collected by the lens 14 and converted into an electric signal by the photodiode 16. In this embodiment, since the detector can be shared in the fluorescence light receiving optical system, it can contribute to miniaturization of the apparatus.

  In the fluorescence measuring apparatus shown in FIG. 25, an excitation light filter 30 capable of transmitting two wavelength bands is used as the excitation optical system in the same manner as in the embodiment of FIG. 20, and the embodiment shown in FIG. Similarly, the fluorescence transmitted through the lens 10b is separated into two fluorescent light paths according to the wavelength of the fluorescence using a normal dichroic filter 28a having one transmission region and one reflection region, and each of the fluorescent light paths has a single transmission wavelength. Fluorescent filters 34a and 34b having bands may be arranged respectively. In this embodiment, since the number of excitation light filters can be reduced in the excitation optical system, it can contribute to downsizing of the apparatus.

It is a figure which shows the spectrum by the base film structure of the short wavelength transmission filter for excitation light filters. It is a figure which shows the spectrum after the optimization design of the filter. It is a figure which shows the spectrum by the base film structure of the notch filter for excitation light filters. It is a figure which shows the spectrum after the optimization design of the filter. It is a figure which shows the spectrum of the excitation light filter of one Example which consists of the short wavelength transmission filter optimized and the notch filter optimized. It is a figure which shows the spectrum by the base film structure of the long wavelength transmission filter for fluorescence filters. It is a figure which shows the spectrum after the optimization design of the filter. It is a figure which shows the spectrum by the base film structure of the notch filter for fluorescence filters. It is a figure which shows the spectrum after the optimization design of the filter. It is a figure which shows the spectrum of the fluorescence filter of one Example which consists of the long wavelength transmission filter optimized and the notch filter optimized. It is a figure which shows the spectrum by the base film structure of the long wavelength transmission filter for dichroic filters. It is a figure which shows the spectrum after the optimization design of the filter. It is a figure which shows the spectrum by the base film structure of the notch filter for dichroic filters. It is a figure which shows the spectrum after the optimization design of the filter. It is a figure which shows the spectrum of the dichroic filter of one Example which consists of the long wavelength transmission filter optimized and the notch filter optimized. It is a figure which shows the spectrum of the filter set produced in the Example. It is a figure of the spectrum which compares the optical characteristic of the excitation light filter and fluorescence filter which were manufactured in the Example with the conventional existing product. It is a figure which shows the spectrum of an example of 2 types of LED and the absorption and emission wavelength range of a fluorescence reagent. It is a schematic block diagram which shows the conventional fluorescence detection apparatus. It is a schematic block diagram which shows the fluorescence detection apparatus using the filter set of an Example. It is a figure which shows the spectrum of two types of LED and a filter set. It is a figure which shows the absorption spectrum and emission spectrum of two types of fluorescent reagents. It is a figure which shows the measurement result of the DNA sample which the invader reaction was complete | finished. It is a schematic block diagram which shows the other Example of the fluorescence detection apparatus using the filter of an Example. It is a schematic block diagram which shows the further another Example of the fluorescence detection apparatus using the filter of an Example.

Explanation of symbols

4a, 4b, 10, 10a, 10b, 14 Lens 12 Sample 16 Photodiode 20, 22 LED
28, 32 Dichroic filter 30 Excitation light filter 34 Fluorescence filter

Claims (11)

A substrate that is transparent in the wavelength range used;
It is formed on one surface of the substrate and has a laminated structure in which a high refractive index layer and a low refractive index layer are alternately laminated sequentially, and one of the short wavelength side and the long wavelength side from the predetermined wavelength is a transmission region and the other A transmission filter in which is a reflection region,
A wavelength region that is formed on the other surface of the substrate and has a laminated structure in which a high refractive index layer and a low refractive index layer are alternately laminated in sequence, within a transmission region of the transmission filter and away from the predetermined wavelength And a notch filter having a reflection region,
An optical filter having two transmission regions and two reflection regions that are discontinuous in the spectrum. The transmission filter is a short wavelength transmission filter in which a shorter wavelength side than the predetermined wavelength is a transmission region,
The optical filter according to claim 1, wherein the transmission region and the reflection region are a first transmission region, a first reflection region, a second transmission region, and a second reflection region in order from the short wavelength side. The transmission filter is a long wavelength transmission filter having a transmission region on the longer wavelength side than the predetermined wavelength,
The optical filter according to claim 1, wherein the transmission region and the reflection region are a first reflection region, a first transmission region, a second reflection region, and a second transmission region in order from the short wavelength side. In a filter set consisting of a set of filters used in an apparatus that performs analysis using a fluorescent reagent,
An excitation light filter in which two transmission regions are set so as to transmit two excitation wavelengths for exciting the fluorescent reagent;
A fluorescent filter in which two transmission regions are set so as to transmit two fluorescent wavelengths generated from the fluorescent reagent;
The single filter comprising the optical filter according to any one of claims 1 to 3, which is set so as to have two reflection regions that reflect the two excitation wavelengths and two transmission regions that transmit the two fluorescence wavelengths. A filter set comprising a single dichroic filter for two wavelength bands. The one or both of the said excitation light filter and the fluorescence filter are the filters for single 2 wavelength bands which consist of the optical filter as described in any one of Claim 1 to 3 set so that it may have two permeation | transmission area | regions. The filter set according to claim 4. In a filter set consisting of a set of filters used in an apparatus that performs analysis using a fluorescent reagent,
An excitation light filter in which two transmission regions are set so as to transmit two excitation wavelengths for exciting the fluorescent reagent;
A fluorescent filter in which two transmission regions are set so as to transmit two fluorescent wavelengths generated from the fluorescent reagent,
The one or both of the said excitation light filter and the fluorescence filter are the filters for single 2 wavelength bands which consist of the optical filter as described in any one of Claim 1 to 3 set so that it may have two permeation | transmission area | regions. A filter set characterized by that. The filter set according to any one of claims 4 to 6, wherein the reflection region of the two-wavelength band excitation light filter and the two-wavelength band fluorescence filter intersects at a reflectance of 90% or more. The filter set according to any one of claims 5 to 7, wherein the two-wavelength band fluorescent filter has a wide transmission region so as to cover an emission spectrum of the fluorescent reagent. A fluorescence measuring apparatus for measuring a sample labeled with two types of first and second fluorescent reagents,
A first excitation light source that generates light including a first excitation wavelength capable of exciting the first fluorescent reagent and a second excitation that generates light including a second excitation wavelength capable of exciting the second fluorescent reagent. An excitation optical system having a light source and switching light from each light source so that the light can be arranged on a common excitation light optical axis;
An excitation light filter disposed in the excitation optical system and set to have two transmission regions each including the first excitation wavelength and the second excitation wavelength;
The optical filter according to any one of claims 1 to 3, wherein two reflection regions each including the first excitation wavelength and the second excitation wavelength and two fluorescence wavelengths generated from the fluorescence reagent are transmitted. A single two-wavelength band dichroic filter having two transmission regions, arranged to reflect excitation light from the excitation optical system toward the sample, and to receive and transmit fluorescence from the sample;
A fluorescence filter that is set to have two transmission regions each including two fluorescence wavelengths generated from the fluorescence reagent, and is arranged at a position to receive and transmit the fluorescence transmitted through the dichroic filter;
A photodetector disposed at a position for receiving the fluorescence transmitted through the fluorescence filter;
Fluorescence measuring device with The one or both of the said excitation light filter and the fluorescence filter are the filters for single 2 wavelength bands which consist of the optical filter as described in any one of Claim 1 to 3 set so that it may have two permeation | transmission area | regions. The fluorescence measuring apparatus according to claim 9. A fluorescence measuring apparatus for measuring a sample labeled with two types of first and second fluorescent reagents,
A first excitation light source that generates light including a first excitation wavelength capable of exciting the first fluorescent reagent and a second excitation that generates light including a second excitation wavelength capable of exciting the second fluorescent reagent. An excitation optical system having a light source, switching the light from each light source and irradiating the sample by placing it on a common excitation light optical axis;
An excitation light filter disposed in the excitation optical system and set to have two transmission regions each including the first excitation wavelength and the second excitation wavelength;
A detector for detecting fluorescence generated from the sample;
A fluorescence receiving optical system arranged at a position for receiving fluorescence from the sample on the opposite side of the excitation optical system with respect to the sample, and guiding the received fluorescence to the detector;
A fluorescence filter disposed in the fluorescence receiving optical system and set to have two transmission regions each including two fluorescence wavelengths generated from the fluorescence reagent,
The one or both of the said excitation light filter and the fluorescence filter are the filters for single 2 wavelength bands which consist of the optical filter as described in any one of Claim 1 to 3 set so that it may have two permeation | transmission area | regions. A fluorescence measuring apparatus characterized by that.

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