JP4017472B2 - microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microscope, and more particularly to a new high-performance and high-performance optical microscope that obtains high spatial resolution by illuminating a stained sample with light of a plurality of wavelengths from a highly functional laser light source.
[0002]
[Prior art]
Optical microscope technology is old and various types of microscopes have been developed. In recent years, more advanced microscope systems have been developed due to advances in peripheral technologies such as laser technology and electronic image technology.
[0003]
In such a background, for example, in JP-A-8-184552, chemical analysis as well as control of contrast of an image obtained by using a double resonance absorption process generated by illuminating a sample with light of a plurality of wavelengths. A high-performance microscope that has made it possible is also proposed.
[0004]
This microscope selects specific molecules using double resonance absorption and observes absorption and fluorescence due to specific optical transitions. This principle will be described with reference to FIGS. FIG. 4 shows the electronic structure of the valence electron orbit of the molecules constituting the sample. First, the electrons in the valence electron orbit of the molecule in the ground state (S0 state) shown in FIG. 4 are excited by light of wavelength λ1. Thus, the first electronic excitation state (S1 state) shown in FIG. Next, the second electron excitation state (S2 state) shown in FIG. 6 is obtained by exciting similarly with light of another wavelength λ2. In this excited state, the molecule emits fluorescence or phosphorescence and returns to the ground state as shown in FIG.
[0005]
In the microscope method using the double resonance absorption process, an absorption image and a light emission image are observed using the absorption process of FIG. 5 and the fluorescence and phosphorescence emission of FIG. In this microscope method, first, the molecules constituting the sample are excited to the S1 state as shown in FIG. 5 with light having a resonance wavelength λ1 by laser light or the like. At this time, the number of molecules in the S1 state in the unit volume is It increases as the intensity of light to be applied increases.
[0006]
Here, since the linear absorption coefficient is given by the product of the absorption cross section per molecule and the number of molecules per unit volume, in the excitation process as shown in FIG. The coefficient depends on the intensity of the light having the wavelength λ1 irradiated first. That is, the linear absorption coefficient with respect to the wavelength λ2 can be controlled by the intensity of the light with the wavelength λ1. This indicates that the contrast of the transmitted image can be completely controlled with the light of the wavelength λ1 when the sample is irradiated with the light of the two wavelengths of the wavelength λ and the wavelength λ2 and a transmission image with the wavelength λ2 is taken.
[0007]
Further, when the deexcitation process by fluorescence or phosphorescence in the excited state of FIG. 6 is possible, the emission intensity is proportional to the number of molecules in the S1 state. Therefore, image contrast can be controlled when used as a fluorescence microscope.
[0008]
Furthermore, the microscope method using a double resonance absorption process enables not only the above-described image contrast control but also chemical analysis. That is, since the outermost valence electron orbit shown in FIG. 4 has an energy level unique to each molecule, the wavelength λ1 varies depending on the molecule, and the wavelength λ2 is also unique to the molecule.
[0009]
Here, even when illuminating with a conventional single wavelength, it is possible to observe an absorption image or fluorescence image of a specific molecule to some extent, but generally the wavelength regions of absorption bands of several molecules overlap. It is impossible to accurately identify the chemical composition of the sample.
[0010]
On the other hand, in the microscope method using the double resonance absorption process, the molecules that absorb or emit light are limited by the two wavelengths of λ1 and λ2, so that the chemical composition of the sample can be identified more accurately than the conventional method. Become. In addition, when valence electrons are excited, only light having a specific electric field vector with respect to the molecular axis is strongly absorbed. Therefore, if the polarization direction of the wavelengths λ1 and λ2 is determined and an absorption or fluorescence image is taken, the same Even molecules can be used to identify the orientation direction.
[0011]
Recently, for example, in Japanese Patent Application Laid-Open No. 2001-100102, a fluorescence microscope having a high spatial resolution exceeding the diffraction limit using a double resonance absorption process has been proposed.
[0012]
FIG. 8 is a conceptual diagram of a double resonance absorption process in a molecule. A molecule in the ground state S0 is excited by light having a wavelength λ1 to S1, which is a first electronically excited state, and further excited by a second electron by light having a wavelength λ2. The state of being excited by the state S2 is shown. FIG. 8 shows that the fluorescence from S2 of certain molecules is extremely weak.
[0013]
In the case of molecules having optical properties as shown in FIG. 8, a very interesting phenomenon occurs. FIG. 9 is a conceptual diagram of the double resonance absorption process, as in FIG. 8. The horizontal X axis represents the spread of the spatial distance, and the light of the wavelength region λ2 and the light of the wavelength region λ2 is not irradiated. A space area A0 is shown.
[0014]
In FIG. 9, in the spatial region A0, a large number of molecules in the S1 state are generated by excitation of light of wavelength λ1, and at this time, fluorescence emitted at wavelength λ3 is seen from the spatial region A0. However, in the spatial region A1, since the light of wavelength λ2 is irradiated, most of the molecules in the S1 state are immediately excited to the higher S2 state and no S1 state molecule exists. Such a phenomenon has been confirmed by several molecules. Thereby, in the spatial region A1, the fluorescence of the wavelength λ3 is completely lost, and the fluorescence from the S2 state is not originally present. Therefore, in the spatial region A1, the fluorescence itself is completely suppressed (fluorescence suppression effect), and only from the spatial region A0. Fluorescence will be emitted.
[0015]
This is extremely important when considered from the field of application of a microscope. That is, in a conventional scanning laser microscope or the like, a laser beam is condensed on a micro beam by a condensing lens and scanned on an observation sample. The size of the micro beam at that time is determined by the numerical aperture and wavelength of the condensing lens. The diffraction limit is determined by, and in principle, no further spatial resolution can be expected.
[0016]
However, in the case of FIG. 9, two types of light of wavelength λ1 and wavelength λ2 are spatially superposed and the fluorescent region is suppressed by irradiation with light of wavelength λ2, for example, the light of wavelength λ1. Focusing on the irradiation region, the fluorescent region can be made 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, in Japanese Patent Application Laid-Open No. 11-95120, a fluorescent labeler molecule for fully utilizing the function of the super-resolution microscope and two light beams having wavelengths λ1 and λ2 to be used are used. The timing of irradiation of the sample is disclosed. In this prior art, there are at least three quantum states including the ground state, and the transition when the de-excitation from the higher energy state excluding the first electronic excited state to the ground state is a thermal relaxation process rather than the relaxation process by fluorescence. A sample in which a fluorescent labeler molecule for staining various dominant molecules and a biomolecule subjected to biochemical staining technology are chemically bonded is excited to the S1 state with light of wavelength λ1 that excites the molecule to be stained. Subsequently, the fluorescence from the S1 state is suppressed by immediately exciting to a higher quantum level with light of wavelength λ2. As described above, the spatial resolution can be improved by artificially suppressing the spatial fluorescent region by utilizing the optical properties of the molecules.
[0018]
The optical properties of such molecules can be explained from a quantum chemical standpoint. That is, in general, in a molecule, each atom constituting the molecule is connected by a σ or π bond. In other words, the molecular orbitals of the molecules have σ molecular orbitals or π molecular orbitals, and the electrons existing in these molecular orbitals play an important role in bonding each atom. Among them, the electrons in the σ molecular orbitals strongly bond each atom, and determine the interatomic distance in the molecule that is the skeleton of the molecule. On the other hand, the electrons in the π molecular orbitals hardly contribute to the bonding of each atom, but rather are bound to the whole molecule by a very weak force.
[0019]
In many cases, when electrons in the σ molecular orbital are excited by light, the atomic spacing of the molecules changes greatly, and a large structural change including molecular dissociation occurs. As a result, most of the energy that light gives to molecules changes to thermal energy due to atomic kinetic energy and structural changes. Therefore, excitation energy is not consumed in the form of light called fluorescence. In addition, since the structure change of molecules occurs at a very high speed (shorter than picoseconds), even if fluorescence occurs in the process, the lifetime is extremely short.
[0020]
On the other hand, electrons in the π molecular orbitals do not substantially change the molecular structure even when excited, stay in the high-level quantum discrete level for a long time, and emit fluorescence in the order of nanoseconds to be de-excited. It has properties.
[0021]
According to quantum chemistry, having a π molecular orbital is equivalent to having a double bond, and it is necessary to select a molecule with abundant double bonds as the fluorescent labeler molecule to be used. It becomes. This is confirmed that even in a molecule having a double bond, the fluorescence from the S2 excited state is extremely weak in a 6-membered ring molecule such as benzene and pyrazine (for example, M. Fujii et.al.Chem.Phys. Lett. 171 (1990) 341).
[0022]
Therefore, if a molecule containing a six-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 further, the fluorescence from the molecule is excited by excitation from the S1 state to the S2 state by photoexcitation. Can be easily suppressed, so that super-resolution can be effectively utilized. In other words, if the fluorescent labeler molecules are stained and observed, 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, Since only a specific chemical tissue can be selectively stained, even a detailed chemical composition of a sample can be analyzed.
[0023]
In general, the double resonance absorption process occurs only when two light wavelengths, polarization states, and the like satisfy specific conditions. By using this, the molecular structure 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 two wavelengths of light and the orientation direction of molecules form a specific angle, the double resonance absorption process is It happens strongly. Therefore, by simultaneously irradiating the sample with two wavelengths of light and rotating the polarization direction of each light, the degree of disappearance of the fluorescence changes, so information on the spatial orientation of the tissue to be observed from that state 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 the above Japanese Patent Application Laid-Open No. 11-95120, it can be seen that in addition to super-resolution, it has a high analytical capability. Furthermore, by devising the irradiation timing of the two lights of wavelength λ1 and wavelength λ2, S / N can be improved and fluorescence suppression can be effectively caused, and super-resolution is more effectively expressed. It becomes possible to do.
[0025]
As a specific example of such super-resolution microscopy, for example, in Japanese Patent Laid-Open No. 2001-100102, light having a wavelength λ1 (especially laser light) that excites a fluorescent labeler molecule from the S0 state to the S1 state is used as pump light. As shown in FIG. 10, the light of wavelength λ2 that is excited from the S1 state to the S2 state is used as the erase light, and the pump light is emitted from the light source 81 and the erase light is emitted from the light source 82, respectively, and the pump light is reflected by the dichroic mirror 83. After that, it is condensed on the sample 115 by the annular optical system 84, and the erase light is converted into a hollow beam by the phase plate 86, and then transmitted through the dichroic mirror 83 and spatially overlapped with the pump light to be annular optical system. 84 is proposed to be condensed on the sample 115.
[0026]
According to this microscope, fluorescence other than the vicinity of the optical axis where the intensity of the erase light becomes zero is suppressed. As a result, a region narrower than the spread of the pump light (Δ <0.61 · λ1 / NA, where NA is an annular zone) 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. 11, a configuration in which a phase difference π is given at a point-symmetrical 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, the fluorescence detection type microscope including the conventionally proposed super-resolution microscopy has a problem that the fluorescence detection sensitivity is low and a good fluorescence microscope image cannot be obtained.
[0028]
That is, the conventional super-resolution microscope can perform spatial measurement with nanometer-order resolution, but on the other hand, the absolute amount of fluorescent labeler molecules present in the observation region is reduced, and in the extreme case, one molecule, that is, a single molecule A situation arises where molecules must be measured. A pulse counting method is generally used for the detection of this single molecule. This pulse counting method focused on the phenomenon of light emission depending on the fluorescence yield (φ) at the time when a molecule was excited to the first electronic excited state mainly by irradiation of pump light from a pulse light source. It is a measurement method. Here, the fluorescence yield is a process in which a molecule in an excited state is deexcited to a ground state by a fluorescence emission process. For example, the fluorescence yield is 0.9 for rhodamine 6G molecules in methanol, and most of them. In the fluorescence process, one photon is emitted and de-excited.
[0029]
In the pulse counting method, the number (N) of occurrence events of the fluorescence emission phenomenon when the pump light is irradiated for a certain time is measured. Therefore, in the case of rhodamine 6G molecule, since the fluorescence lifetime is 3 nsec, an optical pulse with a pulse width of 3 nsec is intermittently observed by the detector. Here, if there is one molecule per unit volume and the number of generated events within a unit time is no, the number of events is m · no if there are m molecules per unit volume. In this way, under the condition that there are few molecules in the measurement region and the light pulse is irradiated intermittently, the spatial distribution of molecules existing in the micro region, that is, a fluorescence microscope image can be obtained by measuring the number of occurrence events. .
[0030]
However, in super-resolution microscopy, erase light is simultaneously irradiated in addition to pump light, and the intensity of this erase light, in other words, the number of photons is much larger than that of fluorescent photons emitted from molecules. There is a problem that the S / N at the time of measuring the number of occurrences of the light emission phenomenon is lowered. For this reason, conventionally, a filter or the like is arranged in the optical path of the detection optical system to remove the photons of the erase light. However, even if such measures are taken, a phenomenon in which a fluorescence process occurs in the detector The photons of erase light are incident at a frequency that cannot be ignored compared to the number, resulting in a fluorescence image that does not correspond to the spatial distribution of the fluorescent molecules to be detected, and a good fluorescence microscope image cannot be obtained. .
[0031]
Accordingly, an object of the present invention made in view of the above point is that a microscope configured appropriately so that a S / N can be improved by a pulse counting method, and a desired microscopic light emitting molecule can be reliably detected and a good microscopic image can be obtained. Is to provide.
[0032]
[Means for Solving the Problems]
  The invention of a microscope according to claim 1 that achieves the above object is provided for exciting molecules in a sample from a ground state to an excited state.pulseA pulsed light source that generates excitation light;
  the abovepulseA condensing optical system for condensing the excitation light on the sample;
  the abovepulseFirst conversion means for receiving excitation light and converting it into an electrical pulse signal;
  the abovepulseSecond conversion means for receiving light emitted when molecules in the sample excited by the excitation light are deexcited and converting them into electric pulse signals;
  Phase of electric pulse signal from the first conversion meansWhenElectric pulse signal from the second conversion meansSo that the phase of the electric pulse signal from the first conversion means is the same asAdjusting means for adjusting;
  the aboveBy adjusting meansPhaseAdjusted electrical pulse signalWhenElectric pulse signal from the second conversion meansOutput of logical operation withMeasuring means for measuring
  And a microscope image of the sample is obtained based on the output of the measuring means.
[0033]
  Furthermore, the invention of the microscope 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 pulsed first light that causes the molecule to transition from a ground state to a first electronically excited state;
  A second light source that generates a second light that causes the molecule to transition from a 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 the irradiation region of the first light and the second light,
  First conversion means for receiving the first light and converting it into an electrical pulse signal;
  Second conversion means for receiving light emitted when the molecule is deexcited from the first electronically excited state to the ground state and converting it into an electric pulse signal;
  Phase of electric pulse signal from the first conversion meansWhenElectric pulse signal from the second conversion meansSo that the phase of the electric pulse signal from the first conversion means is the same asAdjusting means for adjusting;
  the aboveBy adjusting meansPhaseAdjusted electrical pulse signalWhenElectric pulse signal from the second conversion meansOutput of logical operation withMeasuring means for measuring
  And a microscope image of the sample is obtained based on the output of the measuring means.
[0034]
  According to a third aspect of the present invention, in the microscope according to the first or second aspect, the measuring means is a logical product of the electric pulse signal adjusted by the adjusting means and the electric pulse signal from the second converting means. And a pulse counter that counts the output pulses of the logic operator.
According to a fourth aspect of the present invention, in the microscope according to the first aspect, the measurement means is configured such that the second conversion means is based on de-excitation of the molecule with reference to the light emission time of the light source at the exit of the pulse light source. The phase is adjusted by delaying the propagation time until light emission is received and the electric pulse signal from the first conversion means.
According to a fifth aspect of the present invention, in the microscope according to the second aspect, the measurement unit is configured such that the second conversion unit is configured to remove the molecule on the basis of the light emission time of the light source at the exit of the first light source. It is characterized in that the phase is adjusted by delaying the propagation time until light emission by excitation is received and the electric pulse signal from the first conversion means.
[0035]
The principle of the present invention will be described below with reference to FIG.
[0036]
FIG. 1 is a time chart showing the operation when the erase light is a CW light source in the super-resolution fluorescence microscope. FIG. 1A shows the oscillation timing of the pump light laser pulse, and FIG. The fluorescence emission timing of the fluorescent labeler molecule is shown, FIG. 1 (c) shows the fluorescence output signal of the detector that receives the fluorescence, and FIG. 1 (d) shows the timing of the detection gate pulse.
[0037]
As shown in FIG. 1 (a), if the pulse width of the pump laser pulse, that is, the emission time is t and the emission time interval is T, the fluorescent labeler molecule is shown in FIG. 1 (b). The light is emitted at a time interval T in synchronization with the pump light laser, delayed by a propagation time d1 until the light is collected on the sample by the microscope optical system with reference to the laser emission time at the emission port. However, since the fluorescence lifetime of the fluorescent labeler molecule generally has a finite value τ (several nanoseconds), the emission time of the fluorescent labeler molecule coincides with t when the emission time t of the pump laser is longer than τ. , T is shorter than τ.
[0038]
As shown in FIG. 1 (c), the fluorescence from the fluorescent labeler molecule reaches the detector with a further delay of propagation time d2 by the detection optical system, so that it is eventually delayed by (d1 + d2) time from the exit of the pump light laser. In this state, light is received in synchronization with the pump light laser pulse. Here, a detector that receives fluorescence generally has a unique response time r. Therefore, if r is larger than the fluorescence emission time, the pulse width of the output fluorescence output signal is r. It becomes the light emission time itself.
[0039]
In the pulse counting method, the number of pulses of the fluorescence output signal within a unit time in FIG. 1C is measured. In the super-resolution fluorescence microscope, strong erase light is emitted in addition to the pump light. Light enters the detector and causes noise. For example, when the erase light is a CW light source, a pulse-like pseudo signal as indicated by the oblique lines in FIG. 1C is output from the detector. In addition, most of the pseudo signals are light emission in which pump light is not generated because the light emission time interval T of the pump light laser pulse is overwhelmingly longer than the light emission time t and the fluorescence light emission time of the fluorescent labeler molecule. Occurs during time interval T. For this reason, this pseudo signal is counted, and the measurement accuracy, that is, the S / N is deteriorated. Further, even if no erase light is generated, a dark current signal is statistically generated, and similarly the measurement accuracy is deteriorated.
[0040]
Therefore, in the present invention, as shown in FIG. 1 (d), the phase of the fluorescence output signal completely coincides with the pump light laser pulse shown in FIG. 1 (a) by the first converting means and adjusting means. A detection gate pulse is generated, and in the measuring means, the correlation between the detection gate pulse and the fluorescence detection signal, for example, a logical product is taken, and the output pulse, that is, the fluorescence detection signal when the detection gate pulse is at a high level. Measure the pulse. In this way, it is possible to measure only the pulses caused by the pump light irradiation with good S / N by excluding the pseudo signal pulse due to the erase light irradiation from the pulse train of the fluorescence detection signal. Even when only one molecule exists spatially, a fluorescence detection signal resulting from irradiation of pump light from that molecule can be reliably detected, and a good fluorescence microscope image can be obtained.
[0041]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, an embodiment of a microscope according to the present invention will be described with reference to the drawings with reference to FIGS.
[0042]
FIG. 2 is a diagram showing the configuration of the microscope, and FIG. 3 is a block diagram showing an example of the configuration of the signal processing circuit. The microscope of the present embodiment is a laser scanning super-resolution fluorescence microscope in which erase light is converted into a hollow beam to develop super-resolution to improve spatial resolution, and a biological sample stained with rhodamine 6G is observed. To do.
[0043]
Here, rhodamine 6G has a maximum absorption in the wavelength band of 530 nm, and an absorption band that can be excited from the first electronic excited state (S1) to a higher electronically excited state has a wavelength of 600 to 650 nm. Exists in the area.
[0044]
Therefore, in this embodiment, a laser diode (LD) excitation type mode-locked Nd: YAG laser 1 is used as the first light source, and its double harmonic (532 nm) is used as pump light. This LD-pumped mode-locked Nd: YAG laser 1 can oscillate picosecond laser pulses at a repetition rate on the order of MHz by adjusting design parameters of the laser resonator. For example, the GE-100 series manufactured by Time-Bandwidth, Switzerland, is a standard specification and can oscillate pulsed light having a pulse width of 6 psec with an average output of 50 mW at a repetition frequency of 100 MHz. This corresponds to 500 pJ of energy of one pulse. Further, by changing the design of the resonator, the repetition frequency can be adjusted between 25 MHz and 1 GHz.
[0045]
The second light source uses a commercially available, inexpensive and highly reliable CW He-Ne laser 2 and its fundamental wave (633 nm) is used as erase light.
[0046]
In FIG. 2, the pump light from the LD-pumped mode-locked Nd: YAG laser 1 is split into two by a beam sampler 3, one of which is received by a Pin-photodiode 4, and the other is a cuvette-type half. The light is incident on a beam combiner 5 made of a mirror. Further, the erase light from the CW He-Ne laser 2 passes through the phase plate 6 as shown in FIG. 11 to form a hollow beam, is incident on the beam combiner 5, and is synthesized coaxially with the pump light. The pump light and the erase light are transmitted through a beam separator 7 composed of a cuvette type half mirror, and then sequentially reflected by galvanometer mirrors 8 and 9 that can swing around axes orthogonal to each other. The sample is condensed on the surface of the biological sample 11 stained with 6G, and the condensing point is moved by swinging the galvanometer mirrors 8 and 9 to perform two-dimensional scanning on the sample surface. In FIG. 2, in order to simplify the drawing, the galvanometer mirrors 8 and 9 are shown so as to swing around axes parallel to each other. The phase plate 6 is coated with a vapor deposition film so as to give the phase distribution as shown in FIG. 11 to the erase light. As a result, the light intensity is canceled on the optical axis and shaped into a hollow beam.
[0047]
On the other hand, the fluorescence emitted from the sample 11 by the irradiation of the pump light and the erase light is collimated by the objective lens 10, followed by a path opposite to the incident optical path, and incident on the beam separator 7 through the galvanometer mirrors 9 and 8. The fluorescence reflected by the beam separator 7 is condensed on the pinhole 13 by the projection lens 12.
[0048]
The pinhole 13 is disposed at a confocal position with respect to the fluorescence emission point of the sample 11, and the fluorescence transmitted through the pinhole 13 is transmitted through the pump light cut notch filter 14 and the erase light cut notch filter 15, respectively. After removing the pump light and erase light, the photomultiplier 16 receives the light and obtains a fluorescence output signal.
[0049]
Next, the signal processing circuit shown in FIG. 3 will be described. An output electric signal from the Pin-photodiode 4 that receives the pump light from the Nd: YAG laser 1 branched by the beam sampler 3 is supplied to a discriminator 21 to be a square electric pulse signal, for example, a TTL clock. Convert to signal. The discriminator 21 not only squarely shapes the response signal of the pulsed light, but also converts only an optical pulse having an intensity equal to or higher than a certain peak height threshold. Remove the pseudo electrical response caused by the cause. The Pin-photodiode 4 and the discriminator 21 constitute first conversion means.
[0050]
The clock signal output from the discriminator 21 is supplied to the gate generator 23 through the delay circuit 22, and the appropriate pulse width and peak value shown in FIG. 1D are synchronized with the input clock signal in the gate generator 23. The detection gate pulse is generated, and this detection gate pulse is supplied to one input terminal of the logical operation unit 24. Here, the delay circuit 22 and the gate generator 23 constitute adjustment means.
[0051]
The fluorescence output signal from the photomultiplier tube 16 is converted into a square electric pulse signal, for example, a TTL clock signal by the discriminator 25 and supplied to the other input terminal of the logic unit 24. Here, the photomultiplier tube 16 and the discriminator 25 constitute a second conversion means.
[0052]
The logical operation unit 24 calculates a logical product (AND). In this embodiment, the detection gate pulse from the gate generator 23 and the clock signal from the discriminator 25 are both positive logic, that is, both pulses are When input simultaneously, one pulse is output. The timing of the detection gate pulse with respect to the clock signal from the discriminator 25 is adjusted by the delay circuit 22, and the pulse width is adjusted by the gate generator 23.
[0053]
The output of the logic unit 24 is supplied to a pulse counter 26, where the number of pulses input per unit time is counted, and the data is stored in the frame memory of the personal computer 27. Here, the logic unit 24 and the pulse counter 26 constitute a measuring means. In this manner, the data of each pixel is stored in the frame memory while the galvanometer mirrors 8 and 9 are swung in synchronization with the video rate, and the fluorescence two-dimensional image of the sample 11 is displayed on the monitor 28 such as a CRT in real time. indicate.
[0054]
According to the present embodiment, among the pulses corresponding to the fluorescence output signal from the photomultiplier tube 16, the pulse having no causal relationship with the pump light pulse is removed by the logic unit 24, and the observation sample 11 is detected by the pump light pulse. Since only the pulse corresponding to the fluorescence signal at the time of excitation is pulse-counted, a weak fluorescence signal can be imaged with an extremely high S / N.
[0055]
In addition, this invention is not limited only to the said embodiment, Many deformation | transformation or a change is possible. For example, the present invention is not limited to the super-resolution fluorescence microscope using the pump light pulse and the erase light as in the above embodiment, and the molecule in the sample is excited from the ground state to the excited state by the pulse light source, and the molecule The present invention can also be effectively applied to a microscope that detects light emission when is deexcited.
[0056]
【The invention's effect】
  As described above, the present inventionAccording to the pulseThe S / N ratio by the counting method can be improved, and a desired microscopic light emitting molecule can be reliably detected and a good microscopic image can be obtained.
[Brief description of the drawings]
FIG. 1 is a diagram for explaining the principle of the present invention.
FIG. 2 is a diagram showing a configuration of a microscope according to an embodiment of the present invention.
3 is a block diagram showing a configuration of an example of a signal processing circuit of the microscope shown in FIG.
FIG. 4 is a conceptual diagram showing an electronic structure of a valence electron orbit of a molecule constituting a sample.
5 is a conceptual diagram showing a first electronic excited state of the molecule of FIG.
FIG. 6 is also a conceptual diagram showing a second electron excited state.
FIG. 7 is also a conceptual diagram showing a state in which the second electron excited state returns to the ground state.
FIG. 8 is a conceptual diagram for explaining a double resonance absorption process in a molecule.
FIG. 9 is also a conceptual diagram for explaining a double resonance absorption process.
FIG. 10 is a diagram illustrating a configuration of an example of a conventional super-resolution microscope.
11 is a plan view showing the configuration of the phase plate shown in FIG.
[Explanation of symbols]
1 Nd: YAG laser
2 He-Ne laser
3 Beam sampler
4 Pin-photodiode
5 Beam combiner
6 Phase plate
7 Beam separator
8,9 Galvano mirror
10 Objective lens
11 samples
12 Projection lens
13 pinhole
14 Pump light cut notch filter
15 Erase light cut notch filter
16 photomultiplier tubes
21, 25 Discriminator
22 Delay circuit
23 Gate generator
24 logic unit
26 Pulse counter
27 Personal computer
28 Monitor

Claims (5)

A pulsed light source that generates pulsed excitation light for exciting molecules in the sample from the ground state to the excited state;
A condensing optical system for condensing the pulsed excitation light on the sample;
First conversion means for receiving the pulse excitation light and converting it into an electric pulse signal;
Second conversion means for receiving light emitted when molecules in the sample excited by the pulse excitation light are deexcited and converting them into electric pulse signals;
As the phase of the electric pulse signal from the phase and the second converting means of the electric pulse signal from said first converting means coincide, to adjust the phase of the electric pulse signal from said first conversion means Adjusting means;
Measuring means for measuring the output of the logical operation of the electric pulse signal from the electrical pulse signal and the second conversion means whose phase is adjusted by the adjustment means,
And a microscope configured to obtain a microscope image of the sample based on the output of the measuring means. 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 pulsed first light that causes the molecule to transition from a ground state to a first electronically excited state;
A second light source that generates a second light that causes the molecule to transition from a 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 the irradiation region of the first light and the second light,
First conversion means for receiving the first light and converting it into an electrical pulse signal;
Second conversion means for receiving light emitted when the molecule is deexcited from the first electronically excited state to the ground state and converting it into an electric pulse signal;
As the phase of the electric pulse signal from the phase and the second converting means of the electric pulse signal from said first converting means coincide, to adjust the phase of the electric pulse signal from said first conversion means Adjusting means;
Measuring means for measuring the output of the logical operation of the electric pulse signal from the electrical pulse signal and the second conversion means whose phase is adjusted by the adjustment means,
And a microscope configured to obtain a microscope image of the sample based on the output of the measuring means.   The measuring means includes a logical operator that calculates a logical product of the electric pulse signal adjusted by the adjusting means and the electric pulse signal from the second converting means, and a pulse that counts output pulses of the logical operator. The microscope according to claim 1, further comprising a counter. The measurement means is based on the light emission time of the light source at the emission port of the pulse light source, the propagation time until the second conversion means receives light emission due to deexcitation of the molecule, and the time from the first conversion means. The microscope according to claim 1, wherein the phase is adjusted by delaying the electric pulse signal. The measurement means is based on the light emission time of the light source at the exit of the first light source, the propagation time until the second conversion means receives light emitted by deexcitation of the molecule, and the first conversion means. The microscope according to claim 2, wherein the phase is adjusted by delaying an electric pulse signal from.

Images