JP2004029345A - Microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope capable of realizing complete observation of an image by preventing discoloration of an observation molecule. <P>SOLUTION: The microscope has an exciting light source 1 generating exciting light for exciting the molecule in a sample 9 from a base state to a 1st excitation state, a condensing optical system 8 condensing the exciting light from the light source 1 on the sample 9, and a detection means 14 detecting emitted light when the molecule in the excited sample 9 is deexcited, and the light source 1 is a pulse light source. The microscope is constituted to satisfy ν<1/τ and 1/(I<SB>0</SB>×σ<SB>0</SB>)<T<τ when it is assumed that the repeating frequency of the exciting light from the pulse light source is ν, pulse width is T, the life of the molecule in the sample 9 in the 1st excitation state is τ, absorption cross section obtained when the molecule in the sample 9 is excited from the base state to the 1st excitation state is σ<SB>0</SB>, and photon flux on the condensed surface of the sample by the exciting light is I<SB>0</SB>. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a microscope, and particularly to a new high-performance and high-performance optical microscope that illuminates a stained sample with light of a plurality of wavelengths from a highly functional laser light source to obtain high spatial resolution.
[0002]
[Prior art]
The technology of optical microscopes is old, and various types of microscopes have been developed. In recent years, further advanced microscope systems have been developed with advances in peripheral technologies such as laser technology and electronic imaging technology.
[0003]
Against this background, for example, in Japanese Patent Application Laid-Open No. 8-184552, not only control of the contrast of an obtained image but also chemical analysis is performed using a double resonance absorption process generated by illuminating a sample with light of a plurality of wavelengths. There has been proposed a high-performance microscope that has made it possible.
[0004]
This microscope uses a double resonance absorption to select a specific molecule, and observes absorption and fluorescence caused by a specific optical transition. This principle will be described with reference to FIGS. FIG. 3 shows the electronic structure of the valence orbits of the molecules constituting the sample. First, the electrons of the valence orbits of the molecules in the ground state (S0 state) shown in FIG. 3 are excited by light of wavelength λ1. Then, the first electronically excited state (S1 state) shown in FIG. 4 is set. Next, it is similarly excited by light of another wavelength λ2 to be in the second electronically excited state (S2 state) shown in FIG. Due to this excited state, the molecule emits fluorescence or phosphorescence, and returns to the ground state as shown in FIG.
[0005]
In the microscopy using the double resonance absorption process, an absorption image and an emission image are observed using the absorption process in FIG. 4 and the emission of fluorescence and phosphorescence in FIG. In this microscopy, molecules constituting the sample are first excited to the S1 state as shown in FIG. 4 by laser light or the like at a resonance wavelength λ1. At this time, the number of molecules in the S1 state in a unit volume is: It increases as the intensity of the irradiated light increases.
[0006]
Here, the linear absorption coefficient is given by the product of the absorption cross-sectional area per molecule and the number of molecules per unit volume. Therefore, in the excitation process as shown in FIG. The coefficient will depend on the intensity of the light of the wavelength λ1 which is first irradiated. That is, the linear absorption coefficient for the wavelength λ2 can be controlled by the intensity of the light having the wavelength λ1. This indicates that the contrast of the transmitted image can be completely controlled by the light of the wavelength λ1 if the sample is irradiated with the light of the two wavelengths of the wavelength λ and the wavelength λ2 and the transmission image of the wavelength λ2 is taken.
[0007]
When the de-excitation process by fluorescence or phosphorescence in the excited state in FIG. 5 is possible, the emission intensity is proportional to the number of molecules in the S1 state. Therefore, when used as a fluorescence microscope, it is possible to control the image contrast.
[0008]
Furthermore, microscopy using the double resonance absorption process allows not only control of the image contrast as described above, but also chemical analysis. That is, since the outermost valence orbit shown in FIG. 3 has an energy level unique to each molecule, the wavelength λ1 differs depending on the molecule, and the wavelength λ2 also becomes unique to the molecule.
[0009]
Here, even when illuminating with a conventional single wavelength, it is possible to observe an absorption image or a fluorescence image of a specific molecule to some extent, but in general, the wavelength regions of the absorption bands of some molecules overlap, so However, accurate identification of the chemical composition of a sample is not possible.
[0010]
On the other hand, in the microscopy using the double resonance absorption process, molecules that absorb or emit at two wavelengths, wavelength λ1 and wavelength λ2, are limited, so that it is possible to identify the chemical composition of a sample more accurately than the conventional method. Become. Also, when exciting valence electrons, only light having a specific electric field vector with respect to the molecular axis is strongly absorbed. Therefore, if the polarization directions of the wavelengths λ1 and λ2 are determined and the absorption or fluorescence images are taken, the same results can be obtained. Even molecules can be used to identify the orientation direction.
[0011]
Recently, for example, Japanese Patent Application Laid-Open No. 2001-100102 has proposed a fluorescence microscope having a high spatial resolution exceeding a diffraction limit by using a double resonance absorption process.
[0012]
FIG. 7 is a conceptual diagram of a double resonance absorption process in a molecule, in which a molecule in a ground state S0 is excited by light having a wavelength λ1 into S1, which is a first electronically excited state, and further, is excited by a light having a wavelength λ2. The state of being excited by the state S2 is shown. FIG. 7 shows that the fluorescence from S2 of a certain molecule is extremely weak.
[0013]
In the case of a molecule having optical properties as shown in FIG. 7, a very interesting phenomenon occurs. FIG. 8 is a conceptual diagram of the double resonance absorption process as in FIG. 7, in which the X axis on the horizontal axis represents the expansion of the spatial distance, and the spatial region A1 irradiated with light of wavelength λ2 and the light of wavelength λ2 are not irradiated. A spatial area A0 is shown.
[0014]
In FIG. 8, in the spatial region A0, a large number of molecules in the S1 state are generated by the excitation of the light having the wavelength λ1, and at this time, fluorescence emitted at the wavelength λ3 is seen from the spatial region A0. However, in the spatial region A1, since the light of the wavelength λ2 is irradiated, most of the molecules in the S1 state are immediately excited to the higher-order S2 state, and the molecules in the S1 state no longer exist. Such a phenomenon has been confirmed by several molecules. Thereby, in the spatial region A1, the fluorescence of the wavelength λ3 completely disappears, and furthermore, the fluorescence from the S2 state is not originally present. Therefore, in the spatial region A1, the fluorescence itself is completely suppressed, and the fluorescence is emitted only from the spatial region A0. Become.
[0015]
This is extremely important when viewed from the microscope application field. That is, in a conventional scanning laser microscope or the like, a laser beam is condensed into a microbeam by a condensing lens to scan over an observation sample. At this time, the size of the microbeam depends on the numerical aperture of the condensing lens and the wavelength. , And a higher spatial resolution cannot be expected in principle.
[0016]
However, in the case of FIG. 8, the two types of light of the wavelength λ1 and the wavelength λ2 are spatially well overlapped, and the fluorescent region is suppressed by irradiation with the light of the wavelength λ2. Focusing on the irradiation area, the fluorescent area can be narrower than the diffraction limit determined by the numerical aperture and wavelength of the condenser lens, and the spatial resolution can be substantially improved. Therefore, by utilizing this principle, it becomes possible to realize a super-resolution microscope, for example, a fluorescence microscope, using a double resonance absorption process exceeding the diffraction limit.
[0017]
Further, in order to enhance the super-resolution of the microscope, for example, Japanese Patent Application Laid-Open No. H11-95120 discloses a fluorescent labeler molecule for fully utilizing the function of the super-resolution microscope, and two light beams of wavelengths λ1 and λ2 to be used. And the irradiation timing of the sample. This prior art has at least three quantum states including a ground state, and the transition when de-excited from a higher energy state excluding the first electronically excited state to the ground state is more a thermal relaxation process than a fluorescence relaxation process. A sample in which a fluorescent labeler molecule that stains various dominant molecules and a biomolecule that has been subjected to biochemical staining technology are chemically bonded is excited into the S1 state by light having a wavelength λ1 that excites the molecule to be stained. Subsequently, the light of wavelength λ2 is immediately excited to a higher quantum level to suppress the fluorescence from the S1 state. As described above, the spatial resolution can be improved by artificially suppressing the spatial fluorescent region using the optical properties of molecules.
[0018]
The optical properties of such molecules can be explained from a quantum chemical standpoint. That is, generally, each atom constituting the molecule is connected by σ or π bond. In other words, the molecular orbital of the molecule has a σ molecular orbital or a π molecular orbital, and electrons existing in these molecular orbitals play an important role of bonding each atom. Among them, the electron of the σ molecular orbit strongly bonds each atom, and determines the distance between atoms in the molecule which is the skeleton of the molecule. On the other hand, electrons in the π molecular orbital make little contribution to the bonding of each atom, but are rather bound to the whole molecule by an extremely weak force.
[0019]
In many cases, when an electron in a σ molecular orbital is excited by light, the interatomic distance of the molecule changes greatly, causing a large structural change including dissociation of the molecule. As a result, most of the energy given to the molecule by light changes to thermal energy due to the kinetic energy and structural change of the atom. Therefore, the excitation energy is not consumed in the form of light called fluorescence. In addition, since the structural change of a molecule occurs at a very high speed (less than picosecond), even if fluorescence occurs in the process, the life is extremely short.
[0020]
On the other hand, the electrons in the π molecular orbital structure hardly change even when excited, stay at a high quantum discrete level for a long time, emit fluorescence in the order of nanoseconds, and excite them. Has properties.
[0021]
According to quantum chemistry, a molecule has a π molecular orbit and a double bond is equivalent, and it is necessary to select a molecule with a large number of double bonds for the fluorescent labeler molecule used. It becomes. It has been confirmed that the fluorescence from the S2 excited state is extremely weak in a 6-membered ring molecule such as benzene and pyrazine even in a molecule having a double bond (for example, M. Fujii et. Al. Chem. Phys. Lett., 171 (1990) 341).
[0022]
Therefore, if a molecule containing a 6-membered ring molecule such as benzene or pyrazine is selected as the fluorescent labeler molecule, the fluorescence lifetime from the S1 state is long, and the light is excited from the S1 state to the S2 state by photoexcitation, so that the fluorescence from the molecule is increased. Can be easily suppressed, so that the super-resolution can be effectively used. In other words, if observation is performed by staining with these fluorescent labeler molecules, not only can the fluorescence image of the sample be observed with high spatial resolution, but also by adjusting the chemical groups of the side chains of the molecule, the biological sample can be analyzed. Since only a specific chemical tissue can be selectively stained, even the detailed chemical composition of the sample can be analyzed.
[0023]
In general, the double resonance absorption process occurs only when the two light wavelengths, polarization states, and the like satisfy specific conditions. Therefore, by using this, the structure of a molecule can be known in detail. That is, there is a strong correlation between the polarization direction of light and the orientation direction of molecules, and when the polarization direction of each of the 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 with light of two wavelengths and rotating the polarization direction of each light, the extent of the disappearance of the fluorescence changes. Can also be obtained. This is also possible by adjusting the wavelengths of the two lights.
[0024]
As described above, according to the technique described in Japanese Patent Application Laid-Open No. H11-95120, it can be seen that the technique has high analytical performance in addition to super-resolution. Further, by devising the irradiation timing of the two lights of the wavelength λ1 and the wavelength λ2, the S / N can be improved and the fluorescence can be effectively suppressed, and the super-resolution is more effectively exhibited. It is possible to do.
[0025]
As a specific example of such super-resolution microscopy, for example, Japanese Patent Application Laid-Open No. 2001-100102 discloses that light (particularly laser light) having a wavelength λ1 that excites a fluorescent labeler molecule from the S0 state to the S1 state is used as a pump light. As shown in FIG. 9, pump light is emitted from a light source 81 and erase light is emitted from a light source 82, and the pump light is reflected by a dichroic mirror 83, as shown in FIG. After that, the light is condensed on the sample 95 by the annular optical system 84, the erase light is converted into a hollow beam by the phase plate 86, then transmitted through the dichroic mirror 83 and spatially overlapped with the pump light to form the annular optical system. There has been proposed a device for focusing light on a sample 95 according to 84.
[0026]
According to this microscope, fluorescence other than near the optical axis where the intensity of the erase light becomes zero is suppressed, and as a result, a region narrower than the spread of the pump light (Δ <0.61 · λ1 / NA, NA Only the fluorescent labeler molecules present in the (numerical aperture of the optical system 84) are observed, and as a result, super-resolution is developed. As the phase plate 86 for converting the erase light into a hollow beam, for example, as shown in FIG. 10, a structure in which a phase difference π is provided at a point symmetric position with respect to the optical axis, or a liquid crystal surface is used. A liquid crystal spatial modulator can be used.
[0027]
[Problems to be solved by the invention]
However, in a fluorescence detection type microscope including super-resolution microscopy, there is a problem of fading of a fluorescent dye. In particular, in a commercially available laser microscope, a continuous wave (CW) laser is mainly used as an excitation light source, that is, a pump light source. However, a fading phenomenon often occurs during observation, and the contrast is reduced before sufficient image observation is performed. There is a problem of lowering.
[0028]
Therefore, an object of the present invention made in view of the above point is to provide a microscope capable of preventing fading of observed molecules and performing sufficient image observation.
[0029]
[Means for Solving the Problems]
The microscope according to claim 1, which achieves the above object, comprises an excitation light source that generates excitation light for exciting molecules in a sample from a ground state to a first excitation state,
A focusing optical system that focuses the excitation light from the excitation light source on the sample,
Detecting means for detecting light emission when molecules in the excited sample are de-excited. The excitation light source is a pulse light source, and the repetition frequency of the excitation light from the pulse light source is ν, and the pulse width is T. Τ is the lifetime of the molecule in the sample in the first excited state, and σ is the absorption cross section when the molecule in the sample is excited from the ground state to the first excited state. ₀ And the photon flux of the excitation light on the sample focusing surface ₀ When
[Equation 3]
ν <1 / τ (1)
1 / (I ₀ ・ Σ ₀ ) <T <τ (2)
It is characterized by having been configured to satisfy the following.
[0030]
The invention according to claim 2 is a microscope for observing a sample including a molecule having at least three electronic states including a ground state,
A first light source that generates first light that causes the molecule to transition from a ground state to a first electronically excited state;
A second light source that generates second light that causes the molecule to transition from the first electronically excited state to a second electronically excited state having a higher energy level;
An optical system for irradiating the sample with at least a part of an irradiation area of the first light and the second light overlapped;
Detecting means for detecting light emission when the molecule is de-excited from the first electronically excited state to the ground state,
The first light source is a pulsed laser, and the repetition frequency of the first light from the pulsed laser is ν, the pulse width is T, the lifetime of the first electronically excited state of the molecule is τ, and the molecule is shifted from the ground state. The absorption cross section when excited to the first electronic excited state is σ ₀ , The photon flux of the first light on the sample focusing surface is I ₀ In this case, the configuration is such that the expressions (1) and (2) are satisfied.
[0031]
According to a third aspect of the present invention, in the microscope according to the first aspect, a repetition frequency of the excitation light from the pulse light source is higher than a sampling frequency for scanning the sample.
[0032]
According to a fourth aspect of the present invention, in the microscope according to the second aspect, a repetition frequency of the first light from the pulse laser is higher than a sampling frequency for scanning the sample.
[0033]
The invention according to claim 5 is the microscope according to claim 2 or 4, wherein the second light source is a pulse laser, and a pulse width of the second light from the pulse laser is different from the first light source. Is longer than the pulse width of the first light.
[0034]
The invention according to claim 6 is the microscope according to claim 2 or 4, wherein the second light source is a continuous wave laser.
[0035]
Hereinafter, the principle of the present invention will be described with reference to FIG.
[0036]
FIG. 2 is an energy diagram showing a general quantum level of a molecule. For example, when the molecule is first excited from the ground state S0 to the first electronically excited state S1 at the wavelength of resonance, the molecule emits fluorescence with a predetermined fluorescence yield and relaxes to the ground state. Here, assuming that the life remaining in the S1 state is τ, the relaxation rate is given by 1 / τ. For fluorescent molecules, τ is typically on the order of nanoseconds in the aggregate phase and the relaxation rate is ⁸ -10 ⁹ / Sec.
[0037]
At this time, when molecules in the S1 state are irradiated with erase light having a wavelength that satisfies the conditions of the double resonance absorption process, the molecules in the S1 state decrease at a rate of σI. Here, σ is the absorption cross section of molecules in the S1 state with respect to the erase light, and I is the photon flux of the erase light. The transition destination may be an S2 state or a higher quantum state capable of transition, or a triplet state, but in any case, transition to a quantum state with an extremely small fluorescence yield. This is the double resonance absorption process. In general, molecules do not emit fluorescence from a state other than the S1 state, and thus the fluorescence from the molecule is suppressed when the double resonance absorption process occurs. As described above, the super-resolution microscope is based on this principle.
[0038]
When the dye molecules are irradiated with the pump light for a long time, the dye molecules are discolored. This is also a kind of double resonance absorption phenomenon caused by the single irradiation of the pump light. That is, a phenomenon occurs in which the molecules in the S1 state further absorb the pump light and are ionized. This is a chemical reaction involving molecular dissociation, and the molecule no longer loses its previous optical properties. In particular, in the case of a fluorescent molecule, the fluorescent yield decreases and the fluorescent molecule does not function as a fluorescent labeler.
[0039]
In general, the time required to complete the molecular excitation process is only a few picoseconds, and most molecules emit fluorescence within several nanoseconds after excitation and return to the S0 state. This process will be described in more detail. The absorption cross section when the molecule is excited from the S0 state to the S1 state by the pump light is σ ₀ , The irradiation time of the pump light is T, and the photon flux of the pump light is I ₀ Then, T> 1 / (I ₀ ・ Σ ₀ Under the conditions that satisfy (), one molecule is excited to the S1 state with a probability of 63% or more. For example, in the case of rhodamine 6G, σ ₀ Is 10 ^-17 cm ² Therefore, if weak light having a wavelength of 532 nm and a light intensity of 1 nW is condensed to 1 μm as pump light, for example, T> 10 ^-17 The condition of sec is realized, and the molecule is instantaneously excited.
[0040]
Therefore, in principle, a commercially available pulse laser having a femtosecond or picosecond pulse width can be used as the pump light source. However, when the pump light is incident during the excitation lifetime τ after the excitation, molecules may be ionized and discolored. Therefore, it is desirable that the pulse width of the pump light source be as short as possible than the excitation lifetime τ of the molecule.
[0041]
Further, if the pump light is re-irradiated after the excitation lifetime at which the fluorescence ends, the molecules are excited to the S1 state without fading, so that the fluorescence is emitted at a very high frequency. Therefore, it is desirable that the time interval of the next pump light to be irradiated is longer than τ. In particular, if the pump light is irradiated with a femtosecond or picosecond pulse width at the τ interval, the molecular light will have a very low probability of fading. Can always be maintained in the S1 state, thereby maximizing the probability of occurrence of fluorescence on a time average, and enabling sufficient image observation.
[0042]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, an embodiment of a microscope according to the present invention will be described with reference to the drawings.
[0043]
FIG. 1 is a diagram illustrating a configuration of a microscope according to an embodiment of the present invention. The microscope according to the present embodiment is a laser scanning microscope in which erase light is formed into a hollow beam to develop super-resolution to improve spatial resolution, and observes a biological sample stained with rhodamine 6G. .
[0044]
Here, the absorption of rhodamine 6G is maximized in the wavelength band of 530 nm, and the absorption band capable of being excited from the first electronic excited state (S1) to a higher energy electronically excited state has a wavelength of 600 to 650 nm. Exists in the area of
[0045]
Therefore, in the present embodiment, a mode-locked Nd: YAG laser 1 of a laser diode (LD) excitation type is used as the first light source, and its second harmonic (532 nm) is used as the pump light. The LD-excited mode-locked Nd: YAG laser 1 can oscillate picosecond laser pulses at a repetition frequency on the order of MHz by adjusting the design parameters of the laser resonator. For example, the GE-100 series manufactured by Time-Bandwidth, Switzerland, can oscillate pulse light having a pulse width of 6 psec with an average output of 50 mW at a repetition frequency of 100 MHz as a standard specification. This corresponds to the energy of one pulse corresponding to 500 pJ. Also, the repetition frequency can be adjusted between 25 MHz and 1 GHz by changing the design of the resonator.
[0046]
As the second light source, a commercially available and inexpensive and highly reliable CW He-Ne laser 2 is used, and its fundamental wave (633 nm) is used as erase light.
[0047]
The pump light from the LD-excitation mode-locked Nd: YAG laser 1 is made incident on a beam combiner 3 composed of a cuvette-type half mirror. The erase light from the CW He-Ne laser 2 passes through a phase plate 4 as shown in FIG. 10 to form a hollow beam, is incident on the beam combiner 3, and is synthesized coaxially with the pump light. After the pump light and the erase light are transmitted through a beam separator 5 composed of a cuvette-type half mirror, they are sequentially reflected by galvano mirrors 6 and 7 which can swing about axes orthogonal to each other, and then are subjected to rhodamine by an objective lens 8. Light is condensed on the surface of the biological sample 9 stained with 6G, and the light converging point is moved by the swinging of the galvanometer mirrors 6 and 7 to two-dimensionally scan the sample surface. In FIG. 1, for simplification of the drawing, the galvanometer mirrors 6 and 7 are shown to swing about axes parallel to each other. The phase plate 4 is coated with a vapor-deposited film so as to give the erase light a phase distribution as shown in FIG. Thereby, the light intensity is canceled at the optical axis and the beam is shaped into a hollow beam.
[0048]
On the other hand, the fluorescence emitted from the sample 9 by the irradiation of the pump light and the erase light is collimated by the objective lens 8, then follows a path opposite to the incident optical path, and enters the beam separator 5 through the galvanometer mirrors 7 and 6. The fluorescence reflected by the beam separator 5 is condensed on a pinhole 11 by a projection lens 10.
[0049]
The pinhole 11 is arranged at a confocal position with respect to the fluorescence emission point of the sample 9, and the fluorescence transmitted through the pinhole 11 is transmitted through the pump light cut notch filter 12 and the erase light cut notch filter 13, respectively. After removing the pump light and the erase light, the light is received by the photomultiplier 14 to obtain a fluorescence signal.
[0050]
A fluorescent signal obtained from the photomultiplier 14 is A / D converted and stored in a video frame memory of a computer (not shown) for controlling a microscope, and the video signal stored in the video frame memory is D / A converted. And a two-dimensional fluorescence image of the sample 9 is displayed in real time on a monitor (not shown) such as a CRT. The galvanometer mirrors 6 and 7 swing in synchronization with the video rate of the fluorescent two-dimensional image displayed on the monitor.
[0051]
In the present embodiment, since the excitation lifetime (τ) of rhodamine 6G that stains the biological sample 9 is 3 nsec, the above equation (1) is satisfied, so that the LD excitation type mode-locked Nd: YAG laser 1 The repetition frequency (ν) of the pump light is set to 333 MHz or less.
[0052]
The objective lens 8 has a numerical aperture (NA) of 0.9. In this case, the pump light (wavelength λ) ₁ = 532 nm) from the Rayleigh equation:
(Equation 4)
d = 1.22 · λ ₁ / NA
= 0.72 (μm)
And the focusing area is about 4 × 10 ^-9 cm ² It becomes.
[0053]
Therefore, the number of photons passing through the sample condensing surface per unit time and unit area, that is, the photon flux I ₀ Is 6.5 × 10 ²⁸ photons / cm ² / Sec. In addition, the absorption cross section (σ) of the molecule from the S0 state to the S1 state with respect to the pump light of rhodamine 6G. ₀ ) Is 10 ^-17 cm ² Therefore, 1 / (σ in this case ₀ ・ I ₀ ) Is about 1.5 psec. Therefore, in the present embodiment, in order to satisfy the above equation (2), the pulse width (T) of the pump light generated from the LD-excitation mode-locked Nd: YAG laser 1 is set to 6 psec (> 1.5 psec). I do.
[0054]
In this way, the rhodamine 6G molecule in the ground state can be excited to the S1 state with a sufficient probability, and the pulse width of the pump light (T = 6 psec) can be adjusted to the excitation lifetime (τ = 3 nsec) of the rhodamine 6G molecule. Since it is much shorter than this, no re-excitation occurs and the effects of bleaching can be neglected.
[0055]
When a CRT is used as a monitor for displaying a two-dimensional fluorescence image of the sample 9, the CRT generally has 525 scanning lines, and if one frame is 1/30 sec, the scanning period of one line is 63. 0.5 μsec. Therefore, in order to digitize the fluorescent signal obtained from the photomultiplier 14 and store it in the video frame memory of the computer, it is necessary to A / D convert the fluorescent signal in synchronization with the scanning period. It is necessary to swing the galvanometer mirrors 6 and 7 completely in synchronization with the video rate.
[0056]
Here, for example, when the aspect ratio of the CRT pixel is set to 1: 1, it is necessary to sample one line scanning period, that is, 63.5 μsec, to 525 pixels, and this sampling frequency corresponds to the video rate (ω). In this case, the frequency is 8.27 MHz. Therefore, in this case, if the repetition frequency (ν) of the pump light is not higher than 8.27 MHz, one or more pulses of the pump light cannot be irradiated per pixel, so that a good fluorescent signal with little fluctuation can be obtained. This makes it difficult to display a microscope image on a CRT in real time.
[0057]
For this reason, in this embodiment, the repetition frequency (ν) of the pump light is set to 100 MHz which is 333 MHz or less and satisfies ω <ν. In this manner, since 10 or more pulses of the pump light can be emitted per pixel, a good fluorescent signal with extremely small statistical fluctuation obtained by integrating the fluorescent light by each pulse can be obtained. A simple microscope image can be displayed on a CRT in real time.
[0058]
Further, in this embodiment, since the CW He-Ne laser 2 is used as the erase light source, the pump light and the erase light can completely overlap in the time domain. Therefore, the fluorescence suppression effect, which is the basis of the super-resolution in the super-resolution microscope, can be very effectively induced, and can effectively contribute to the improvement of the spatial resolution.
[0059]
It should be noted that the present invention is not limited only to the above-described embodiment, and various modifications or changes can be made. For example, in the above embodiment, rhodamine 6G is used as the dye for the sample 9, but other coumarin dyes or fluorescent labels such as fluorescent proteins can also be used. Further, the erase light source is not limited to the CW laser, and a pulse laser can also be used. In this case, the repetition frequency of the erase light from the pulse laser is synchronized with the repetition frequency of the pump light, and the pulse width of the erase light is made longer than that of the pump light. In this way, the erase light and the pump light can be completely overlapped in the time domain, so that the fluorescence suppression effect can be very effectively induced as in the above embodiment, and the spatial resolution can be improved. Can contribute to the future.
[0060]
【The invention's effect】
As described above, according to the present invention, the repetition frequency of the excitation light from the pulsed light source is set as the pulsed light source that generates the excitation light for exciting the molecules in the sample from the ground state to the first excited state. , The pulse width T, the lifetime of the molecule in the sample in the first excited state is τ, and the absorption cross section when the molecule in the sample is excited from the ground state to the first excited state is σ. ₀ And the photon flux of the excitation light on the sample focusing surface ₀ Where ν <1 / τ and 1 / (I ₀ ・ Σ ₀ ) <T <τ, so that fading of the observed molecules can be prevented and sufficient image observation can be performed.
[Brief description of the drawings]
FIG. 1 is a diagram showing a configuration of a microscope according to an embodiment of the present invention.
FIG. 2 is a diagram for explaining the principle of the present invention.
FIG. 3 is a conceptual diagram showing an electronic structure of a valence orbit of molecules constituting a sample.
FIG. 4 is a conceptual diagram showing a first electronically excited state of the molecule of FIG. 3;
FIG. 5 is a conceptual diagram showing a second electronically excited state.
FIG. 6 is a conceptual diagram showing a state of returning from a second electronically excited state to a ground state.
FIG. 7 is a conceptual diagram for explaining a double resonance absorption process in a molecule.
FIG. 8 is a conceptual diagram for explaining a double resonance absorption process.
FIG. 9 is a diagram showing a configuration of an example of a conventional super-resolution microscope.
FIG. 10 is a plan view showing a configuration of a phase plate shown in FIG. 9;
[Explanation of symbols]
1 Nd: YAG laser
2 He-Ne laser
3 Beam combiner
4 Phase plate
5 Beam separator
6,7 Galvanomirror
8 Objective lens
9 samples
10. Projection lens
11 Pinhole
12 Pump light cut notch filter
13 Erase light cut notch filter
14 Photomultiplier tube

Claims (6)

An excitation light source for generating excitation light for exciting molecules in the sample from a ground state to a first excited state;
A focusing optical system that focuses the excitation light from the excitation light source on the sample,
Having detection means for detecting light emission when molecules in the excited sample are de-excited,
The excitation light source is a pulsed light source. The repetition frequency of the excitation light from the pulsed light source is ν, the pulse width is T, the lifetime of the first excited state of the molecule in the sample is τ, and the molecule in the sample is shifted from the ground state to the first state. When the absorption cross section at the time of excitation to one excitation state is σ ₀ and the photon flux of the excitation light on the sample focusing surface is I ₀ ,

A microscope characterized by satisfying the following. A microscope for observing a sample including a molecule having at least three electronic states including a ground state,
A first light source that generates first light that causes the molecule to transition from a ground state to a first electronically excited state;
A second light source that generates second light that causes the molecule to transition from the first electronically excited state to a second electronically excited state having a higher energy level;
An optical system for irradiating the sample with at least a part of an irradiation area of the first light and the second light overlapped;
Detecting means for detecting light emission when the molecule is de-excited from the first electronically excited state to the ground state,
The first light source is a pulsed laser. The repetition frequency of the first light from the pulsed laser is ν, the pulse width is T, the lifetime of the molecule in the first electronically excited state is τ, and the molecule is shifted from the ground state. When the absorption cross section when excited to the first electronically excited state is σ ₀ and the photon flux of the first light on the sample focusing surface is I ₀ ,

A microscope characterized by satisfying the following. The microscope according to claim 1, wherein a repetition frequency of the excitation light from the pulse light source is higher than a sampling frequency for scanning the sample. The microscope according to claim 2, wherein a repetition frequency of the first light from the pulse laser is higher than a sampling frequency for scanning a sample. 3. The apparatus according to claim 2, wherein the second light source is a pulse laser, and a pulse width of the second light from the pulse laser is longer than a pulse width of the first light from the first light source. Or the microscope according to 4. The microscope according to claim 2, wherein the second light source is a continuous wave laser.

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