JP2004251814A - Method and analyzer for fluorometric analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enhance sensitivity and resolution in fluorometric measurement using a DNA chip, or a protein chip, making the best use of a basic technique for an existing femto-second confocal microscope. <P>SOLUTION: A measuring system of high detection S/N ratio is realized, using two kinds of fluorescences different in fluorescence spectrum, in which the fluorescence spectra are crossed not in peak positions of the fluorescences but in wavelengths sided to foots thereof, and using as a light source for an excitation beam a femto-second pulse laser beam of which the wavelength is varied periodically around a crossing position as the center, and high-resolution of observation is attained using a two-photon or multiple-photon absorbing process. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a fluorescence analysis method and a fluorescence analysis device, and more specifically, to a fluorescence analysis method and a fluorescence analysis device that cause less damage to a subject, have good detection sensitivity, and are inexpensive.
[0002]
The Human Genome Project was completed, and the nucleotide sequence containing human genetic information was decoded. In the future, this information will be used to reform medical technologies such as personalized medicine, drug discovery, and regenerative medicine. Among such movements in the life science field, there is a field related to a device for analyzing DNA or protein. At present, DNA and proteins are prepared by arranging a large number of different standard DNAs in a grid pattern on a substrate (also called a microarray chip) called a DNA chip or a protein chip, and collecting a sample DNA from a cell or the like. Information is obtained by reacting with.
[0003]
In order to realize genomic drug discovery and the like that have attracted industrial attention, DNA and protein information must be collected using various techniques in the future. However, each person has about 3 billion base sequences, and at present, it is estimated that scanning a single human base sequence costs about 30 billion yen, resulting in extremely large inspection costs. It is known to happen. Therefore, in order to make the genome application industry a reality, a cost reduction technique for information analysis is strongly desired, and the present invention relates to an analysis method and an analyzer that realize the cost reduction.
[0004]
[Prior art]
Nucleotide sequences containing the entire human genetic information (human genome) are almost being determined, and movements to elucidate the structure and function of proteins aimed at utilizing such genetic information concretely have become active. Have been. Amid these trends, the cost of analyzing individual genetic information has become a problem.
[0005]
In order to realize genomic drug discovery that has attracted attention from an industrial perspective, it is necessary to collect basic information on proteins from various angles using various methods in the future. However, as mentioned above, the base sequence of each person is about 3 billion bases. For example, the genome information of 1000 people is about 3 tera bases, and the information collection cost is assumed to be 1 yen per base. However, huge inspection costs are incurred. Therefore, in order to realize genomic drug discovery and make personalized medicine a reality, it is essential to develop measurement methods in consideration of cost reduction in parallel with basic research.
[0006]
As part of this, research and development have been promoted, such as increasing the density of microarray chips or making them three-dimensional. The strategy is to reduce the cost by increasing the analysis density of one chip. However, when the density is increased by the conventional method,
(1) The wavelengths of the excitation light and the fluorescence are close to each other, so that weak fluorescence cannot be detected. However, as the density increases, the intensity of the excitation light must be increased. Such conflicting issues arise.
(2) In addition, since the wavelength of visible light is directly excited, damage to the sample must be considered. In parallel with the increase in the density of microchips, the challenges of light sources must be overcome.
[0007]
On the other hand, confocal fluorescence microscope systems utilizing the two-photon absorption effect or the multi-photon absorption effect have recently become widespread as devices utilizing the characteristics of femtosecond light pulses. A brief summary of the features of this method is as follows.
{Circle around (1)} Since the wavelength of the femtosecond pulse is generally in the near-infrared region, there is little destruction of cells.
{Circle around (2)} Since the focal point where the multiphoton absorption process occurs is smaller than the part where the fundamental wave is condensed, observation with higher resolution than the resolution determined by the diffraction limit of the fundamental wave becomes possible.
{Circle around (3)} Since the minute portion can be excited without being affected by the confocal depth of the condenser lens, scanning in the depth direction (three-dimensional scanning) is also possible.
[0008]
As described above, high-precision observations that could not be measured with a conventional microscope can be performed. At present, the technical features mentioned here are limited to fluorescence microscopes, but by applying this technology to microchip array analysis with high density, dramatic improvement in sensitivity, shortening of analysis time, and eventually It can be expected to reduce analysis costs.
[0009]
As a typical example of the prior art relating to these analyses, Japanese Patent Application Laid-Open No. 2000-500874, “System for Identifying Fluorescent Molecules Using Time-Resolved Fluorescence Measurement” (hereinafter referred to as Document 1), Japanese Patent Application Laid-Open No. 2001-100102, Japanese Patent Application Laid-Open No. 2001-100102, "Flow Fluorescence Method and Apparatus" (hereinafter referred to as Document 2), Japanese Patent Application Laid-Open No. 10-318924, "Optical Device with Pulsed Laser" (hereinafter referred to as Document 3). "Microscope system" (hereinafter, referred to as Reference 4) and Japanese Patent Publication No. 9-507579, "Signal processing method and apparatus for chemical analysis of a sample" (hereinafter, referred to as Reference 5) can be given.
[0010]
Document 1 describes the use of at least two groups of molecules that bind analyte molecules and have different fluorescent properties.
[0011]
Document 2 describes an analyzer using two-photon excitation.
[0012]
Literature 3 describes a fluorescence detection device using a sub-picosecond pulse laser as a light source and utilizing two-photon absorption and multi-photon absorption.
[0013]
Literature 4 discloses a first laser light as a pump light having a first wavelength for exciting a molecule from a ground state to a first excited state, and an excitation from the first excited state to a second excited state. A super-resolution microscope system using a double resonance absorption process is described by irradiating a sample with a second laser light as an erase light having a second wavelength for performing the first wavelength and the second wavelength. The wavelength 2 is generated by wavelength-modulating the laser light.
[0014]
Document 5 describes that a sample is irradiated with two different wavelengths of laser light having wavelengths separated by 3 to 40 nm generated by using wavelength modulation to measure fluorescence.
[0015]
These documents 1 to 5 describe various attempts to improve the accuracy, sensitivity, and protection of the sample when measuring the fluorescence emitted from the sample irradiated with the laser beam. The common technical idea is to utilize the peak value of the absorption spectrum or the fluorescence emission spectrum of the sample, and the laser light is not a laser light that periodically fluctuates in a narrow wavelength region, but rather. That is, a stable laser beam is used.
[0016]
[Problems to be solved by the invention]
However, conventional fluorescence analysis methods and apparatuses have high analysis costs, are not sufficiently accurate in analysis, and do not sufficiently consider the damage to the sample. It is strongly desired.
[0017]
An object of the present invention is to make use of the basic technology of the existing femtosecond confocal microscope to improve the sensitivity and resolution in fluorescence measurement using a DNA chip or a protein chip. It is an object of the present invention to provide a device that introduces laser technology and relatively improves observation technology using a plurality of fluorescent labels.
[0018]
Here, a femtosecond laser refers to a pulsed light laser having a pulse width on the order of femtoseconds narrower than 1 picosecond.
[0019]
From the social background and the industrial point of view, promoting the spread of equipment is the biggest issue. For that purpose, it is necessary to develop a technology to reduce the cost of femtosecond laser light sources, which determines most of equipment costs. It becomes important. In order to reduce the cost of the light source, it is necessary to reduce the output intensity of the femtosecond laser light source to the minimum necessary for power saving and cost reduction by optimizing the oscillation conditions. In addition, if the system is considered as a device, considering the flow of R & D on microchip arrays, signals from fluorescent dyes will be sufficient even when responding to array densification and excitation with reduced laser output. The idea is to devise a signal detection technique so that it can be detected by S / N.
[0020]
The technical items are summarized as follows.
As a part of reducing the cost of testing genetic information such as DNA chips, the density of chips has been increased. In a conventional analyzer, a normal laser is used as an excitation light source. In this technique, a reduction in resolution when the dot size is increased to 1 micron or less is inevitable.
[0021]
Further, when the dot size becomes 1 micron or less, the fluorescence intensity from each dot becomes weak. Therefore, it is attempted to maintain the sensitivity by increasing the intensity of the excitation laser. However, in the conventional method, there is a problem of separation between the laser to be intensified and basically weak fluorescence.
[0022]
By realizing precise dispersion compensation inside the laser cavity and controlling the optimal mode-locking conditions, an energy-saving light source necessary and sufficient to excite a fluorescent dye or a fluorescent protein can be realized.
[0023]
For that purpose, a multilayer evaporation mirror having a function of continuously changing the dispersion amount is required.
[0024]
[Means for Solving the Problems]
The present invention has been made to solve many of these problems.
[0025]
Particularly salient features of the present invention are as follows.
[0026]
(1) The present invention takes into account that usually two types of fluorescent dyes, which emit red and green, are used as labels in a DNA chip or protein chip inspection apparatus, and the characteristics thereof are utilized as they are. That is, the detection sensitivity is improved by differentially amplifying the two types of fluorescence.
[0027]
In other words, by using two types of fluorescent labels, the wavelength modulation using the slope of the absorption spectrum of each fluorescent label and the intersection of each absorption spectrum as a center wavelength with the wavelength corresponding to the vicinity of the intersection of the absorption spectra as the center wavelength. Is used as the excitation light, 180 degrees of phase difference information is added to each fluorescence, and after the fluorescence from each fluorescent label is wavelength-separated, each fluorescence is separated by a photodetector. The present inventors have proposed a fluorescence detecting unit that converts electric signals corresponding to fluorescence, guides each electric signal to a differential amplifier, and removes common-mode noise mainly caused by mixing of excitation light with a common-mode noise removal ratio of about 100 dB.
[0028]
(2) In the example of the present invention, if a femtosecond laser is used as an excitation light source and the two-photon absorption effect is used, the excitation wavelength can be set in the infrared region. It is possible to avoid a decrease in detection sensitivity due to mixing of scattered light and the like due to the proximity of the fluorescent wavelengths.
[0029]
(3) In the example of the present invention, in a two-photon or multi-photon absorption process caused by irradiating a femtosecond light pulse, the focal portion is smaller than the portion focusing the fundamental wave. Observation with higher resolution than the focusing resolution determined by the wave diffraction limit is possible.
[0030]
Further, when a molecular sample in which the above two types of fluorescent labels are simultaneously bound is used, a place where the fluorescent labels emit light simultaneously within the light-collecting region is specified. Higher resolution than measurement by photon absorption process.
[0031]
By increasing the electrical sensitivity and introducing a femtosecond laser beam that can make use of the binding of the molecular size of the fluorescent label, it is possible to cope with an increase in the density of array dots.
[0032]
(4) In the example of the present invention, in order to achieve downsizing by optimizing the oscillation conditions and comprehensive cost reduction, a conventional dispersion compensating mirror using a vapor-deposited thin film and a dispersion compensating amount variable type mirror using oblique vapor deposition are manufactured. By realizing precise dispersion compensation inside the laser resonator and controlling the optimal mode-locking conditions, a low-cost and small femtosecond laser beam can be realized.
[0033]
Hereinafter, features of the present invention will be described in detail.
[0034]
In order to achieve the object of the present invention, an example of the fluorescence analysis method of the present invention is a method of irradiating a biomolecule sample with excitation light so that the biomolecule sample or a phosphor added as a label to the biomolecule sample is used. A fluorescent analysis method for determining the components of a biomolecule sample by detecting the intensity and spectrum of the fluorescence emitted from a fluorescent material, wherein the vertical axis defined as the absorption spectrum for the excitation light as the fluorescent substance is the excitation light absorption of the fluorescent substance. At least two types of phosphors having different excitation light absorption intensity-wavelength curves represented by a graph with the intensity taken and the horizontal axis representing the excitation light absorption wavelength of the phosphor are used, and the two types of phosphors have their absorption spectra. Is not at the peak position of each absorption spectrum, the slope of the absorption spectrum of one phosphor is positive and the slope of the absorption spectrum of the other phosphor is negative Intersecting phosphors, wherein the excitation light is excitation light that is wavelength-modulated so that the wavelength periodically changes over at least a part of the time during irradiation of the sample. And
[0035]
Then, in the example of the fluorescence analysis method of the present invention, after the fluorescence from the fluorescent label is wavelength-separated, each wavelength-separated fluorescence is converted into an electric signal corresponding to each fluorescence by a photodetector, It is characterized in that fluorescence detection means for guiding a signal to a differential amplifier and removing common-mode noise is used.
[0036]
An example of the fluorescence analysis method of the present invention is such that, when λ1 and λ2 are positive numbers, the excitation light is between the respective wavelengths λ3 and λ4 that give respective peak values of the absorption spectra of the two kinds of phosphors. The pump light is wavelength-modulated pumping light that changes between λ0 + λ1 and λ0-λ2 with the specific wavelength λ0 interposed therebetween, and each of the wavelengths λ0 + λ1 and λ0-λ2 is a shorter one of the wavelengths λ3 and λ4. The wavelength is longer than the longer wavelength of the wavelengths λ3 and λ4.
[0037]
In an example of the fluorescence analysis method of the present invention, the wavelength modulation that changes between λ0 + λ1 and λ0-λ2 across the specific wavelength λ0 is applied during the entire time period during which the sample is irradiated with the excitation light. It is characterized by:
[0038]
An example of the fluorescence analysis method of the present invention is characterized in that the change in the wavelength of the excitation light is a sine wave having a specific wavelength λ0 as a central wavelength.
[0039]
An example of the fluorescence analysis method of the present invention is characterized in that the detected fluorescence is a method of measuring fluorescence emission due to a multiphoton absorption effect in a biomolecule sample.
[0040]
An example of the fluorescence analysis method of the present invention is characterized in that the sample is a sample containing a biomolecule to which an organic dye or a fluorescent protein having a multiphoton absorption effect is added as a label.
[0041]
An example of the fluorescence analysis method of the present invention is characterized in that the sample is added with a plurality of organic dyes or fluorescent proteins having a multiphoton absorption effect as labels, and a plurality of components can be analyzed.
[0042]
An example of the fluorescence analysis method of the present invention is characterized in that the multiphoton absorption is two-photon absorption.
[0043]
An example of the fluorescence analysis method of the present invention is characterized in that the fluorescence is detected by performing a phase detection of a fluorescence signal from a fluorescent dye or a fluorescent protein in synchronization with the frequency of the wavelength modulation of the excitation light. And
[0044]
An example of the fluorescence analysis method of the present invention is characterized in that an optical device having a function of separating a fluorescence wavelength, which is constituted by a spatial beam optical system, is used for detecting fluorescence from the sample.
[0045]
An example of the fluorescence analysis method of the present invention is configured such that, when detecting fluorescence from the sample, the fluorescence from the sample is caused to travel in the optical fiber in a direction opposite to the excitation light, and an optical fiber coupler and an optical fiber. The present invention is characterized in that an optical device having a function of separating a fluorescence wavelength, which is constituted by a fiber filter module, is used.
[0046]
An example of the fluorescence analysis method of the present invention is characterized in that the optical device has a disk-shaped lens array that can scan the entire plane of the sample.
[0047]
In an example of the fluorescence analysis method of the present invention, the signal based on the two types of fluorescence input to the differential amplifier has the same amplitude and the opposite phase to each other at any stage before receiving the differential amplification. The signal is controlled by the control means so that the signal has the following relationship.
[0048]
An example of the fluorescence analysis method of the present invention is characterized in that the control means is means for changing a resistance value of an electric resistance element by an electric means.
[0049]
An example of the fluorescence analysis method of the present invention is that the dispersion compensation of the light source of the excitation light used to excite the sample is performed by using a dispersion compensation element using a multilayer film manufactured using an oblique deposition technique. Features.
[0050]
And the example of the fluorescence analysis method of the present invention is characterized by having some of the features of the present invention.
[0051]
In order to achieve the object of the present invention, an example of the fluorescent analyzer of the present invention is a method of irradiating a biomolecule sample with excitation light to thereby add the biomolecule sample or a phosphor added as a label to the biomolecule sample. A fluorescence analyzer that can determine the components of the biomolecule sample by detecting the intensity and spectrum of the fluorescence emitted from the apparatus, wherein the apparatus is, as the phosphor, a vertical axis defining an absorption spectrum for excitation light. It is possible to use at least two types of phosphors having different excitation light absorption intensity-wavelength curves, which are plotted by taking the excitation light absorption intensity of the phosphor and taking the excitation light absorption wavelength of the phosphor on the horizontal axis. Or a condition setting means corresponding to the phosphor, wherein the two kinds of phosphors have a position where the absorption spectrum is not the peak position of each absorption spectrum. A phosphor that crosses such that the slope of the absorption spectrum of one phosphor is positive and the slope of the absorption spectrum of the other phosphor is negative, and the excitation light is applied to the sample. The pump light is characterized in that the excitation light is wavelength-modulated so that the wavelength periodically changes over at least a part of the time.
[0052]
In the example of the fluorescence analyzer of the present invention, the fluorescence analyzer, after wavelength-separating the fluorescence from the fluorescent label, converts each wavelength-separated fluorescence into an electrical signal corresponding to each fluorescence by a photodetector. And a fluorescence detection unit for guiding each of the electric signals to a differential amplifier to remove common-mode noise.
[0053]
In the example of the fluorescence analyzer according to the present invention, when the excitation light has λ1 and λ2 as positive numbers, the excitation light is between two wavelengths λ3 and λ4 which give respective peak values of the absorption spectra of the two kinds of phosphors. The pump light is wavelength-modulated pumping light that changes between λ0 + λ1 and λ0-λ2 with the specific wavelength λ0 interposed therebetween, and each of the wavelengths λ0 + λ1 and λ0-λ2 is a shorter one of the wavelengths λ3 and λ4. The wavelength is longer than the longer wavelength of the wavelengths λ3 and λ4.
[0054]
In the example of the fluorescence analyzer of the present invention, the excitation light is subjected to wavelength modulation that changes between λ0 + λ1 and λ0-λ2 across the specific wavelength λ0 during the entire time period during which the sample is irradiated. It is characterized by:
[0055]
The example of the fluorescence analyzer of the present invention is characterized in that the change in the wavelength of the excitation light is a sine wave having a specific wavelength λ0 as a center wavelength.
[0056]
An example of the fluorescence analyzer of the present invention is characterized in that the device is a device for measuring fluorescence emission due to a multiphoton absorption effect in a biomolecule sample.
[0057]
An example of the fluorescence analyzer of the present invention is characterized in that the sample is a sample containing a biomolecule to which an organic dye or a fluorescent protein having a multiphoton absorption effect is added as a label.
[0058]
An example of the fluorescence analyzer of the present invention is an apparatus which can use a sample in which the sample is obtained by adding a plurality of organic dyes or fluorescent proteins having a two-photon absorption effect to the sample as labels, and which can analyze a plurality of components. It is characterized by being.
[0059]
An example of the fluorescence analyzer of the present invention is characterized in that the multiphoton absorption is two-photon absorption.
[0060]
An example of the fluorescence analyzer of the present invention utilizes the fact that the device performs a phase detection in synchronization with the frequency of the wavelength modulation of the excitation light with a fluorescence signal from a fluorescent dye or a fluorescent protein in the detection of the fluorescence. Device.
[0061]
An example of the fluorescence analyzer according to the present invention is characterized in that an optical device having a function of separating a fluorescence wavelength, which is constituted by a spatial beam optical system, is used for detecting fluorescence from the sample.
[0062]
An example of the fluorescence analyzer according to the present invention is configured such that, when detecting fluorescence from the sample, the fluorescence from the sample is caused to travel in the optical fiber in a direction opposite to the excitation light, and an optical fiber coupler and an optical fiber are connected. The present invention is characterized in that an optical device having a function of separating a fluorescence wavelength, which is constituted by a fiber filter module, is used.
[0063]
An example of the fluorescence analyzer of the present invention is characterized in that the optical device has a disk-shaped lens array that can scan the entire plane of the sample.
[0064]
In an example of the fluorescence analyzer of the present invention, the signal based on the two types of fluorescence input to the differential amplifier has the same amplitude and the opposite phase to each other at any stage before receiving the differential amplification. The signal is controlled by the control means so that the signal has the following relationship.
[0065]
An example of the fluorescence analyzer of the present invention is characterized in that the control means is means for changing a resistance value of an electric resistance element by an electric means.
[0066]
An example of the fluorescence analyzer of the present invention is based on the fact that a dispersion compensating element using a multilayer film manufactured using an oblique deposition technique is used for dispersion compensation of a light source of excitation light used for exciting the sample. Features.
[0067]
And the example of the fluorescence analyzer of the present invention is characterized by having some of the features of the present invention.
[0068]
In order to achieve the object of the present invention, an example of the fluorescence analyzer of the present invention is to irradiate a sample such as a biomolecule or a biomolecule to which a fluorescent substance or the like is added as a label with excitation light, and to irradiate the sample with the excitation light. In an analyzer that can determine components such as biomolecules by detecting the intensity or spectrum of luminescence emitted from a phosphor or the like added as a label to the biomolecules or the biomolecules, the excitation light includes: In at least a part of the time period during which the sample is irradiated, when λ1 and λ2 are positive numbers, the wavelength periodically changes between λ0 + λ1 and λ0-λ2 across the specific wavelength λ0. It is characterized by being pump light that has undergone wavelength modulation.
[0069]
In the example of the fluorescence analyzer of the present invention, the excitation light is subjected to wavelength modulation that changes between λ0 + λ1 and λ0-λ2 across the specific wavelength λ0 during the entire time period during which the sample is irradiated. It is characterized by:
[0070]
An example of the fluorescence analyzer according to the present invention is characterized in that the change in the wavelength of the excitation light is a sine wave having a specific wavelength λ0 as a center wavelength.
[0071]
And the example of the fluorescence analyzer of the present invention is characterized by having some of the features of the present invention.
[0072]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings used in the description schematically show the dimensions, shapes, arrangements, and the like of the components so that the present invention can be understood. For convenience of explanation of the present invention, the magnification may be partially changed in the drawings, and the drawings used in the present invention may not necessarily be similar to the real thing or the description of the embodiment. In addition, in each drawing, the same components are denoted by the same reference numerals, and redundant description may be omitted.
[0073]
Hereinafter, embodiments of the present invention will be described.
[0074]
Embodiment 1
1 to 5 are diagrams for explaining the basic technical concept of the present invention. FIG. 1 is a diagram for explaining two kinds of dyes whose two-photon absorption spectra just intersect, and FIG. FIG. 3 is a diagram illustrating an absorption spectrum and a fluorescence spectrum of a dye having a long wavelength, FIG. 3 is a diagram illustrating an absorption spectrum and a fluorescence spectrum of a dye having an absorption at a long wavelength, and FIG. FIG. 5 is a diagram illustrating each fluorescence spectrum of a dye having absorption on a longer wavelength side, and FIG. 5 is a diagram illustrating a modulated excitation light and a fluorescence intensity that changes depending on the modulation.
[0075]
1 to 5, reference numeral 1 denotes a two-photon absorption spectrum of Dye A, 2 denotes a fluorescence spectrum of Dye A, 3 denotes a two-photon absorption spectrum of Dye B, 4 denotes a fluorescence spectrum of Dye B, and 5 denotes an X-shape. A dotted line indicating the position of the crossing point on the wavelength of the two-photon absorption spectrum that intersects, 6 is the excitation light frequency-modulated (± Δλ (ν)) around the wavelength (λ0), and 7 is the fluorescence when it is weakened. Intensity spectrum, 8 is the fluorescence intensity spectrum when intensified, 9 is the state when the modulation of the excitation wavelength moves to the short wavelength side, λ0−Δλ, and 10 is the state when the modulation of the excitation wavelength is on the long wavelength side, λ0 + Δλ. The state at the time of movement, 11 shows the fluorescence intensity spectrum when it becomes strong, and 12 shows the fluorescence intensity spectrum when it becomes weak. The vertical axis of the coordinate axes in each figure is the light intensity, and the horizontal axis is the wavelength.
[0076]
The improvement of the light-gathering resolution and the improvement of the detection sensitivity premised on application to a high-density microchip array are based on a principle called a two-photon absorption phenomenon.
[0077]
In a microchip array such as a DNA chip or a protein chip, a fluorescent dye is used as a label.
[0078]
As the fluorescent dye, dye A and dye B whose two-photon absorption spectra just cross each other as shown in FIG. 1 are selected.
[0079]
As shown in FIG. 1, the long wavelength side of the two-photon absorption spectrum 1 of the dye A and the short wavelength side of the two-photon absorption spectrum 3 of the other dye B have an X-shaped cross point at the wavelength indicated by the dotted line 5. Two types of fluorescent dyes are selected.
[0080]
The dye A and the dye B are irradiated with femtosecond pulse excitation light having a center wavelength (λe) twice as long as the wavelength (λ0) of the crossing point on the two-photon absorption spectrum, and the femtosecond two-photon absorption effect is utilized. The dye A and the dye B are excited, and the fluorescence is measured. At this time, the two types of fluorescent dyes reflect a slight difference in the structure of the molecule to be observed, and the two types of fluorescent dyes are bound together, or only one of them is bound. Create different states.
[0081]
At this time, the relationship between the excitation wavelength λe of the femtosecond pulse excitation light and the wavelength λ0 of the spectrum intersection 5 is expressed by the following equation.
[0082]
λ0 = λe / 2
Conventionally, this state has been distinguished as a state that develops red, a state that develops green, and a state that looks yellow.
[0083]
Here, as shown in FIG. 5, the wavelength of the excitation light source is set at the intersection of the two-photon absorption spectrum.
Is excited by light 6 which gives frequency modulation (also referred to as wavelength modulation) (± Δλ (ν)) around the wavelength (λ0).
[0084]
At the time of modulation in which the excitation wavelength moves to the longer wavelength side, that is, in the state of [lambda] 0+ [Delta] [lambda], code 10, the longer wavelength side of the fluorescent dye (green) (colored light dye A) has a downward-sloping spectrum slope of the absorption spectrum. As a result, the fluorescence intensity is weakened as in the fluorescence intensity spectrum 7, while the short wavelength side of the fluorescent dye (red) (fluorescent dye B) feels a right-up slope of the absorption spectrum and the fluorescence intensity is as shown in the fluorescence intensity spectrum 8. Become stronger.
[0085]
Conversely, when the modulation of the excitation wavelength shifts to the shorter wavelength side, that is, in the state of λ0−Δλ, code 9, the longer wavelength side of the fluorescent dye A feels a slope of the absorption spectrum rising to the right, and the fluorescence intensity is increased. On the other hand, the fluorescence intensity becomes strong as shown in the fluorescence intensity spectrum 11, while the short wavelength side of the fluorescent dye B feels a downward slope of the absorption spectrum and the fluorescence intensity becomes weak as shown in the fluorescence intensity spectrum 12.
[0086]
Therefore, when the modulated excitation light is irradiated, the fluorescence intensity 2 of the fluorescent dye A having a shorter fluorescent wavelength (green) and the fluorescent intensity 4 of the fluorescent dye B having a longer wavelength (red) are the same. The fluorescence intensity changes synchronously with light. That is, when the fluorescent intensity 2 of the fluorescent dye on the short wavelength side (green) increases, the fluorescent intensity 4 of the fluorescent dye on the long wavelength side (red) changes to decrease.
[0087]
Here, the principle of differential amplification using two types of fluorescence will be described with reference to FIG.
[0088]
FIG. 8 is a diagram for explaining the principle of differential amplification using two kinds of phosphors. Reference numeral 57 denotes a ratio inversion amplification input, 58 denotes an inversion amplification input, 59 denotes a differential amplifier, and 60 denotes a differential amplifier. , 61 is a signal waveform connected to the ratio inversion input terminal, 62 is a signal waveform connected to the inversion input terminal, 63 is an in-phase noise signal, 64 is an input signal, 65 is an input signal, and 66 is an output signal.
[0089]
A general differential amplifier 59 has an inverting amplification input 58 and a ratio inverting amplification input 57. The voltage or current applied between these two input terminals is amplified with an appropriate amplification factor, and the output terminal 60 is amplified. Lead to. At this time, as a signal form between the input terminals, a signal waveform 62 connected to the inverted input terminal and a signal waveform 61 connected to the ratio inverted input terminal are considered.
[0090]
Since the differential amplifier has a common-mode noise rejection ratio of about 100 dB, the common-mode noise signal 63 does not appear at the output 60. On the other hand, signals having opposite phases, such as the input signal 64 and the input signal 65, are amplified as the output signal 66.
[0091]
Taking advantage of this feature of the differential amplifier, for example, a detection signal from the fluorescent label of the green fluorescent dye A is input to the inverting input terminal 58, and a detection signal from the fluorescent label of the red fluorescent dye B is input to the ratio inversion input terminal 57. Then, for example, a signal caused by the pump light which is likely to be mixed as the in-phase noise signal 63 is removed.
[0092]
FIG. 6 is a schematic diagram of a high-sensitivity fluorescence measurement system including the excitation light source of the present invention. In FIG. 6, reference numeral 13 denotes a master signal source, 14 denotes a femtosecond laser light source, 15 denotes an optical modulator, 16 denotes a sample to be measured, 16A denotes fluorescence emitted from the sample 16 to be measured, and 17 denotes optics that passes red fluorescence. A filter, 18 is an optical filter for passing green fluorescence, 19 and 20 are photodetectors, 21, 22, and 29 are amplifiers, 23 and 24 are variable resistors having an external control function, 25 is a control device, and 26 and 27. Is an output, and 28 is a phase detector.
[0093]
As shown in FIG. 6, the oscillation wavelength of the femtosecond laser light source 14 is modulated by the optical modulator 15 by the master signal source 13 to generate the modulated excitation light 6.
Then, the light 16A from the sample 16 to be measured, for example, the light that has passed through the optical filter 17 that passes red fluorescence and the filter 18 that passes green fluorescence is detected by the light detector 19 and the light detector 20, respectively. I do. Next, the electric signal from each photodetector is amplified by an amplifier 21 and an amplifier 22, respectively, and is input to a variable resistor 23 having an external control function and a variable resistor 24 having an external control function.
[0094]
The outputs 26 and 27 of the variable resistors 23 and 24 are monitored by the control device 25 and are controlled so that the intensities of the outputs 26 and 27 are always the same. By inputting the fluorescence signal whose intensity has been adjusted in this way and the signal of the master signal source 13 to the differential amplifier 28, synchronization detection synchronized with the frequency (ν) of the master signal source 13 is performed, and the amplifier 29 , An output 30 can be obtained.
[0095]
FIG. 7 shows the state of the operation as described above, with the horizontal axis representing the time axis.
[0096]
FIG. 7 is a diagram for explaining a signal timing chart in the above-described example of the high-sensitivity fluorescence detection apparatus of the present invention. Reference numeral 31 denotes a signal waveform of the master signal source 13, and 32 denotes two signals, that is, signal waveforms of the output 26 and the output 27. Is a composite signal of
[0097]
As shown, all signals are shown in synchronization with the signal waveform 31 of the master signal source 13. For example, when the output 26 and the output 27 having the same fluorescence signal intensity enter the differential amplifier 28, a state like a composite signal 32 of the two signals is created internally, and as a result, the output 30 of the amplifier 29 is generated. Becomes zero as a voltage.
[0098]
In this way, if the two types of wavelengths are detected by the synchronization detection, a zero output state can be generated by adjusting the intensity balance of each wavelength. To realize this zero output state, if the control device 25 knows what control voltage difference has been applied to the variable resistors 23 and 24, the state of the fluorescent light that has passed through the filters 17 and 18 is determined. Can be easily detected.
[0099]
By utilizing this characteristic, three states when two types of fluorescent dyes are used can be distinguished.
[0100]
That is, if the target molecule is in a state where only one of the fluorescent dyes is bound, the balance of the zero voltage is lost, and only one of the fluorescences is measured. The output of the detection signal 30 in such a state does not become zero even when the differential gain of the control device 25 is adjusted.
[0101]
The synchronous detection means as described above makes it possible to detect the binding state of the two types of fluorescent dyes sensitively.
[0102]
In particular, it is possible to further finely separate a state that looks yellow as a mixture of red and green colors.
[0103]
That is, the intensity ratio between red fluorescence and green fluorescence can be measured by this detection method. That is, it is possible to electrically discriminate between a strong yellow of red and a strong yellow of green instead of a mere yellow mixed with fluorescence as in the related art.
[0104]
Embodiment 2
In order to reduce the cost of the light source, it is necessary to reduce the output intensity of the femtosecond laser light source to a necessary minimum. In order to generate a femtosecond optical pulse, it is desirable to adjust the dispersion inside the optical resonator, and ideally, the dispersion inside the resonator is zero over the oscillation wavelength region. However, it is not easy to create such a situation, and it is usually not easy to specify the conditions due to variations in characteristics of individual mirrors and variations in characteristics of crystals.
[0105]
Therefore, as one embodiment of the present invention, a variable dispersion compensation type mirror by oblique evaporation as shown in FIG. 9 is proposed.
[0106]
FIG. 9 is a view for explaining a dispersion compensation amount variable type mirror by oblique vapor deposition as one embodiment of the present invention, wherein reference numeral 33 denotes a substrate glass, 34 denotes a dispersion compensation mirror layer, and 35 denotes a thickness in the in-plane direction. The reference numeral 36 denotes an oblique deposition beam, 37 denotes a dispersion compensation characteristic curve, 38 denotes a horizontal axis representing the location of the dispersion compensation mirror, and the vertical axis denotes the secondary group velocity dispersion. Here, the direction in the plane of incidence means a direction parallel to the plane of incidence of the mirror of each layer constituting the multilayer film. A plane parallel to the plane of incidence of the mirror is assumed in each layer, and the direction of the plane is assumed. It is.
[0107]
The layer 35 whose thickness changes in the in-plane direction is illustrated as the uppermost layer in FIG. 9, but is not limited thereto. It may be formed in this way, or all the layers of the dispersion compensating mirror layer 34 may be formed in this way.
[0108]
When a dispersion compensating mirror is formed by the oblique deposition beam 36, the position of the light beam incident on the mirror and the dispersion compensation characteristics can be adjusted by giving a structure to the substrate 33, appropriately rotating the substrate, or controlling the mask. Can be produced by appropriately adjusting the relationship of the change.
[0109]
The variable dispersion compensation type mirror is formed so that the dispersion compensation characteristic changes stepwise with respect to the change of the incident position of the light beam, and the incident position of the light beam on the multilayer film is adjusted by tilting the mirror with respect to the incident beam, The position can be selected as appropriate by the movement of the position, the movement of the light beam by controlling the optical system, and the like.
[0110]
The relationship between the incident position or tilt of the light beam of the variable dispersion compensation type mirror and the dispersion compensation characteristics of the mirror is examined in advance by experiments, etc., and stored as control data in the memory of the control system to improve control accuracy. Can be used to make it.
[0111]
This mirror is a variable dispersion compensation type mirror having a configuration in which a mirror layer 34 capable of compensating for the second-order group velocity dispersion by a film thickness is formed on a substrate glass 33 by an oblique deposition beam 36. By manufacturing a mirror having such a configuration, the dispersion compensation amount indicated by reference numeral 38 changes the second-order group velocity dispersion-wavelength characteristic depending on the location of the mirror, and the content of dispersion compensation changes. A dispersion compensation characteristic curve as shown by 37 is obtained.
[0112]
By constructing an optical resonator using this dispersion compensation variable type mirror, the dispersion inside the resonator can be continuously adjusted by changing the position of the mirror, and the individual resonator-specific dispersion can be adjusted. Dispersion compensation can be easily and accurately performed. As a result, it is possible to reduce the excess pumping energy generated due to the subtle deviation of the dispersion compensation amount, and ultimately to reduce the cost of the light source.
[0113]
Embodiment 3
FIG. 10 is a diagram schematically showing a high-sensitivity DNA chip fluorescence analyzer using a femtosecond laser light source 10 as an embodiment of the present invention. Reference numeral 39 denotes a microarray, 40 denotes an objective lens, and 41 denotes A dichroic mirror that passes both red and green fluorescent light, 42 is a dichroic mirror that reflects red, 43 and 44 are photodetectors, 45 is a detecting device, 46 is a control computer, 47 is a display device, and 48 is a moving stage for scanning. is there.
[0114]
The laser light 6 generated by the femtosecond laser light source 10 and modulated by the master signal source 9 is reflected by the dichroic mirror 41 that reflects the excitation light, and is reflected by the objective lens 40 onto a microarray 39 such as a DNA chip. It is collected.
[0115]
The fluorescence from the microarray resulting from the excitation light travels on the optical axis of the focused excitation beam, not shown, just opposite to the excitation light and passes through both the red and green fluorescence After passing through the dichroic mirror 41, the light reaches a dichroic mirror 42 that transmits green and reflects red.
[0116]
Green transmitted through the mirror 42 is detected by the photodetector 43, and red reflected by the mirror 42 is detected by the photodetector 44.
[0117]
The detection device 45 including the phase detector 28 shown in FIG. 6 and the moving stage 48 for scanning the microchip in a plane are controlled by a control computer 46, and the fluorescence information from the microchip 39 is displayed on the display device 47. Will be displayed.
[0118]
Embodiment 4
FIG. 11 is a diagram schematically illustrating an example in which an optical fiber (also referred to as a fiber) is used in a high-sensitivity DNA chip fluorescence analyzer using a femtosecond laser light source 10 as an embodiment of the present invention. Reference numeral 49 denotes a condenser collimator lens, 50 denotes a single mode optical fiber, 51 denotes a fiber coupler, 52 denotes a filter module, 53 and 54 denote photodetectors, and 55 denotes a collimating lens.
[0119]
The laser light 6 modulated by the master signal source 13 and emitted from the femtosecond laser light source 10 is coupled to the single mode optical fiber 50 by the collimating lens 55. Thereafter, the light is coupled to the fiber coupler 51, and is condensed on the microchip array 39 by the converging collimator lens 49.
[0120]
Although not shown in the figure, the fluorescence from the microchip array 39 travels on the optical axis of the focused excitation beam in a direction exactly opposite to that of the excitation beam, is separated by the fiber coupler 51, and passes through the single mode optical fiber 50. Via the filter module 52.
[0121]
In the filter module 52, the light is separated into red and green light, and enters the light detector 53 and the light detector 54 via the fiber 50.
[0122]
A detection device 45 including the phase detector 28 shown in FIG. 6 and a moving stage 48 for scanning the microchip array in a plane are controlled by a control computer 46, and the fluorescence information from the microchip array 39 is displayed on a display device. 47.
[0123]
Embodiment 5
FIG. 12 is a view schematically showing an example in which an optical fiber is used in a high-sensitivity DNA chip fluorescence analyzer using a femtosecond laser light source 10 as an embodiment of the present invention. Type micro lens array.
[0124]
The laser light 6 modulated by the master signal source 13 and emitted from the femtosecond laser light source 10 is coupled to the single mode optical fiber 50 by the collimating lens 55. Thereafter, the light is coupled to the fiber coupler 51 and is condensed on the microchip array 39 via the disk-shaped micro lens array 56.
[0125]
Fluorescence (not shown) from the microchip array 39 travels on the optical axis of the focused excitation beam just opposite to the optical path of the excitation light, is separated by the fiber coupler 51, and is separated by the single mode light. A fiber 50 couples to a filter module 52. In the filter module 52, the light is separated into red and green light, and enters a light detector 53 and a light detector 54 via a fiber.
[0126]
The rotation of the detection device 45 including the phase detector 28 and the like shown in FIG. 6 and the disk-shaped micro lens array 56 are controlled by the control computer 46, and the fluorescence information from the micro chip array 39 is displayed on the display device. 47.
[0127]
As can be understood from the above description, unlike the conventional laser excitation means, the present invention avoids a decrease in detection sensitivity due to the mixing of scattered light and the like due to the proximity of the excitation wavelength and the fluorescence wavelength. can do.
[0128]
As described above, the present invention has been described with reference to some embodiments. However, the present invention is not limited to this, and allows various variations.
[0129]
For example, for an unknown test sample, a search was performed to find what kind of fluorescent luminescent material having characteristics that can be used for the fluorescence measurement of the present invention, and the excitation light source and measurement system of the present invention were determined based on the results. It is possible to set various control conditions appropriately, and to automate the dispersion compensation of the excitation light source, and to use a monitor that uses optical coupling etc. in the path until the excitation light irradiates the test sample. The monitoring result can be fed back to the control.
[0130]
The variable dispersion compensation type mirror may be configured such that at least a part thereof is a mirror whose dispersion compensation characteristic continuously changes due to a change in the incident beam position.
[0131]
【The invention's effect】
As described above, the present invention provides a fluorescence analysis method and a fluorescence analysis apparatus which can be applied to biological analysis, are inexpensive, have a high detection S / N ratio, and can perform accurate fluorescence measurement. is there. Moreover, damage to the sample is small.
[0132]
Furthermore, since the fluorescence inspection apparatus using the femtosecond laser light of the present invention as excitation light can be expected to dramatically improve detection sensitivity, the development of light sources and detection systems corresponding to the densification of DNA chips and protein chips has been developed. And greatly contribute to the development of the life science field in the future.
[0133]
Further, the present invention can exert a great effect when used for a light source and a detection method of a flow cytometer (cell sorter).
[0134]
By manufacturing a femtosecond laser light source using a “variable compensation type dispersion mirror by oblique deposition” as in the present invention, it becomes possible to optimize the minimum necessary excitation light intensity and output intensity, and further analyze Cost reduction can be realized.
[0135]
In addition, the practical application of femtosecond laser technology to life sciences according to the present invention will have a significant social spillover effect as the biotechnology industry.
[Brief description of the drawings]
FIG. 1 is a diagram illustrating two types of dyes whose two-photon absorption spectra intersect.
FIG. 2 is a diagram illustrating an absorption spectrum and a fluorescence spectrum of a dye having absorption on a short wavelength side.
FIG. 3 is a diagram illustrating an absorption spectrum and a fluorescence spectrum of a dye having absorption on a long wavelength side.
FIG. 4 is a diagram illustrating the respective fluorescence spectra of a dye having absorption on the short wavelength side and a dye having absorption on the long wavelength side.
FIG. 5 is a diagram for explaining wavelength-modulated excitation light and fluorescence intensity that changes depending on the modulation as an embodiment of the present invention.
FIG. 6 is a diagram illustrating a high-sensitivity fluorescence detection device as an embodiment of the present invention.
FIG. 7 is a diagram illustrating a signal timing chart in the high-sensitivity fluorescence detection device as an embodiment of the present invention.
FIG. 8 is a diagram illustrating the principle of differential amplification using two types of phosphors as an embodiment of the present invention.
FIG. 9 is a view for explaining a dispersion compensation amount variable type mirror by oblique deposition used in the present invention.
FIG. 10 is a diagram illustrating a high-sensitivity DNA chip fluorescence analyzer using a femtosecond laser light source as an embodiment of the present invention.
FIG. 11 is a diagram illustrating a high-sensitivity DNA chip fluorescence analyzer using a femtosecond laser light source as an embodiment of the present invention.
FIG. 12 is a diagram illustrating a high-sensitivity DNA chip fluorescence analyzer using a femtosecond laser light source as an embodiment of the present invention.
[Explanation of symbols]
1,3: 2 photon absorption spectrum
2, 4: Fluorescence spectrum
5: dotted line indicating the position of the crossing point on the two-photon absorption spectrum wavelength that crosses in an X shape
6: Light that has been frequency-modulated (± Δλ (ν)) around the wavelength (λ0)
7, 8, 11, 12: Fluorescence intensity spectrum
9: State when the modulation of the excitation wavelength moves to the shorter wavelength side, λ0-Δλ
10: State when the modulation of the excitation wavelength moves to the longer wavelength side, λ0 + Δλ
13: Master signal source
14: Femtosecond laser light source
15: Optical modulator
16: Sample to be measured
16A: fluorescence emitted from the sample 16 to be measured,
17, 18: Optical filter
19, 20, 43, 44, 53, 54: Photodetector
21, 22, 29: Amplifier
23, 24: Variable resistors having an external control function
25: Control device
26, 27, 30: Output
28: Phase detector
31: signal waveform of the master signal source 13
32: Composite signal of two signals
33: Substrate glass
34: Dispersion compensation mirror layer
35: Layer whose thickness changes in the in-plane direction of incidence
36: Oblique deposition beam
37: dispersion compensation characteristic curve
38: horizontal axis representing the location of the dispersion compensation amount mirror
39: Microchip array
40: Objective lens
41, 42: Dichroic mirror
45: Detector
46: Control computer
47: Display device
48: Moving stage to scan
49: Condensing collimator lens
50: Single mode optical fiber
51: Fiber coupler
52: Filter module
55: Collimating lens
56: Disk type micro lens array
57: Ratio inversion amplification input
58: Inverting amplification input
59: Differential amplifier
60: Output from the difference amplifier
61: Signal waveform connected to ratio inversion input terminal
62: signal waveform connected to inverting input terminal
63: common-mode noise signal
64, 65: input signal
66: output signal

Claims (35)

By irradiating the biomolecule sample with excitation light, the components of the biomolecule sample are determined by detecting the intensity or spectrum of the fluorescence emitted from the biomolecule sample or a fluorescent substance added as a label to the biomolecule sample. In the fluorescence analysis method described above, as the phosphor, the excitation light represented as a graph with the excitation light absorption intensity of the phosphor taken on the vertical axis and the excitation light absorption wavelength of the phosphor taken on the horizontal axis, which is defined as the absorption spectrum for the excitation light At least two types of phosphors having different absorption intensity-wavelength curves are used, and the two types of phosphors have a positive absorption spectrum slope at a position where the absorption spectrum is not at the peak position of each absorption spectrum. And the phosphors intersect so that the slope of the absorption spectrum of the other phosphor is negative, and the excitation light is applied to the sample. Fluorescence analysis method wavelength over at least a portion of the time is characterized in that the excitation light is wavelength-modulated so as to vary periodically between being. The fluorescence analysis method according to claim 1, wherein the fluorescence analysis method separates the wavelength of the fluorescence from the fluorescent label and then converts each wavelength-separated fluorescence into an electric signal corresponding to each fluorescence by a photodetector. And a fluorescence detection unit for guiding each of the electric signals to a differential amplifier to remove common-mode noise. 3. The fluorescence analysis method according to claim 1, wherein, when λ 1 and λ 2 are positive numbers, the excitation light has wavelengths λ 3 and λ 4 that give respective peak values of absorption spectra of the two types of phosphors. 4. The pump light is wavelength-modulated pumping light that changes between λ0 + λ1 and λ0−λ2 with a specific wavelength λ0 therebetween. A fluorescence analysis method, wherein the wavelength is longer than the longer wavelength and shorter than the longer wavelength of the wavelengths λ3 and λ4. The fluorescence analysis method according to any one of claims 1 to 3, wherein the excitation light is between λ0 + λ1 and λ0-λ2 across a specific wavelength λ0 during the entire time period during which the excitation light is irradiated on the sample. A fluorescence analysis method characterized by undergoing changing wavelength modulation. The fluorescence analysis method according to any one of claims 1 to 4, wherein a change in the wavelength of the excitation light is a sine wave having a specific wavelength λ0 as a center wavelength. The fluorescence analysis method according to any one of claims 1 to 5, wherein the detected fluorescence is a method of measuring fluorescence emission due to a multiphoton absorption effect in a biomolecule sample. . The fluorescence analysis method according to any one of claims 1 to 6, wherein the sample is a sample containing a biomolecule to which an organic dye or a fluorescent protein having a multiphoton absorption effect is added as a label. Fluorescence analysis method. The fluorescence analysis method according to claim 7, wherein the sample is added with a plurality of organic dyes or fluorescent proteins having a multiphoton absorption effect as labels, and a plurality of components can be analyzed. Method. The fluorescence analysis method according to any one of claims 6 to 8, wherein the multiphoton absorption is two-photon absorption. The fluorescence analysis method according to any one of claims 1 to 9, wherein a phase of a fluorescence signal from a fluorescent dye or a fluorescent protein is synchronized with a frequency of the wavelength modulation of the excitation light in the detection of the fluorescence. A fluorescence analysis method characterized by using a method. The fluorescence analysis method according to any one of claims 1 to 10, wherein an optical device having a function of separating a fluorescence wavelength configured by a spatial beam optical system is used for detecting fluorescence from the sample. Analysis method. The fluorescence analysis method according to any one of claims 1 to 10, wherein the detection of the fluorescence from the sample is performed so that the fluorescence from the sample proceeds in an optical fiber in a direction opposite to the excitation light. A fluorescence analysis method using an optical device having a fluorescence wavelength separating function constituted by an optical fiber coupler and an optical fiber filter module. 13. The fluorescence analysis method according to claim 12, wherein the optical device has a disk-shaped lens array that can scan the entire plane of the sample. The fluorescence analysis method according to any one of claims 1 to 13, wherein the signal based on the two types of fluorescence input to the differential amplifier has an amplitude at any stage before being subjected to differential amplification. The fluorescence analysis method is controlled by control means so that the signals have the same phase and the phases are opposite to each other. 15. The fluorescence analysis method according to claim 14, wherein said control means is means for changing a resistance value of an electric resistance element by an electric means. The fluorescence analysis method according to any one of claims 1 to 15, wherein dispersion compensation of a light source of excitation light used for exciting the sample is performed by using a multilayer film manufactured using an oblique deposition technique. A fluorescence analysis method, wherein an element is used. By irradiating the biomolecule sample with excitation light, the components of the biomolecule sample are determined by detecting the intensity or spectrum of the fluorescence emitted from the biomolecule sample or a fluorescent substance added as a label to the biomolecule sample. In the fluorescence analyzer that can be used, the apparatus uses the phosphor as the phosphor, taking the excitation light absorption intensity of the phosphor on the vertical axis, which is defined as the absorption spectrum for the excitation light, and taking the excitation light absorption wavelength of the phosphor on the horizontal axis. Or at least two types of phosphors having different excitation light absorption intensity-wavelength curves represented in a graph or having condition setting means corresponding to the phosphors are provided. When the absorption spectrum is not at the peak position of each absorption spectrum, the slope of the absorption spectrum of one phosphor is positive and that of the other A phosphor crossing such that the slope of the absorption spectrum is negative, wherein the excitation light has a wavelength such that the wavelength periodically changes over at least a part of the time during irradiation of the sample. A fluorescence analyzer, wherein the excitation light is modulated. 18. The fluorescence analyzer according to claim 17, wherein the fluorescence analyzer, after wavelength-separating the fluorescence from the fluorescent label, converts each wavelength-separated fluorescence into an electric signal corresponding to each fluorescence by a photodetector. And a fluorescence analyzer for guiding each of the electric signals to a differential amplifier to remove in-phase noise. 19. The fluorescence analyzer according to claim 17, wherein, when λ1 and λ2 are positive numbers, the excitation light has both wavelengths λ3 and λ4 that give respective peak values of absorption spectra of the two kinds of phosphors. The pump light is wavelength-modulated pumping light that changes between λ0 + λ1 and λ0−λ2 with a specific wavelength λ0 therebetween. A wavelength longer than the longer wavelength and shorter than the longer one of the wavelengths λ3 and λ4. 20. The fluorescence analyzer according to any one of claims 17 to 19, wherein the excitation light is between λ0 + λ1 and λ0-λ2 across a specific wavelength λ0 during the entire time period during which the excitation light is irradiated on the sample. A fluorescence analyzer characterized by undergoing changing wavelength modulation. 21. The fluorescence analyzer according to claim 17, wherein a change in the wavelength of the excitation light is a sine wave having a specific wavelength λ0 as a center wavelength. 22. The fluorescence analyzer according to any one of claims 17 to 21, wherein the device is a device that measures fluorescence emission due to a multiphoton absorption effect in a biomolecule sample. 23. The fluorescence analyzer according to any one of claims 17 to 22, wherein the sample is a sample containing a biomolecule to which an organic dye or a fluorescent protein having a multiphoton absorption effect is added as a label. Fluorescence analyzer. 24. The fluorescence analyzer according to claim 23, wherein the apparatus can use a sample in which a plurality of organic dyes or fluorescent proteins having a two-photon absorption effect are added as labels to the sample, and a plurality of components can be analyzed. A fluorescence analyzer, which is an apparatus. The fluorescence analyzer according to any one of claims 22 to 24, wherein the multiphoton absorption is two-photon absorption. 26. The fluorescence analyzer according to any one of claims 17 to 25, wherein the device synchronizes a fluorescence signal from a fluorescent dye or a fluorescent protein with the frequency of the wavelength modulation of the excitation light in detecting the fluorescence. An apparatus for fluorescence detection, wherein the apparatus utilizes detection. The fluorescence analyzer according to any one of claims 17 to 26, wherein an optical device having a function of separating a fluorescence wavelength configured by a spatial beam optical system is used for detecting fluorescence from the sample. Analysis equipment. 27. The fluorescence analyzer according to any one of claims 17 to 26, wherein the fluorescence from the sample is caused to travel in an optical fiber in a direction opposite to the excitation light for detecting the fluorescence from the sample. A fluorescence analyzer comprising an optical device having a function of separating a fluorescence wavelength, which is constituted by an optical fiber coupler and an optical fiber filter module. 29. The fluorescence analyzer according to claim 28, wherein the optical device has a disk-shaped lens array that can scan the entire plane of the sample. 30. The fluorescence analyzer according to any one of claims 17 to 29, wherein the signal based on the two types of fluorescence input to the differential amplifier has an amplitude at any stage before being subjected to differential amplification. The fluorescence analyzer is controlled by the control means so that the signals have the same phase and the phases are opposite to each other. 31. The fluorescence analyzer according to claim 30, wherein said control means is means for changing a resistance value of an electric resistance element by an electric means. 32. The fluorescence analyzer according to any one of claims 17 to 31, wherein dispersion compensation of a light source of excitation light used to excite the sample is performed using a multilayer film manufactured using an oblique deposition technique. A fluorescence analyzer using an element. A sample such as a biomolecule or a biomolecule to which a fluorescent substance or the like is added as a label is irradiated with excitation light, and emitted from the biomolecule or the like or a fluorescent substance or the like added as a label to the biomolecule or the like by the excitation light irradiation. In an analyzer that can determine components such as biomolecules by detecting emission intensity or spectrum, the excitation light has a wavelength in at least a part of a time period during which the sample is irradiated. , Λ1 and λ2 are positive numbers, and the excitation light is wavelength-modulated excitation light that periodically changes between λ0 + λ1 and λ0−λ2 across the specific wavelength λ0. 28. The fluorescence analyzer according to claim 27, wherein the excitation light is subjected to wavelength modulation that changes between λ0 + λ1 and λ0-λ2 across a specific wavelength λ0 during the entire time period during which the sample is irradiated. A fluorescence analyzer. 29. The fluorescence analyzer according to claim 27, wherein the change in the wavelength of the excitation light is a sine wave having a specific wavelength λ0 as a center wavelength.

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