Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/636—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6445—Measuring fluorescence polarisation
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
• • G—PHYSICS
  • G21—NUCLEAR PHYSICS; NUCLEAR ENGINEERING
  • G21K—TECHNIQUES FOR HANDLING PARTICLES OR IONISING RADIATION NOT OTHERWISE PROVIDED FOR; IRRADIATION DEVICES; GAMMA RAY OR X-RAY MICROSCOPES
  • G21K7/00—Gamma- or X-ray microscopes

Definitions

• the invention of this application relates to a microscope system. More specifically, the invention of this application provides a high-performance, high-performance, new microscope that can obtain a high-quality image with high spatial resolution by illuminating a stained sample with light of a plurality of wavelengths. This is related to the microscopic system.
• This optical microscope can select a specific molecule using double resonance absorption, and observe the absorption and fluorescence resulting from a specific optical transition.
• the electrons in the valence orbital 2 of the molecule in the base state illustrated in FIG. 1 are excited to the valence orbital 3 which is a free orbit by the light source as illustrated in FIG. This state is the first excited state.
• the electrons in the valence orbit 1 are excited into the vacancies generated in the valence orbit 2. This state is the second excited state.
• the molecule returns to the ground state from the second excited state by emitting fluorescence or phosphorescence as illustrated in FIG. Then, an absorption image and an emission image are observed using the absorption process in FIG. 2 and the emission of fluorescence and phosphorescence in FIG.
• the linear absorption coefficient is the absorption per molecule Since it is given by the product of the cross-sectional area and the number of molecules per unit volume, in the excitation process as shown in Fig. 3, the linear absorption coefficient for the subsequently irradiated resonance wavelength ⁇ 2 is the intensity of the ⁇ 1 light initially irradiated. Depends on.
• the linear absorption coefficient for the wavelength ⁇ 2 can be controlled by the intensity of the light of the wavelength ⁇ 1. This means that if the sample is illuminated with light of two wavelengths, wavelength 1 and wavelength I2, and a transmission image at wavelength 2 is taken, the contrast of the transmission image is completely reduced by the amount of light at wavelength ⁇ 1. Indicates that control is possible.
• the emission intensity is proportional to the number of molecules in the first excited state. Therefore, it is possible to control the image contrast even when used as a fluorescence microscope.
• this conventional optical microscope enables not only control of contrast but also chemical analysis. Since the outermost valence orbit in Fig. 1 has an energy level unique to each molecule, the wavelength ⁇ 1 differs for each molecule. At the same time, the wavelength ⁇ 2 is also unique to the molecule. Therefore, the molecules that absorb or emit light can be limited by the two wavelengths of the wavelength ⁇ 1 and the wavelength ⁇ 2, and the chemical composition of the sample can be accurately identified. Furthermore, when valence electrons are excited, only light having a specific electric field vector with respect to the molecular axis is strongly absorbed, so the polarization directions of wavelengths ⁇ 1 and ⁇ 2 are determined, and the absorption image or fluorescence is determined. If an image is taken, the orientation of the same molecule can be identified.
• FIG. 5 is a conceptual diagram illustrating a double resonance absorption process in a molecule, in which a molecule in a ground state is excited to a first excited state by light having a wavelength of ⁇ 1, and is further excited to a second excited state by light having a wavelength of ⁇ 2. Which indicates that the fluorescence from this second excited state is extremely weak for some molecules.
• Figure 6 illustrates the spread of the spatial distance in the double resonance absorption process with the X axis on the horizontal axis.
• a spatial region A 1 irradiated with light of wavelength ⁇ 2 and a spatial region A 0 not irradiated with light of wavelength ⁇ 2 are shown.
• fluorescence emitted at the wavelength ⁇ 3 is seen from the spatial region AO.
• the spatial region A 1 most of the molecules in the first excited state are immediately excited to the second higher excited state due to irradiation with light of wavelength ⁇ 2, and the molecules in the first excited state do not exist.
• the two types of wavelengths of ⁇ ⁇ and ⁇ 2 are spatially well overlapped, so that, for example, the irradiation region of ⁇ 1 Focusing on this, the fluorescent region is smaller than the size determined by the numerical aperture and wavelength of the condenser lens, and the spatial resolution is substantially improved. Therefore, if this principle is used, a fluorescence microscope exceeding the diffraction limit will be possible. This is a super-resolution microscope using a double resonance absorption process.
• optical properties of such molecules can be explained as follows from a quantum chemical standpoint.
• a molecule is composed of ⁇ or; You.
• molecular orbitals have ⁇ or ⁇ molecular orbitals, and electrons existing in these molecular orbitals play an important role in bonding each atom.
• the electron of ⁇ molecular orbit strongly bonds each atom and determines the distance between atoms in the molecule which is the skeleton of the molecule.
• electrons in ⁇ molecular orbitals hardly contribute to the bonding of each atom, but are bound to the whole molecule with extremely weak force.
• the molecular structure itself hardly changes, stays at a high quantum discrete level for a long time, emits fluorescence in the order of nanoseconds, and excites. Has nature.
• molecules According to quantum chemistry, molecules have 7 ⁇ molecular orbitals and are equivalent to having double bonds, and it is necessary to select a molecule with a large amount of double bonds for one fluorescent labeler molecule to be used. It is a necessary condition. Also, it has been confirmed that the fluorescence from the second excited state is extremely weak in molecules having a double bond, such as benzene-pyrazine, in a six-membered ring molecule (for example, M. Fujii et. A. Chem. Phys. And ett. 171 (1990) 341).
• the fluorescent labeler molecule if a molecule containing a 6-membered ring, such as benzene-pyrazine, is selected as the fluorescent labeler molecule, the fluorescence lifetime from the first excited state is long, and the light is excited to change from the first excited state to the second excited state. Excitation can easily suppress fluorescence from molecules, so that the super-resolution of the microscope can be used effectively.
• the double resonance absorption process is such that two light wavelengths and polarization states satisfy certain conditions. This can only be used to determine very detailed molecular structures. In other words, there is a strong correlation between the plane of polarization of light and the orientation direction of molecules, and when the polarization plane of each of two wavelengths of light and the orientation direction of molecules form a specific angle, the double resonance absorption process occurs. It happens strongly. Therefore, by simultaneously irradiating the sample surface with light of two wavelengths and rotating the respective polarization planes, the degree of the disappearance of the fluorescence changes, and the state of the tissue to be observed from that state Information on spatial orientation can also be obtained. This can also be achieved by adjusting the two wavelengths of light. As described above, it can be seen that the conventional optical microscope using the double resonance absorption process has high analytical capability as well as super-resolution.
• FIG. 7 illustrates the timing of irradiating a sample with two types of light having wavelengths ⁇ 1 and ⁇ 2.
• a pulse light shorter than the time r during which one fluorescent labeler molecule emits fluorescence, that is, the lifetime r of the first excited state is first irradiated with light having a wavelength ⁇ 1 for a time t, and then the wavelength ⁇ 2 light is emitted.
• a pulse light of wavelength ⁇ 1 which is sufficiently shorter than the lifetime of the first excited state of the fluorescent labeler molecule, is irradiated for a time t to generate a molecule in the first excited state in the familiar region. .
• a region not required for observation is irradiated with pulsed light having a wavelength ⁇ 2, which is also sufficiently shorter than the lifetime of the first excited state, to excite molecules in the first excited state to the second excited state, Suppress fluorescence.
• the excitation process can be described by the following rate equation. That is, the number of molecules per unit volume of the molecules stained on the sample is ⁇ . Let the photon flux of light of wavelength ⁇ 1 be I. Let N be the number of molecules in the ground state after a lapse of time t after irradiation with light of wavelength ⁇ 1. Then, assuming that the lifetime of the first excited state is r and the absorption cross section when the light of wavelength ⁇ 1 transitions from the ground state to the first excited state is ⁇ 0 , the rate equation is as follows: Become. : : NI ⁇ ( N Q- N
• Equation 3 when the irradiation time of light of wavelength ⁇ 1 is shorter than the life of the molecule in the first excited state, and when the photon flux of light of wavelength ⁇ 1 is small, ⁇ is the irradiation time. It is almost proportional to t.
• n (l 0 ⁇ 0I N 0 t) -e T
• Equation 6 shows the number of molecules in the first excited state per unit volume in the region where fluorescence is suppressed
• Equation 7 shows the number of molecules in the first excited state per unit volume in the region where fluorescence is not suppressed. Indicates a number.
• the fluorescence intensity F 1 from the region where the fluorescence is suppressed and the fluorescence intensity F 2 from the region where the fluorescence is not suppressed are given by the following Expressions 8 and 9, respectively. .
• FIG. 8 illustrates a timing for measuring the intensity of the fluorescent light emitted from the parental region.
• the timing of measuring the fluorescence intensity is to sufficiently measure the intensity of the fluorescence emitted from the parental region after the irradiation of the light of the wavelength ⁇ 2 is completed. With such measurement timing, fluorescence from the parental observation area can be measured very well with little fluorescence from the suppressed area.
• FIGS. 9 and 10 show examples of the timing of irradiating the sample with two types of light having the wavelengths ⁇ 1 and ⁇ 2 and the timing of measuring the fluorescence intensity from the observation region, respectively.
• the super-resolution microscope can also be effectively realized by the timings illustrated in FIGS. 9 and 10.
• t and ⁇ ⁇ ⁇ need to be shorter (t, T ⁇ ) in any of the cases shown in Figs. 8 to 10.
• t, T ⁇ the time at which the molecules in the first excited state are de-excited to the ground state during irradiation with the two types of light of ⁇ 2, and the fluorescence itself from the observation region is lost.
• t, ⁇ ⁇ as shown in Fig. 11, two types of light, ⁇ 1 and ⁇ 2, are radiated at the same time, and the fluorescence emitted from the parental area simultaneously.
• two types of strong excitation light of ⁇ 1 and ⁇ 2 may be mixed into the detector during the fluorescence measurement.
• the fluorescence lifetime is not more than 1 ns sec, the fluorescence phenomenon from the parental region ends before the detector and the measurement circuit become active, and measurement becomes impossible.
• one fluorescent labeler molecule that stains the sample needs to have a fluorescence lifetime of 1 ns sec or more.
• the fluorescence intensity from the time when the number of molecules in the first excited state immediately after being excited by the light of ⁇ 1 is sufficient is measured. According to Equation 9 above, the number of excited molecules decays exponentially with a time constant determined by the excitation lifetime.
• the pulse widths t and ⁇ of the light are sufficiently shorter than the molecular lifetime of the first excited state, the first excited state immediately after being excited by the light of ⁇ 1 Fluorescence of a sufficiently high intensity, that is, the effective signal intensity, can be measured from the molecule of the state.
• t and T are the molecular lifetimes of the first excited state! : About 1/10, the number of molecules in the first excitation state is 90% of the number of molecules immediately after being excited by the light of ⁇ 1, and sufficient signal intensity from the effective fluorescent region is obtained. can get.
• a wavelength ⁇ 2 light (hereinafter, this light) having a sufficient intensity to excite from the first excited state to the second excited state and suppress the fluorescence from the first excited state Is called an erase light, and the light of wavelength ⁇ 1 that excites from the ground state to the first excited state is called a pump light).
• I high intensity laser light of several TWZ cm 2 in fluorescence microscopy of laser scanning using a resonant two-photon absorption process re also a slightly small strength, because it is very strong, the effect of the biological sample It was a problem.
• Such a high-intensity laser is light that is too intense for the living cells of the sample, and particularly when long-term measurement is required, the effects of heat storage and multiphoton absorption of the sample are remarkable. The impact must be reduced as much as possible.
• the wavelengths of the pump light and the erase light are out of the absorption wavelength band of living cells.
• the beam of the erase light condensed on the sample surface must be a beam having an intensity distribution with zero at the center and an axisymmetric shape. (Such a beam is hereinafter referred to as a hollow beam). This is because disturbances in the intensity distribution directly lead to degradation of the resolution of the microscope.
• a laser is used as the light source of the erasing light.
• a major premise is that the beam profile of the laser is not good. Not be. That is, it is desirable that the intensity distribution of the beam is symmetric with respect to the optical axis.
• dye lasers conventionally used as light sources have beam shapes It is close to a triangle and its intensity distribution is not uniform. Therefore, the beam shape converged on the sample surface is not the expected hollow beam, but a broken beam pattern, and the resolution and image quality of the microscope image are degraded.
• using such an annular aperture makes it possible to align the optical axis, focus, etc.
• the adjustment time required to obtain a good image is extremely long, and a skill for that purpose is also required.
• the light source to be used has a fixed wavelength, has a simple configuration, and is inexpensive.
• This micromanipulator technology for example, focuses high-intensity laser light on dielectric particles of polyethylene particles, causing polarization and attracting the particles to the region where the electric field is strongest. It captures and moves.
• this technology it is known that it is good to stably capture a limited number of specific particles and to apply one laser beam to the particles from various directions as much as possible.
• irradiating a laser beam from various directions requires a large number of laser light sources and a complicated mirror optical system, so even if it is possible to capture in one space, the moving operation is extremely difficult. It was difficult.
• the invention of this application has been made in view of the above circumstances, and uses an easy and compact optical system to generate erase light for exciting molecules in the first electron excited state to the second electronic excited state.
• the objective is to provide a new forehead microscopy system that can focus light with an excellent beam profile, has high stability and operability, and has excellent super-resolution. .
• the invention of this application is directed to a microscope system including an adjusted sample and a microscope main body, which solves the above-mentioned problem.
• the adjusted sample includes at least three electron states including a ground state.
• the microscope body is composed of a light source of wavelength ⁇ 1, which excites the molecule from the ground state to the first electronically excited state, and a molecule that is in the first electronically excited state.
• a wavelength that excites a two-electron excited state or a higher electronic excited state ; a light source of 12 light; and a condensing optical system that condenses a wavelength ⁇ 1 light and a wavelength ⁇ 2 light on a conditioned sample.
• Claims 1 comprising a conditioned sample and a microscope body, wherein the conditioned sample is stained with molecules having at least three electronic states including a ground state.
• the microscope body has a light source of wavelength ⁇ 1 that excites the molecules from the ground state to the first electronic excited state, and excites the molecules in the first electronic excited state to the second electronic excited state or a higher electronic excited state
• a light source of wavelength ⁇ 2 light a condensing optical system for condensing the wavelength ⁇ 1 light and the wavelength ⁇ 2 light on the adjusted sample, an irradiation area of the wavelength ⁇ 1 light on the adjusted sample and the wavelength ⁇ 2 Light irradiation area
• a microscope system (Claim 2) characterized by having a phase distribution that is out of phase by ⁇ at a symmetric position with respect to the optical axis in a plane to be adjusted, and an adjusted sample and a microscope main body.
• the conditioned sample has at least three electronic states including the ground state, and the excitation wavelength band from the first electron excited state to the second electronic excited state is From the electronically excited state to the ground state vibration level It is stained with molecules that overlap the fluorescence wavelength band when de-excited by the process.
• the main body of the microscope is a light source of wavelength ⁇ 1 that excites the molecules from the ground state to the first electron excited state.
• a wavelength ⁇ 1 light and a wavelength ⁇ 2 light through the superimposing means to emit light when the molecule is de-excited from the first electronic excited state to the ground state. It suppresses the area and has two wavelengths.
• the beam obtained by condensing the beam has a phase distribution shifted by ⁇ ⁇ at a symmetric position with respect to the optical axis in a plane orthogonal to the optical axis.
• the excitation wavelength band from the first electronic excited state to the second electronic excited state is different from the excitation wavelength band from the ground state to the first electronic excited state ( Claim 4)
• the molecule is a molecule containing one or more six-membered rings (Claim 5), and the six-membered ring is a benzene ring or a purine base (Claim 6).
• the six-membered ring derivative is a benzene derivative or a purine derivative (Claim 8)
• the molecule is a xanthine-based derivative.
• the molecule is 2,2 ''-Dimethyl-p-terpheny 1: P-terpheny I (PTP): 3.3 ', 2,3-Tetramethylp-quaterphenyl: 2,2-Dimethyl-P-quaterphenyl: 2-M ethy I— 5-1 buty I -p-quaterpheny I: 2-(4-B ⁇ heny 1 y I) -1 5— (4-t-out y I pheny I) -1, 3, 4-ox i azol (BPBD-365): 2- (4-B i pheny I y I) -pheny I -1, 3, 4-oxadiazol: 2, 5.2 '' ', 5 '' -Tetramethy l
• the obtained beam has a phase distribution that continuously changes from 0 to 27 ° when it makes one rotation around the optical axis in a plane orthogonal to the optical axis (claim range 1 2 ), A phase distribution in which a beam obtained by condensing light with a wavelength of ⁇ 2 changes discontinuously from 0 to 2 ⁇ when rotated once around the optical axis in a plane perpendicular to the optical axis.
• the beam obtained by condensing the light of wavelength ⁇ 2 is a Bessel beam (claim 14), and the Bessel beam is a primary Bessel beam (claim 1).
• the wavelength ⁇ 2 The beam obtained by condensing the light is a laser beam having any one of the Gaussian, Laguerre, or Hermite oscillation modes (claim 16).
• a gas laser, solid-state laser, or semiconductor laser must be provided as the light source for ⁇ 1 light (claim 1), and an oscillation wavelength of one of the gas laser, solid-state laser, or semiconductor laser Is a wavelength ⁇ 1 (claim “! 8”), and a harmonic of an oscillation wavelength of a gas laser, a solid-state laser, or a semiconductor laser is a wavelength ⁇ 1 (claims).
• the sum frequency or difference frequency of the oscillation wavelength of any one of a gas laser, a solid-state laser, and a semiconductor laser and its harmonic is a wavelength ⁇ ((claim 20), and a wavelength ⁇ 2 light
• a light source one of a gas laser, a solid laser, and a semiconductor laser must be provided (claim 21), and the oscillation wavelength of the gas laser, the solid laser, or the semiconductor laser must be Wavelength ⁇ 2 (claim 22), and the harmonic or difference frequency of any of the oscillation wavelengths of a gas laser, a solid-state laser, and a semiconductor laser is wavelength ⁇ 2 (claim 23).
• the gas laser is a mode-locked type (claim 26), and the solid-state laser is an Nd: YAG laser.
• the solid-state laser is a semiconductor laser excitation type (Claim 28)
• the solid-state laser is a mode-locked type (claim 29); and the microscope body is a nonlinear medium or a wavelength modulation element for performing wavelength conversion of laser light from a gas laser, a solid-state laser or a semiconductor laser.
• the nonlinear medium or the wavelength modulation element is a nonlinear crystal (claim 31), the nonlinear medium or the wavelength modulation element is a Raman shifter.
• the wavelength ⁇ t is that the fundamental wave of a gas laser or a fixed laser is wavelength-modulated by a nonlinear medium or a wavelength modulation element (claim 3 3), and the wavelength is The ⁇ 1 light is obtained by wavelength-modulating a harmonic of a gas laser or a fixed laser with a non-linear medium or a wavelength modulation element (claim 34), and the wavelength ⁇ 2 light is a gas laser or a solid-state laser.
• the fundamental wave is wavelength-modulated by a non-linear medium or a wavelength modulating element (claim 35), and the wavelength ⁇ 2 light is obtained by converting a harmonic of a gas laser or a solid laser into a non-linear medium or
• the wavelength is modulated by a wavelength modulation element (claim 36), and the condensing optical system of the two wavelengths is orthogonal to the beam obtained by condensing the wavelength ⁇ 2 with respect to the optical axis.
• Phase distribution in the plane A phase plate having a refractive index distribution or an optical path difference distribution (claim 37), and the condensing optical system for the wavelength ⁇ 2 light comprising an annular optical system (claim).
• the condensing optical system for wavelength ⁇ 2 light includes a diffractive optical system (claim 39), and the condensing optical system for wavelength ⁇ 2 light includes an axicon.
• a ring-shaped annular mirror, annular diffraction grating, Fresnel zone plate, annular aperture, or orthogonal to the optical axis At least one phase plate that provides a phase difference of ⁇ from the electric field axially symmetric with respect to the in-plane electric field is provided (Claim 41).
• Emission light condensing optical system that condenses light emitted from And it has (range 4 2 claims), the emission light converging optical system is provided with a sharp force Tsu preparative filter (claims 43)
• the light-emitting and condensing optical system has a notch filter.
• the light-emitting and condensing optical system has a band-pass filter.
• the band-pass filter does not transmit the wavelength ⁇ 1 light and the wavelength ⁇ 2 light but transmits the light emitted from the molecule (claim 46).
• the sealed sample is sealed by a sealing means made of a substance that transmits ⁇ 1 light and wavelength ⁇ 2 light (claim 47), and the adjusted sample transmits wavelength ⁇ ⁇ light and wavelength ⁇ 2 light.
• the material is synthetic quartz S i 0 2, C a F 2, N a F, N a 3 AIF s, L i F, M g F, S i O 2, L a F 3, n d F 3, AI 2 0 3, C e F 3, P b F 2, M g O, T b 0 2, S n O 2 , L a 2 ⁇ 3 , or S i O
• the main body of the microscope is provided with a continuous wave laser separately from the light source of the wavelength ⁇ 1 light and the wavelength ⁇ 2 light, and the continuous wave laser is adjusted on the sample.
• the beam obtained by converging the light into the microscope has a phase distribution in which the phase is shifted only at a symmetric position with respect to the optical axis in a plane orthogonal to the optical axis (claim 50). Is independent of the beam obtained by condensing the continuous wave laser on the conditioned sample and the beam obtained by condensing the wavelength ⁇ 1 light and the beam obtained by condensing the wavelength ⁇ 2 light.
• the provision of a means for relatively scanning the adjusted sample (claim 51) is also provided as an embodiment thereof.
• Figure 1 is a conceptual diagram illustrating the electronic structure of a molecule.
• FIG. 2 is a conceptual diagram illustrating excitation of the molecule of FIG. 1 to a first excited state by a wavelength ⁇ 1.
• FIG. 3 is a conceptual diagram illustrating the excitation of the molecule of FIG.
• FIG. 4 is a conceptual diagram illustrating a de-excitation process involving light emission from the second electronically excited state to the ground state in FIG.
• FIG. 5 is a conceptual diagram illustrating the principle of a long-resolution microscope for molecules having a small luminescence yield in the second excited state.
• FIG. 6 is a conceptual diagram illustrating a double resonance absorption process.
• FIG. 7 is a diagram showing an example of the irradiation timing of the wavelength-incident 1 light and the wavelength ⁇ 2 light and the number of molecules in the first excited state.
• FIG. 8 is a diagram showing an example of irradiation timing and measurement timing of the wavelength-input 1 light and the wavelength ⁇ 2 light.
• FIG. 9 is a diagram showing another example of irradiation timing and measurement timing of the wavelength ⁇ 1 light and the wavelength ⁇ 2 light.
• FIG. 10 is a diagram showing another example of irradiation timing and measurement timing of the wavelength ⁇ 1 light and the wavelength ⁇ 2 light.
• FIG. 11 is a diagram showing another example of the irradiation timing and the measurement timing of the wavelength ⁇ 1 light and the wavelength ⁇ 2 light.
• FIG. 12 is a diagram illustrating a de-excitation process of a molecule from a highly excited state.
• FIG. 3 is a diagram illustrating an energy diagram of a molecule having an overlap in FIG.
• FIG. 14 is a diagram exemplifying the relationship between each cross-sectional area and the wavelength as the molecular structural formula and optical properties of rhodamine 6G.
• FIG. 15 is a diagram illustrating a coordinate system for expressing a Bessel beam.
• FIG. 16 is a diagram illustrating the phase distribution of the beam plane of the Bessel beam.
• FIG. 17 is a diagram illustrating a two-dimensional intensity distribution of the primary Bessel beam.
• FIG. 18 is a diagram showing an example of a phase distribution given to the condensed beam of the erase light by the phase plate.
• FIG. 19 is a diagram showing an example of the numerical aperture of the condensing optical system.
• FIG. 20 is a diagram showing an example of the intensity of the condensed light of the erase light and the intensity of the fluorescent light in the microscope main body of the microscope system according to the embodiment 1 of the present invention.
• FIG. 21 is a conceptual diagram illustrating a phase plate having a refractive index distribution that changes discontinuously around the optical axis.
• I22 is a conceptual diagram exemplifying a phase distribution given to the erase light by the phase plate in a phase plate having a refractive index distribution that changes discontinuously by dividing the optical axis circumference into four parts.
• FIGS. 23A and 23B are a plan view and a side view, respectively, illustrating the structure and optical parameters of the phase plate.
• FIG. 24 is a diagram illustrating an example of the focused light intensity of the erase light and the fluorescence intensity in the microscope main body of the microscope system according to the second embodiment of the present invention.
• FIG. 25 is a diagram showing an example of the focused light intensity of the erase light and the fluorescence intensity in the microscope main body of the microscope system according to the third embodiment of the present invention.
• FIG. 26 is a main part configuration diagram showing an example of the microscope system of the present invention.
• FIG. 27 is a diagram showing an example of an annular optical system in which an annular annular slit is combined with a normal glass lens.
• FIG. 28 is a diagram illustrating a reflective objective lens as an example of an annular optical system.
• FIG. 29 is a diagram exemplifying a Wa Iter type lens which is an example of an annular optical system.
• FIG. 30, (a) and (b) are I, each illustrating a transmission type and reflection type Fresnel zone plate as an example of a diffractive optical system.
• FIG. 31 is a diagram illustrating a transmission diffraction grating having grooves on concentric circles.
• FIG. 32 is a diagram showing an example of an axicon optical system.
• FIG. 33 is a main part configuration diagram showing another example of the microscope system of the present invention.
• FIG. 34 is a main part configuration diagram showing another example of the microscope system of the present invention.
• FIG. 35 is a main part configuration diagram showing an example of a laser resonator that can directly oscillate a primary Bessel beam.
• FIG. 36 is a main part configuration diagram showing an example of the microscope system of the present invention when the Nd: YAG laser has the laser-resonator illustrated in FIG.
• FIG. 9 is a diagram illustrating an amplitude distribution and an intensity distribution of a third higher-order beam.
• FIG. 38 is a diagram illustrating the relationship between the wavelength characteristics of various filters and the detection light wavelength characteristics.
• 1139 is a main part configuration diagram showing an example of the microscope system of the present invention provided with a filter optical system.
• FIG. 40 is a main part configuration diagram showing an example of an electric system corresponding to the Ying microscope system of the present invention shown in FIG. The symbols in the figure indicate the following.
• 1 Nd YAG laser
• the conditioned sample has at least three electronic states including the ground state, and the excitation wavelength band from the first electronic excited state to the second electronic excited state is It is re-stained by molecules that overlap with the fluorescence wavelength band when de-excited from the one-electron excited state to the ground state vibration level by the fluorescence process.
• this molecule a so-called fluorescent labeler molecule, will be described in detail by considering the de-excitation process from its high electronic excitation state.
• Figure 12 conceptually illustrates the de-excitation process of a molecule.
• it is directly radiatively deactivated in SO by vibrational relaxation, that is, internal conversion, but in some cases, it reaches T 1, which has a very long life with different spin multiplicity, that is, crossover between systems. Then, phosphorescence is emitted from T 1 and returns to SO.
• the fluorescence emission yield from the excited state higher than S 2 is extremely low. This is because many of the molecules of S 2 directly radiatively deactivate S 0, or are internally converted to S 1, and then radiatively deactivate S 0 at a considerable rate. Also, since the molecules that have reached T 1 do not contribute to the fluorescence process, only some of the molecules that have reached S 1 emit fluorescence. This is called the Krash law. In particular, fluorescence from S2 is hardly measured in gas-phase benzene derivative molecules.
• a super-resolution microscope using a double resonance absorption process utilizes the extremely low fluorescence yield of molecules from a high-order electronically excited state of S 2 or higher as described above.
• some of the molecules have a wavelength band when transitioning from S 1 to S n (n is 2 or more), and the vibration level from S 1 to S 0. It has a structure that overlaps the fluorescence emission wavelength band at the time of deexcitation by the fluorescence process.
• a phenomenon occurs in which the fluorescence suppression effect by the erase light is substantially enhanced.
• molecules whose wavelength band at the transition from S 1 to Sn overlaps with the fluorescence emission wavelength band from S 1 are defined as molecules that stain the sample in the microscope system of the present invention.
• the fluorescence suppression effect contributed by the stimulated emission described above is added, so that the super-resolution of the main microscope can be further improved.
• the intensity of the erase light is low, the fluorescence can be easily suppressed, and the damage to the parent sample can be reduced.
• molecules having the above-mentioned optical properties include, for example, xanthine-based molecules.
• Rhodamine-based molecule there is a Rhodamine-based molecule to which it belongs.
• Figure 14 illustrates the molecular structure of Rhodamine6G, one of the rhodamine-based molecules, and the relationship between each cross-sectional area and wavelength as its optical characteristics (E. Sahar & D. Treves: IEEE J. Quantum Electron., QE-13, 962 (1977)).
• ⁇ 3 is the absorption cross section from S 0 to S 1
• ⁇ 3 ′ is the absorption cross section from S 1 to Sn
• ⁇ ⁇ represents the absorption cross section from ⁇ 1 to ⁇ ⁇ .
• the resonance wavelength from S 0 to S 1 extends before and after about 530 ⁇ m (see ⁇ ), but the resonance wavelength from S 1 to S ⁇ is about It spreads around 500-600 nm (see ⁇ , *). Then, the fluorescence emission band extends to a region of about 530 to 650 nm overlapping with the resonance wavelength region from S 1 to Sn (see ⁇ ,). Furthermore, it can be seen that there is a special wavelength band at about 530 to 600 nm. In other words, light in this wavelength band cannot excite a molecule from S 0 to S 1, but enables a double resonance absorption process from S 1 to S ⁇ and stimulated emission.
• Rhodamine6G and other rhodamine-based molecules have a wavelength at the transition from S1 to Sn, and a fluorescence wavelength band at the time of deexcitation by the fluorescence process from the vibration level of S1 to S0. Overlaps with
• the wavelength band of the coumarin-based molecule at the transition from S 1 to S ⁇ overlaps the fluorescence emission wavelength band from S 1.
• Comnarin 500 a coumarin-based molecule, has a resonance wavelength from S0 to S1 around 260 nm, a resonance wavelength from S1 to Sn around 3.555 nm, and a fluorescent emission region. It spreads in the region of 320 to 450 nm.
• molecules having such optical properties include six-membered rings such as benzene rings and nitrogen bases (that is, purine salt groups). Contains one or more 6-membered ring derivatives such as benzene derivatives and purine derivatives.
• 6-membered ring derivatives such as benzene derivatives and purine derivatives.
• P-terpheny I P-terpheny I (PTP): 3, 3 ', 2, 3''' -Tetramethy I -P-quaterpheny I: 2, 2 '-0 ⁇ me thy I- P-quater rpheny i: 2-Methy I -5-t-buty I -pq uater phenyl: 2-(4-B i pheny I y I)-5-(4-1 buty heny I)-1, 3 , 4-ox iazol (BPBD-36 5): 2- (4-Bi heny iyl) -phenyl-1, 3, 4-oxadiazol: 2, 5, 2 '', 5 '-Tet ramethy I -p-qu i nquepheny I: 3, 5, 3,, 5-Tet ra— t-buty I -p-qu i nquepheny I: 2, 5-D
• coumarin molecule is a 7-Ethylamino-4-tr if l uo rmethy l coumar in id 2 H ,. N0 2 F 3 : Goumar in 500)
• the excitation wavelength band from SO to S 1 is around 2666 nm
• the excitation wavelength band from S 1 to S 2 is around 532 nm
• the fluorescence wavelength band is It is 532 nm.
• These 266 nm and 532 nm are the fourth harmonic of the YAG laser, respectively.
• the second harmonic so that the fundamental wave and harmonics can be wavelength-modulated by a nonlinear medium that can convert the wavelength or a nonlinear crystal such as a BBO crystal or KTP crystal as a wavelength modulation element. By doing so, it can be easily generated.
• the sample staining molecule is a coumarin-based molecule, and excellent super-resolution is obtained by using a YAG laser as a pump light source and an erase light source in the microscope body. Not only can it be used, but it also has superior workability and optical performance compared to conventional microscopes using a dye laser.
• the wavelength to be used can be basically determined simply by initializing the angle of the nonlinear crystal, it is possible to easily generate a desired excitation wavelength without complicated wavelength adjustment work.
• there is no fluctuation or decrease in laser power due to deterioration of the dye and the oscillation efficiency is good.Therefore, there is no need to use a high-power laser, and the light source can be made small and inexpensive. At the same time, damage to the biological sample can be further reduced.
• a YAG laser for example, a commercially available mode-locked YAG laser having a pulse width of 20 psec or less and a repetition frequency of 100 MHz can be used.
• staining molecule is a xanthine-based or rhodamine-based molecule
• pump light and erase light can be generated in the same manner using a YAG laser and a nonlinear crystal.
• the resonance wavelength from S0 to S1 extends around 530 nm
• the resonance wavelength from S1 to Sn is 50,000 to 600 nm. It spreads before and after.
• Pump light around 530 nm can be handled by the second harmonic of ⁇ AG laser.
• Erase light of about 500 to 600 nm can be easily generated using the Raman effect of a nonlinear crystal (a so-called Raman shifter).
• Raman shifter a so-called Raman shifter
• the conversion efficiency is 20%.
• laser light having a sum frequency or a difference frequency between a fundamental wave and a harmonic of a YAG laser generated using a nonlinear crystal can be used as pump light or erase light.
• a fixed-wavelength gas laser, a solid-state laser, or a semiconductor laser various nonlinear media, or a wavelength modulation element can be used according to each of the above-described molecules.
• a gas laser of all, excimer one The primary, copper vapor laser, Al Gonre one
• an Nd : YAG laser, a Ti sapphire laser, a YLF laser, a ruby laser, or the like can be used.
• the mode-locked laser has a high repetition frequency and a pulse amplitude of several 10 psec or less.
• the super-resolution fine microscope body of the microscope system of the present invention is used. This is a more suitable light source.
• each gas laser, fixed laser, or semiconductor laser as described above as the light source of the wavelength ⁇ 1 pump light and the light source of the wavelength ⁇ 2 light its oscillation wavelength, its harmonics, or the sum of the oscillation wavelength and its harmonics
• the pump light and the erase light are generated by setting the frequency or the difference frequency to the wavelength ⁇ 1 and the wavelength ⁇ 2.
• the wavelengths of the fundamental wave and the harmonics of these various light sources can be modulated by various nonlinear media such as a nonlinear crystal or a wavelength modulation element to generate the wavelength ⁇ 1 pump light and the wavelength ⁇ 2 erase light.
• the beam obtained by focusing the erase light having the wavelength ⁇ 2 on the sample surface is used.
• the shape where the light intensity at the center becomes zero, and the center of the irradiation area It is necessary to leave a fluorescent region in the part.
• the beam obtained by condensing the erase light is shaped such that the light intensity at the center becomes zero, so that the beam is located at a target position with respect to the optical axis in a plane orthogonal to the optical axis.
• a Bessel beam is suitable as a beam having such a phase distribution.
• the Bessel beam can be represented by the following equation.
• E (x, y) is the electric field vector
• E 0 is the amplitude of the electric field vector.
• m 1
• the above equation becomes a first-order Bessel beam.
• This first-order Bessel beam has a singular point on the optical axis where the electric field intensity is zero. In fact, it can be obtained by solving the following wave equation of electromagnetic waves.
• Equation 1 2 Given an axisymmetric boundary condition for a certain axis, Equation 1 2 can be rewritten in the (r, ⁇ , ⁇ ) cylindrical seat ⁇ system as ⁇ 1 d E d 2 E
• k indicates the wave number
• the primary vessel The beam has a phase distribution that continuously changes from 0 to 27 ° when it makes one rotation around the optical axis in a plane perpendicular to its optical axis. Since it is shifted by 7 °, the electric fields completely cancel each other on the optical axis and become zero, indicating that there is a singular point where the electric field intensity is zero on the optical axis.
• the microscope system of the present invention uses a condensed beam of erase light as a Bessel beam, particularly a primary Bessel beam using a condensing optical system, for example.
• a Bessel beam particularly a primary Bessel beam using a condensing optical system, for example.
• This Bessel beam is a pseudo non-diffracted beam that does not seem to be diffused like the two-dimensional intensity distribution profile illustrated in FIG.
• a focused beam of erase light as an example of a Bessel beam having the above-mentioned optical characteristics that is, a phase distribution that has a boundary condition that is axially symmetric with respect to the optical axis and is shifted by 7 ° in the symmetric position with respect to the optical axis.
• the formation of a condensed beam having an optical element can be easily performed by a condensing optical system using existing optical elements.
• the condensing optical system is, for example, an annular optical system such as a reflective objective lens having a ring ⁇ , that is, an optical system having a ring aperture, or a Fresnel zone plate. It is preferable to provide a diffractive optical system such as the above or an axicon.
• the condensing optical system uses a refractive index distribution or an optical path that provides a phase difference distribution in a plane orthogonal to the optical axis.
• a phase plate having a difference distribution may be provided.
• a complete first-order Bessel beam like a complete first-order Bessel beam, it has a phase distribution that continuously changes from 0 to 27 ° when it makes one rotation around the optical axis in a plane perpendicular to the optical axis.
• the phase plate when the phase plate makes one rotation in a direction that rotates around the optical axis in a plane orthogonal to the optical axis of the beam, as shown in FIG.
• the optical path difference distribution is formed by continuously changing from 0 to 27 °.
• a condensed beam that has a phase distribution that changes discontinuously from 0 to 27 ° when it makes one rotation around the optical axis in a plane perpendicular to the optical axis. Then, it can be formed even if the refractive index distribution or the optical path difference distribution changes discontinuously from 0 to 2 ⁇ .
• the light source of the erase light may have a function of forming such a condensed beam.
• a transmission-type orbicular-type diffraction grating, a ring-shaped orbicular mirror, a Fresnel zone plate, a orbicular aperture, and an in-plane perpendicular to the optical axis are provided in the laser resonator as the erase light source. It is also possible to insert a phase plate that gives a phase difference that is shifted by ⁇ between the plates that are axially symmetric with respect to the electric field, and give the above-mentioned boundary conditions to directly convert the beam itself into a primary Bessel beam. It is possible.
• an axisymmetric mode pattern with zero intensity on the optical axis such as ⁇ ⁇ ⁇ 11 is formed.
• a laser beam of a Gaussian type, a Laguerre type, or a Hermitian type oscillation mode having a higher-order mode pattern can be formed. It may be a focused beam of erase light.
• the microscope body of the microscope system of the present invention can further have a micromanipulator function capable of capturing and moving sample particles with a hollow beam.
• the sample particles are attracted to the region with the highest laser intensity, and the stable point becomes the hollow portion at the focal point of the hollow beam, which is completely sealed and captured in the hollow beam. Become so.
• the sample to be captured is hardly irradiated with one laser beam, so that damage to the sample can be suppressed.
• the spatial movement can be realized by beam scanning using an optical system such as a galvanometer mirror, like a normal laser beam.
• appropriate laser intensity capture known to be about a few 1 OMWZ cm 2 Since this intensity is almost equal to the maximum intensity of the erase light used in the main body of the microscope in the microscope system of the present invention, the above-described light source of the erase light and the condensing optical system are used as they are. it can.
• the sample can be captured and moved without causing damage by light irradiation, and has a sophisticated micromanipulator function. Can be.
• the adjusted sample which has been stained by Rhod am in e 6G
• the microscope body the pump light (i.e., the hodamine6G from the ground state S 0 to a first excited states S 1 source and i race light having a wavelength lambda 1 of the light) to excite (i.e., the Rhoda m i ne 6G of the first electronic excited state S 1 as a light source of the second electronic excitation light of the wavelength lambda 2 that excites to state S 2)
• the pump light i.e., the hodamine6G from the ground state S 0 to a first excited states S 1 source and i race light having a wavelength lambda 1 of the light
• excite i.e., the Rhoda m i ne 6G of the first electronic excited state S 1 as a light source of the second electronic excitation light of the wavelength lambda 2 that excites to state S 2
• a Raman shifter is provided as a wavelength modulation element of the
• the beam obtained by condensing the erase light by the condensing optical system provided in the microscope main body is out of phase by ⁇ at a symmetric position with respect to the optical axis in a plane orthogonal to the optical axis, and It is assumed that it has a phase distribution that continuously changes from 0 to 27 ° when it makes one revolution.
• the condensing optical system is provided with a phase plate having a refractive index distribution or an optical path difference distribution as illustrated in FIG. 18 that can give such a phase distribution to the condensed beam of the erase light. ing.
• the height of the resolution of the microscope main body in the microscope system according to the present invention is represented by the general formula of the primary Bessel beam described above, which is expressed by Formula 11:
• p 0 is the light blocking ratio of the pupil of the condensing optical system
• ⁇ f is expressed by the following equation.
• ⁇ indicates the numerical aperture of the condensing optical system
• ⁇ indicates the wavelength of the erase light
• Equation 15 ⁇ . Takes a value between 0 and 1, and 0 indicates PSF (X, y) when the condensing optical system is not an annular optical system.
• Equation 17 becomes Equation 18 when the stimulated emission cross section is 6 f.
• fluorescence labeler is one molecule Rhodami n e6G, in the case where the condenser bi one beam of I race light has a phase distribution as described above, this number Using Fig. 18, it is possible to estimate the degree of spatial resolution at which the fluorescence emitted when Rhod amine 6G de-excites from the first excited state to the ground state can be detected.
• Table 2 exemplifies the environmental parameters used at this time. Table 2
• FIG. 20 illustrates the calculated erase light intensity I (X, y) and the fluorescence intensity F, (X, y). In addition, each intensity is normalized by each peak value.
• the erase light intensity is zero at the center, the fluorescence intensity is also high at the center, and the fluorescent region remains only at the center.
• Rayl igh-l itnit of the converging optical system is a 4 5 5 nm, you if fluorescence intensity F, defined as the spatial resolution of the half-width can detect Rhod a rn i n e6G of (X, y) It is 100 nm, which is above the diffraction limit of the focusing optics.
• the microscope body of the microscope system of the present invention in this embodiment has excellent super-resolution.
• an inexpensive, highly stable mode-locked laser can be used as the YAG laser as the light source of the pump light and the erase light.
• a part of the second harmonic 5 2 3 nm light is extracted by a beam splitter. This is converted to a non-linear crystal such as a BBO crystal with good safety, or a wave such as a Raman shifter. And wavelength-converted into 5 6 0 n m in length modulation element or nonlinear medium, and Iresu light.
• the configuration of the light source is extremely simple.
• the YAG laser is a mode-locked type and a semiconductor laser-excited type, it is possible to obtain a short repetition rate of 1 OOMHz and a short pulse of several 1 Opsec, and to use a light source. It can be small in size and free of solid maintenance.
• the intensity of the irradiation light does not need to be low, and the irradiation light wavelength is around 500 nm, which is not the light absorption band of the biological sample, so that the damage to the biological sample can be extremely reduced. .
• Rhodam i ne6G it was a;, 7-Ethy I am i ⁇ -4-tr if I uormethycoum arin (C, 2 H, o N0 2 F 3: Coumar i n500) , as described above, pump light and Iresu light, can respond in double harmonic and quadruple harmonic ⁇ AG laser, the intensity of Iresu light as 1 0 0 MWZ cm 2, the space of the same 1 0 about 0 nm and Rhodamine6G Resolution can be obtained.
• the oscillation wavelength of the AG laser can be adjusted using a nonlinear crystal such as an optical parametric oscillator (OPO).
• OPO optical parametric oscillator
• the condensed beam of the erase light is assumed to have a phase distribution that continuously changes from 0 to 27 T during one rotation around the optical axis.
• the phase distribution has a discontinuously changed phase distribution.
• a refractive index distribution ( ⁇ 1 ⁇ n 8) is used.
• the fluorescent labeler molecule is RhodamineSG, and its optical and environmental parameters are shown in Tables 1 and 2, respectively
• the focusing optical system of the microscope main body is as shown in Fig. 22.
• the optical axis is divided into four parts, and 0, ⁇ 2, ⁇ , 3 ⁇ 4, and a phase plate having a refractive index distribution that gives a discrete discontinuous phase distribution to the condensed beam of the erase light are provided.
• the intensity distribution of the condensed beam of the race light and the intensity distribution of the emitted fluorescent light were determined.
• FIGS. 23 (a) and 23 (b) are a plan view and a side view, respectively, illustrating the structure and optical parameters of the phase plate.
• the phase plate illustrated in FIGS. 23 (a) and 23 (b) is formed by coating a magnesium fluoride film on a glass substrate (BK-7). Since the refractive index of the magnesium fluoride film is 1 ⁇ 38 at a wavelength of 560 nm, a phase difference of ⁇ 4 is provided at a thickness of 350 nm. Therefore, the film thicknesses that can give a phase distribution of 0, ⁇ 2, ⁇ , and (3 ⁇ ) 2 in each area divided into four around the optical axis are illustrated in Fig. 23 (a), respectively. As described above, they are 350 nm, 500 nm, 1050 nm, and 0 nm.
• a glass substrate is directly etched to have an optical path difference that gives a phase in each region.
• a plate may be formed.
• the parallelism of the glass substrate itself is reduced in order to prevent disturbance of the phase plane of the erase light.
• roughness must be smaller than the optical path difference that gives a phase difference of ⁇ 4.
• the disturbance of the optical path difference due to the parallelism and roughness of the glass substrate itself is set to 350 nm or less.
• FIG. 24 exemplifies the intensity of the condensed beam of the erase light and the fluorescence intensity obtained by being condensed by the condensing optical system having the phase plate of FIG. 23 as described above.
• the condensed beam intensity of the erase light has almost the same shape as the condensed beam intensity of the erase light exemplified in 1U20 in the first embodiment. It can be seen that the intensity is zero. In other words, excellent super-resolution is realized even when the focused beam of the emission light has a phase distribution that changes discontinuously around the optical axis.
• a magnesium fluoride film so as to have a refractive index distribution or an optical path difference distribution that changes discontinuously around the optical axis is a continuous change. It is much simpler than doing this, and can reduce manufacturing costs.
• the beam obtained by condensing the erase light having a wavelength of ⁇ 2 be a primary Bessel beam that is a non-diffracting beam.
• the beam obtained by condensing the erase light having a wavelength of ⁇ 2 be a primary Bessel beam that is a non-diffracting beam.
• the beam phase-modulated by the phase plate as illustrated in FIG. 18 or FIG. 22 is converted into a normal condensing optical system, that is, a condensing beam that does not include the annular optical system as in the first embodiment.
• a normal condensing optical system that is, a condensing beam that does not include the annular optical system as in the first embodiment.
• the beam condensed by a normal condensing optical system having a phase plate has a shape in which the intensity at the center is zero, and the fluorescent region is located at the center. Remains.
• the Rayleigh h-I imit of the focusing optics is 455 nm, but if the half-width of the fluorescence intensity F, (X, y) is the spatial resolution at which Rhod amineSG can be detected, then It is 200 nm, exceeding the diffraction limit of the focusing optics. Therefore, it can be seen that excellent super-resolution was obtained.
• FIG. 26 is a main part configuration diagram showing an example of the microscope system of the present invention having a super-resolution microscopic function.
• Rhod am ind. e6G is used.
• the prepared sample (100) was stained with Rhodamine6G.
• a mode-locked Nd: YAG laser (1) is provided as a light source for the pump light having a wavelength of ⁇ 1 and the erase light having a wavelength of ⁇ 2, and a nonlinear crystal as a nonlinear medium for wavelength conversion.
• a BBO crystal (2) is provided, and the fundamental wave of the Nd: YAG laser (1) is wavelength-converted by the BBO crystal (2) to oscillate a second harmonic of 532 nm. Is the pump light.
• a half mirror (3) is provided on the optical path of the pump light, and a part of the second harmonic wave, that is, a part of the pump light is extracted by the half mirror (3), and the half mirror (3) is used as a nonlinear crystal.
• This erase light is illuminated via the mirror (5) to the phase plate (6) exemplified in the second embodiment, and the phase ⁇ (6) causes the electric field intensity at the center to become zero by this hollow beam. Molded into
• the erase light and the pump light formed as this hollow beam are made to pass through the same optical path by the dichroic mirror-(7), and then to the next dichroic mirror.
• the light is focused on the adjusted sample (100), which is set on the two-dimensional moving stage (10) that moves in the direction of the arrow in the figure via (8) and the focusing objective lens (9). Is done.
• the fluorescent light emitted from the sample (100) adjusted by the irradiation of the pump light and the erase light thus collected is reflected by the dichroic mirror (8), and the fluorescent light is collected by the fluorescent light condensing lens.
• the light is condensed on the light receiving surface of the formal (14) via the sharp cut filter (12) and the pinhole (13).
• the dichroic mirror (8) is an interference filter that transmits pump light and erase light and has a reflectance in the fluorescence band. Therefore, it is possible to discriminate the pump light and the erase light from the fluorescent light that is the signal light.
• the sharp cut filter (12) provided between the dichroic mirror (8) and the photomultiplier (14) has a pump that is mixed by surface scattering of the dichroic mirror (8) and the like. This is a bandpass filter that cuts light and erase light.
• the pinhole (13) also functions as a spatial filter that cuts off stray light that has also been spatially diffused. The sharp cut filter (12) and the pinhole (13) improve the fluorescence detection sensitivity and the SZN ratio.
• the fluorescence By monitoring the intensity of the sample, a two-dimensional fluorescence image of the adjusted sample (100) can be obtained.
• the intensity of the pump light and the erase light is monitored by a photomultiplier (15). For example, by applying signal processing, the fluctuation of the image signal for each pixel due to the intensity conversion of the laser light is suppressed. The image quality can be improved.
• an erase light converging beam having zero intensity at the center of the converging point can be formed.
• a primary Bessel beam which is a non-diffracted beam, can be formed.
• annular optical system for example, as shown in FIG. 2F, there is an optical system in which an annular annular slit (16) is combined with a normal glass lens (17).
• an annular orifice slit (16) for example, an etalon (not shown) is arranged in front of the orbicular slit (16), and the first-order diffracted light is used. It is desirable to increase the amount of light passing through.
• annular optical system that originally has an annular zone ⁇ , which itself has the boundary conditions necessary to form a primary Bessel beam.
• annular optical system originally having the annular zone ⁇ for example, there is a reflective objective lens as exemplified in FIG.
• This reflective objective is a Cassegrain or Schwarzschild type optical system, A reflective mirror with a convex curved surface arranged on the side shields the central part of the optical pupil, and has a role substantially similar to that of the annular ring slit (16) described above.
• a reflection type optical system there is also an oblique incidence type W alter type lens as illustrated in FIG. This W a Iter type lens is an extreme orbicular zone optical system, which is equivalent to using an almost ideal annular orbicular zone slit.
• Axisymmetric diffraction optical systems (including transmission and reflection types) also apply symmetrical boundary conditions around the optical axis to the above wave equation (12), so they should be applied as condensing optical systems. Can be.
• the diffractive optical system is, for example, a Fresnel zone plate.
• FIGS. 30 (a) and 30 (b) illustrate transmission type and reflection type Fresnel zone plates, respectively. Since the Fresnel zone plate originally has an imaging capability, it has both the focusing capability and the boundary conditions necessary for forming the primary Bessel beam.
• a diffraction grating in which a groove or a spiral groove is cut on a concentric circle may be provided as illustrated in FIG.
• the axicon is also an axially symmetric optical system, and can image a point light source on an axis over a wide range on the axis.
• Figure 32 shows an example of an axicon.
• the axicon illustrated in FIG. 32 is a reflection lens called a MacLeod having a conical surface and a flat surface.
• a MacLeod having a conical surface and a flat surface.
• the same point on the axis always exists with respect to the point on the axis within a certain range, and the image point exists at a point symmetrical to the point off the axis.
• Such an axicon also provides a symmetric boundary condition orbiting around the optical axis.
• the above optical system can be provided as a converging objective lens (9).
• the fluorescence detector may include a semiconductor detector such as a PIN photodiode or a CCD in addition to a photomultiplier tube such as a photomultiplier. ,.
• Rhodami ne6G is used as the fluorescent labeler molecule
• another light source is available.
• an OPO or a dye laser can be used instead of the Raman shifter (4).
• the erase light is variable in wavelength
• a rhodamine-based molecule such as RhodaminellO can be used as one labeler molecule.
• the two-dimensional moving stage (10) is moved with respect to the focused beam.
• the optical system is directly oscillated by a galvanometer mirror or the like, as in a conventional laser-scanning microscope, and the beam itself is moved. It is also possible to perform two-dimensional scanning on the sample (100) adjusted in step (1).
• FIG. 33 is a main part configuration diagram showing an example of the microscope system of the present invention having a confocal type super-resolution microscopic function.
• the prepared sample (100) is assumed to be stained with Coumarin500.
• a mode-locked Nd: YAG laser (1) is provided, and the fundamental wave (10) of this Nd: YAG laser (1) is provided. 6 4 nm) is split into two systems by a half mirror (2 8).
• One of the branched fundamental waves is converted to a wavelength of 355 nm by a third harmonic generator (18) made of a BBO-1 crystal, and is converted into pump light.
• the other fundamental wave is incident on a second harmonic generator (19) made of a BBO-2 crystal via a mirror (29), and the second harmonic generator (19) produces a wavelength of 3555 nm.
• the wavelength is converted to an erase light.
• the erase light is applied to the phase plate (31) as exemplified in the second embodiment via the mirror (30), and the central portion of the phase plate (31) has zero electric field intensity. Formed into a hollow beam.
• the erase light and the pump light, which have been made into a hollow beam, are used by the dichroic mirror (2).
• a polarizer (21) is disposed between the dichroic mirror (20) and the third harmonic generator (18).
• the polarization plane can be freely rotated.
• the image of the pinhole (23) illuminated by the collected pump light and erase light is used again as a light source for microbeam formation.
• the pump light and the erase light having passed through the pinhole (23) pass through the dichroic mirror (24) and are moved by the objective lens (25) which is a Sparschild type reflection optical system.
• the light is focused on the adjusted sample (100) placed on the (10).
• the light in the region can be imaged without chromatic aberration. Therefore, the pump light and the erase light having different wavelengths can be focused on the adjusted sample (100) with exactly the same imaging performance and high resolution.
• This Sparschild type reflection optical system is an orbicular zone optical system. By adjusting the radius of the convex mirror arranged inside, the light blocking ratio P is adjusted. By setting to a value between 0 and 1, the boundary conditions necessary for the formation of the first-order Bessel beam are given.
• the fluorescence emitted from the sample (100) adjusted by the irradiation of the collected pump light and erase light is reflected by the dichroic mirror (24) through the objective lens (25). .
• the scattered light and stray light of the pump light and the erase light are not reflected by the dichroic mirror (24), so that only the fluorescent light, which is the signal light, can be separated.
• the fluorescent light reflected by the dichroic mirror (24) passes through the pinhole (26), and the afterglow of the pump light and the emission light is cut by the sharp cut filter (27). And is focused on the light receiving surface of the formal (14).
• the optical system of the microscope main body in the microscope system of the present invention illustrated in FIG. 33 is a confocal optical system. That is, the pinholes (23) and (26) are optically at the confocal position centered on the focused point on the adjusted sample (100).
• the SZN ratio is excellent, and a three-dimensional fluorescence image of the adjusted sample (100) can be obtained by moving the two-dimensional moving stage (100) in the optical axis direction.
• polarizer (21) new useful functions are added to the polarizer (21) in addition to the super-resolution function.
• molecules absorb strongly in an electric vector in a specific direction.
• a benzene derivative or a purine derivative absorbs light having an electric vector in the same direction as the molecular plane.
• the polarizer by rotating the polarization direction of the pump light, only molecules that are spatially oriented in a specific direction can be excited to cause fluorescence. Therefore, by taking the fluorescence image while changing the polarization direction of the pump light by the polarizer (21), the spatial orientation characteristics of a specific molecule or tissue of the adjusted sample (100) can be analyzed. Become like Example 6
• FIG. 34 is a main part configuration diagram showing another example of the microscope system of the present invention.
• the microscope system illustrated in FIG. 34 has a micromanipulator function using a hollow microphone aperture beam in addition to the super-resolution microscope function. In addition, it has a normal fluorescence microscopy function, so that it is always possible to monitor the fluorescence image from the adjusted sample (100) in real time without performing one laser scan.
• the prepared sample (100) was stained with Rhodine6G.
• a mode-locked Nd: YAG laser (32) and a non-linear KTP crystal (3) are used as a pump light and erase light source for super-resolution microscopic function and as a nonlinear medium for wavelength conversion. 5) is provided.
• a continuous-wave CW Nd: YAG laser (33) and a KTP crystal (3) 6) is provided as a laser light source for forming a hollow microphone aperture beam for a micromanipulator function and a nonlinear medium for wavelength conversion.
• a continuous-wave CW Nd: YAG laser (33) and a KTP crystal (3) is provided as a mercury lamp (31) is provided as a light source for ordinary fluorescence microscopy.
• the fundamental wave of the Nd: YAG laser (32) is converted in wavelength by the KTP crystal (35), and the second harmonic of 532 nm is oscillated as pump light.
• This pump light Some of the is taken out by the wafer one half mirror one (3 off), via the mirror one (3 8), B a (N 0 3) is incident on a Raman shifter formed of the crystal (3 9), The wavelength is converted to 563 nm by the Raman shifter (39), and the light is converted into erase light.
• the erase light enters the dichroic mirror (40), and the dichroic mirror
• This emission light is further shaped into a hollow beam having a zero electric field strength at the center by the phase plate (6) as exemplified in the second embodiment.
• the erase light and the pump light formed in this hollow beam are identical to the erase light and the pump light formed in this hollow beam.
• a polarizer (2 1) is installed between the half mirror (3F) and the dichroic mirror (4 1), and the polarizer (2 "1) freely rotates the polarization plane of the pump light. You can do it.
• the pump light and the erase light whose optical paths are aligned coaxially by the dichroic mirror (41) are further shaped by the relay lens (42) and then reflected by the half mirror (43) to be reflected by the objective lens (43). 9) incident on the objective lens
• the light is focused on the adjusted sample (100) set on the two-dimensional moving stage (100).
• the fluorescence emitted from the sample (100) adjusted by the irradiation of the pump light and the erase light passes through the half mirrors (43) and (44), and passes through the half mirror (4).
• the light is reflected by 5) in the direction of incidence on the lens (46).
• the light After being focused on the center of the pinhole (47) by 6), the light is incident on the spectrum meter (49) via the lens (48).
• the pinhole (47) functions as a spatial filter, and serves to increase the SZN ratio of the measurement by cutting off the fluorescence emitted from, for example, an optical system other than the adjusted sample (100).
• a spectrometer (49) is provided as a fluorescence detector, so it is not only necessary to measure the fluorescence intensity but also to observe the fluorescence spectrum. And time response to laser irradiation can be measured. Analyze the chemical structure and composition of the sample (100). Furthermore, polarizer
• the spatial orientation information of the composition can be obtained by relatively changing the polarization planes of the pump light and the erase light.
• a Cd Nd: YAG laser (33) is provided as a continuous wave light source. I have.
• the fundamental wave of this N d ⁇ Y A G laser (33) is wavelength-converted by the KTP crystal (36), and a second harmonic of 532 nm is generated.
• the second harmonic is used as a light source for generating a hollow microbeam used in a micromanipulator.
• the second harmonic passed through the half mirror (38) was wavelength-converted to 563 nm by the Raman shifter (39), and further mixed by the dichroic mirror (40) to 532 nm.
• the second harmonic of this is excited, and only light of 563 nm is extracted with good purity.
• This 563 nm light is shaped by the phase plate (6) into a hollow microbeam with an electric field intensity of zero at the center, and passes through the dichroic mirror (41) and relay lens (42). The light is reflected by the half mirror (43) so as to be incident on the condenser objective lens (9). Then, the light is focused on the adjusted sample (100) by the focusing objective lens (9).
• the same optical system as the above-described erase light for the super-resolution function can be used for the 563 nm hollow microbeam for the micromanipulator.
• a mercury lamp (31) is provided as a light source for the fluorescent light function, and the light from the mercury lamp (31) is divided into a half mirror (44) and a half mirror (43).
• the sample is illuminated on the adjusted sample (100) via the focusing objective lens (9).
• the fluorescent image emitted by this light irradiation enters the CCD camera imaging lens (50) again through the converging objective lens (9), the half mirror (43) and the half mirror (44).
• the image is directly formed on the light receiving surface of the CCD camera (51) by the CCD camera imaging lens (50). This fluorescent image can be monitored directly on the CRT at any time.
• the beam obtained by condensing the erase light be a primary Bessel beam in order to make the microscope body excellent in super-resolution and operability.
• the next Bessel beam is formed using the phase plate, orbicular optics, diffractive optics, axicon, etc. provided in the condensing optical system, as described above, and a gas laser as the light source of erase light. 1. Within the cavity of a solid-state laser or a semiconductor laser—by providing the necessary boundary conditions to form the next Bessel beam, the erase light itself can be turned into a primary Bessel beam.
• FIG. 35 is a main part configuration diagram showing an example of a laser-resonator having the boundary condition.
• the laser-resonator illustrated in FIG. 35 includes a lens (53) having a normal focal length f, a phase plate (54), an output mirror (55), and a resonator on an end face.
• a ring-shaped annular mirror (52) is provided as a mirror.
• a boundary condition that is axisymmetric with respect to the beam optical axis is given, and electric fields that are axisymmetric with respect to an electric field in a plane perpendicular to the optical axis are ⁇ 0.
• the primary Bessel beam can be directly generated by providing a phase plate that gives a phase difference shifted by ⁇ to the beam in the laser resonator.
• the structure of the condensing optical system of the main body of the microscope can be simplified, and the alignment can be improved. Can be very simple.
• FIG. 36 is a main part configuration diagram showing an example of the microscope system of the present invention when the Nd: YAG laser (56) has a laser resonator having the configuration shown in FIG. You.
• a mode lock having a laser resonator having the configuration shown in Fig. 35 as a light source A type Nd: YAG laser (56) is provided.
• This Nd: YAG laser (56) can directly generate the fundamental wave of the primary Bessel beam, and the KTP crystal (5F) converts the wavelength of the fundamental wave to the second harmonic of 532 nm.
• the KTP crystal (5F) converts the wavelength of the fundamental wave to the second harmonic of 532 nm.
• a part of the second harmonic is converted to a wavelength of 563 nm by a Raman shifter (4) made of Ba (NO crystal) to obtain an erase light.
• the condensing optical system uses a phase plate (6) in which the erase light is a hollow beam in the above-described embodiment, or a ring optic that uses a primary Bessel beam. It is not necessary to provide an optical system such as a system, and the light is focused on the adjusted sample (100) via the dichroic mirror (7) (8) and the focusing objective lens (9). Can be.
• the focused beam on the adjusted sample (100) is a primary Bessel beam.
• the optical system that forms the primary Bessel beam is firmly integrated into the laser, so that it has a simple configuration, is resistant to misalignment, and has excellent stability. Resolution and operability can be further improved.
• An optical system that can provide boundary conditions for forming a first-order Bessel beam includes the ring-shaped annular mirror (52) described above and an axial pair with respect to an electric field in a plane perpendicular to the optical axis.
• the annular diffraction grating, Fresnel zone plate, annular aperture, etc. Can be prepared.
• a laser can generate a beam pattern having various mode patterns depending on the configuration of its resonator. Therefore, in a Gaussian, Laguerre, or Hermite-type high-order oscillation mode, As exemplified in 37 (a) to (d), there is a pattern where the intensity becomes zero at the center of one laser beam.
• various lasers as light sources include lasers having a Gaussian-type, Laguerre-type, or Hermite-type high-order oscillation mode in addition to the laser-resonator having the configuration shown in Fig. 35.
• lasers having a Gaussian-type, Laguerre-type, or Hermite-type high-order oscillation mode in addition to the laser-resonator having the configuration shown in Fig. 35.
• the SZN ratio of the fluorescence signal from the sample incident on the emission detector can be improved.
• background light exists in addition to the fluorescence from the observed sample to be detected.
• background lights include i) scattered light of pump light, ii) scattered light of erase light, and iii) fluorescence from an optical system other than the sample.
• the i) is the scattered light with the wavelength ⁇ 1 of the pump light, mainly at the surface of the condenser lens and at the interface of the power glass that protects the observation sample.
• i i is scattered light having a wavelength ⁇ 1 of the erase light for the same reason as i). Since the erase light is stronger than the pump light, it interferes with fluorescence detection.
• the glass material of the optical system or the power glass is ideally non-fluorescent quartz or fluorite, there is no fluorescence in the wavelength region of 250 nm or more from these glass materials. However, if the material is poor, there will be fluorescence from them due to impure parts and color centers.
• the background light is mixed with the fluorescence and incident on the detector. It is desirable to prevent this from happening and to improve the SZN ratio.
• background light can be completely removed by a combination of an optical filter and a spatial filter.
• the wavelength ⁇ 1 of the pump light is the shortest, and the wavelength ⁇ 2 of the erase light and the wavelength band of the fluorescence at the time of deexcitation from S I to S0 exist on the longer wavelength side.
• Rhod amine6G the absorption band from S1 to S2 and the fluorescence wavelength band from S1 to S0 overlap, and the wavelength ⁇ 2 is close to the fluorescence wavelength to be detected.
• the removal of the scattered light of the pump light in i) is generally achieved by diffusing an absorbent containing a polymer into the glass substrate, since the wavelength of the pump light and the erase light is relatively far from each other. This can be performed by providing a sharp cut filter formed by coating the glass on a glass substrate.
• the short wavelength side shorter than the wavelength ⁇ 2 can be completely removed by such a sharp cut filter.
• the light at the shorter wavelength side with a wavelength width of about 10 nm is spaced approximately 30 nm before and after the wavelength separation design position. By removing 0%, light on the long wavelength side can be transmitted.
• the fluorescence measurement wavelength region and the pump light can be separated by 40 nm or more, so the scattered light of the pump light can be reduced by a sharp cut filter. It can be separated and removed from the fluorescence from the sample.
• Such a sharp cut filter for example, is installed on the fluorescent light path before the fluorescent greeting device (Fotomaru (14)), as illustrated in FIG. be able to.
• the removal of the erase light in ii) can be performed by providing a notch filter.
• the notch filter 1 is a filter using a dielectric multi-layer film, and as shown in FIG. 38, does not transmit only a specific wavelength.
• By optimizing the film thickness by stacking a large number of dielectric multilayer films it is possible to completely remove the light within the band of about 20 nm around the design wavelength.
• ⁇ 3 In particular, in the case of Rhodamine6G, the fluorescence emission region spreads to the region of 550 to 650 nm, and the wavelength of the erase light is 562 nm. It is possible to prevent the scattered light of the erase light in the vicinity of nm from being incident on the detector.
• the absorption band from S 1 to S 2 and the absorption band from S 1 to S 0 Since the fluorescence wavelength bands overlap, some of the fluorescence from the sample incident on the emission detector will be partially lost. However, by using a notch filter, only the fluorescence in a narrow area of about 20 nm including 562 nm in the area of 550-650 nm is lost. Losses can be minimized. Further, it is preferable to dispose a pan-pass filter in order to more completely remove background light other than fluorescence from the sample.
• a non-pass filter is formed by coating a glass substrate with a dielectric multilayer film. Contrary to a notch filter, it is a filter that includes a specific wavelength and transmits only light in the wavelength region before and after the specific wavelength. Can be.
• the pump light and the erase light are not transmitted through the wavelength and the bandpass filter having the transmission characteristic only in the fluorescence emission wavelength band is provided, in principle, the filter except for the fluorescence from the sample is completely removed.
• the wavelength can be emphasized.
• a sharp cut filter As described above, by combining a sharp cut filter, a notch filter, and a bandpass filter, for example, in a light emission condensing optical system that condenses light emitted from a sample to a light emission detector, a fluorescent labeler Fluorescence emitted when molecules are deexcited can be detected with a very good SZN ratio.
• Such glass materials include, in addition to synthetic quartz, C a F 2 , N a F, N a 3 AIF 6 , L i F, M g F, S i O 2 , L a F. 3, AI 2 ⁇ 3, C e F 3, P b F?, M g O, T h O 2, S n O, L a 2 ⁇ 3, S i O or the like can be used.
• a filter optical system for separating the fluorescence from the sample from the fluorescence from the optical system other than the sample.
• Such a filter-optical system includes a spatial filter such as a slit or a pinhole.
• FIG. 39 is a main part configuration diagram showing an example of the microscope system of the present invention provided with a filter optical system.
• a notch filter (58) and a no-pass filter (5) are provided in front of the notch filter (14) in the light shielding box (62). 9) and a sharp cut filter (60) are arranged in order. Further, the surface of the light-shielding box (62) on the side of the dichroic mirror (24) has a pinhole (61).
• the laser beam irradiation surface side of the adjusted sample (100) is recovered and protected by a cover glass (63).
• the cover glass (63) is formed of, for example, the above-mentioned glass material.
• the pinhole (23) and the pinhole (61) functioning as a spatial filter are connected to the objective lens (64) and the adjusted sample (1).
• the confocal position is at the 0 0) plane.
• ray tracing is performed.
• the fluorescence emitted at a place other than the condensing point of the pump light and the erase light, in other words, at a place other than the sample surface is a pinhole ( 6 After passing through 1), it is not possible to reach the light receiving surface of the photodetector (14), which is the emission detector.
• fluorescent light emitted from the cover glass (63) does not form an image on the pinhole (61) when passing through the objective lens (64). Therefore, the majority of the non-imaging fluorescent light cannot pass through the pinhole (61). Also, the fluorescent light emitted from the objective lens (64) surface does not directly converge on the pinhole (61) by the objective lens (64) lens, so that it does not pass through the pinhole (61), and 4) Does not reach the light receiving surface of.
• F ( ⁇ ) is the fluorescence intensity of the fluorescent labeler molecule
• ⁇ and ⁇ represent the lower limit wavelength and the upper limit wavelength of the sensitivity range of the emission detector.
• this 1 si 8 hail al corresponds to the fluorescence intensity from the sample without background light contamination.
• FIG. 40 shows an example of an electric system corresponding to the microscope system of the present invention illustrated in FIG. 34 in the sixth embodiment.
• the entire system of the microscope system of the present invention is basically controlled by a personal computer (66).
• This personal computer (66) controls the oscillation of the YAG laser and the drive of a two-dimensional moving stage (10) that is a scanning stage of the tuned sample (100) in FIG. 34, for example.
• All system timings are based on the clock of a personal computer (66). This clock is divided by a divider (67) to a frequency at which laser oscillation is possible, and the divided clock signal is delayed and shaped by a gate and delay generator (68) to form a laser. Controls the AG laser as the Q switch signal and flash lamp signal for control.
• the fluorescence spectrum for each laser shot is monitored on a CCD array (69). Specifically, the fluorescence emitted from the sample (100) adjusted by the laser shot is split by the diffraction grating (70), and the fluorescence is emitted by the one-dimensional CCD array (69). Torr.
• the accumulated data of each pixel of the CCD array (69) is transferred to the memory of the personal computer (66) for each laser shot while synchronizing with the movement of the two-dimensional movement stage (1 °) and the laser emission. .
• a two-dimensional scan image of the sample (100) is formed.
• the fluorescence image of the adjusted sample (100) obtained by irradiation with the mercury lamp (31) is simultaneously monitored by the CCD camera (71), and the fluorescence image data is stored in the frame memory (1). 7 2).
• the personal computer (66) controls the CRT (73) and the frame memory (72), and can perform image display and image processing at any time.
• the image data thus formed can be output by, for example, a CRT (73) or a video printer (74).
• an erase beam that excites a molecule in the first electronic excited state to the second electronic excited state is condensed with an excellent beam profile using a simple and compact optical system.
• a new microscope system with high stability, high operability, and excellent super-resolution is provided.
• a new microscope system with a micromanipulator function that can capture and move sample particles using erase light, which is a hollow beam, without damaging the sample. Will also be provided.

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• 1998
  • 1998-04-09 JP JP09792498A patent/JP3350442B2/ja not_active Expired - Fee Related
• 1999
  • 1999-04-09 US US09/445,389 patent/US6667830B1/en not_active Expired - Lifetime
  • 1999-04-09 DE DE19980759T patent/DE19980759T1/de active Granted
  • 1999-04-09 WO PCT/JP1999/001904 patent/WO1999053356A1/ja active Application Filing
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