WO2006061947A1 - 蛍光顕微鏡及び観察方法 - Google Patents
蛍光顕微鏡及び観察方法 Download PDFInfo
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- 238000000034 method Methods 0.000 title claims description 45
- 230000001678 irradiating effect Effects 0.000 claims abstract description 12
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Classifications
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- 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
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- 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
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- 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"
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/069—Supply of sources
- G01N2201/0696—Pulsed
- G01N2201/0697—Pulsed lasers
Definitions
- the present invention relates to a fluorescence microscope and an observation method, and in particular, irradiates a sample with excitation light.
- the present invention relates to a fluorescence microscope and an observation method for performing observation by detecting fluorescence from a sample.
- a fluorescence microscope is widely used as a useful tool for medical and biological research.
- excitation light from a light source is irradiated onto a fluorescent material in a sample to generate fluorescence from the fluorescent material.
- the fluorescence image of the sample is observed by detecting this fluorescence separately from the excitation light.
- the localization can be observed by using a fluorescent substance as a fluorescent probe and labeling various cells and proteins.
- a laser scanning fluorescence microscope is used (for example, see Patent Document 1 and Patent Document 2).
- a sample is scanned with a laser beam to capture a fluorescent image.
- the spatial resolution is limited by the diffraction limit. Therefore, the conventional microscope has a problem that the spatial resolution cannot be improved beyond a certain level if the wavelength and numerical aperture of light are determined.
- Patent Document 1 Japanese Patent Laid-Open No. 2003-057554
- Patent Document 2 Japanese Unexamined Patent Publication No. 2003-344776
- the conventional microscope has a problem in that the spatial resolution cannot be improved.
- the present invention has been made in view of the above-described problems, and an object thereof is to provide a fluorescence microscope and an observation method that can improve spatial resolution.
- the fluorescence microscope according to the first aspect of the present invention is a laser that emits laser light serving as excitation light.
- One the light source for example, the light source 10 according to the embodiment of the present invention
- an objective lens for condensing the laser light and irradiating the sample for example, the objective lens 13 according to the embodiment of the present invention
- a detector that detects fluorescence generated in the sample by the laser light for example, the detector 22 that applies force to the embodiment of the present invention
- a stroking means for example, a stage 15 which is useful in the embodiment of the present invention for stroking, and when the intensity of the laser light is maximum, saturation of fluorescence occurs, thereby increasing the intensity of the laser light.
- the sample is irradiated with the laser light with the intensity of the laser light changed so that the relationship with the fluorescence intensity becomes non-linear, and the fluorescence corresponding to the intensity of the laser light is detected by the detector. Observation is performed based on the fluorescence saturation component. As a result, the spatial resolution can be improved.
- the fluorescence microscope according to the second aspect of the present invention is the above-described fluorescence microscope, wherein the modulator that modulates the intensity of the laser light to change with time (for example, implementation of the present invention). Further comprising a modulator 16) which is powerful in form, and the sample is irradiated with the intensity of the fluorescence in a non-linear region at the time when the laser beam reaches a peak, scanned while being modulated by the modulator, and generated by the sample.
- the detected fluorescence is detected by the detector, and from the fluorescence detected by the detector, a harmonic component with respect to the frequency at the modulator is extracted and observed. Thereby, the spatial resolution can be improved with a simple configuration.
- the fluorescence microscope according to the third aspect of the present invention is the above-described fluorescence microscope in which the harmonic component is extracted by a lock-in amplifier. This makes it possible to detect with high sensitivity.
- a fluorescent microscope according to the fourth aspect of the present invention is the above-described fluorescent microscope, in which a laser light source is used as the laser light source. This can prevent sample damage and fluorescence fading.
- the fluorescence microscope according to the fifth aspect of the present invention is the above-described fluorescence microscope, wherein the intensity of the laser light is the first intensity at which the fluorescence becomes the nonlinear region, and the first The sample is irradiated with at least two intensities of a second intensity different from the intensity, and scanning is performed in a state where the sample is irradiated with the laser light at the first intensity and the second intensity, respectively. , The intensity of the fluorescence at the first intensity and the intensity of the fluorescence at the second intensity Based on the above, the saturation component of the fluorescence from the sample is calculated. As a result, the spatial resolution can be improved with a simple configuration.
- the fluorescence microscope according to the sixth aspect of the present invention is a multi-photon excitation light source in which the laser light source generates multi-photon excitation in the above-described fluorescence microscope. Thereby, detection with higher sensitivity can be performed.
- the fluorescence microscope according to the seventh aspect of the present invention is the above-described fluorescence microscope, further comprising separation means for separating fluorescence from the laser beam based on a difference in wavelength, and separated by the separation means.
- the detected fluorescence is detected by the detector. As a result, the spatial resolution can be improved.
- a fluorescence microscope according to the eighth aspect of the present invention is the above-described fluorescence microscope, further comprising a focal position changing unit that changes a focal position of the object lens along an optical axis direction. . From this, three-dimensional observation can be performed.
- the fluorescence microscope according to the ninth aspect of the present invention is the above-described fluorescence microscope, wherein the fluorescence generated by irradiating the sample with the laser light is detected by the detector via a confocal optical system. Is. As a result, the spatial resolution can be further improved.
- An observation method emphasizing the tenth aspect of the present invention is an observation method in which a sample is irradiated with laser light that is excitation light, and fluorescence from the sample is detected to observe the sample.
- the intensity of the laser beam is maximum, the intensity of the laser beam is changed so that the relationship between the laser beam intensity and the fluorescence intensity becomes nonlinear due to the saturation of the fluorescence.
- the laser beam with the changed intensity is condensed, irradiated to the sample, scanned so as to change the relative position between the sample and the laser beam, and the fluorescence generated in the sample by the laser beam is laser-induced. Fluorescence separated from light and separated from the laser light is detected, and observation is performed based on a saturation component of the fluorescence from the detected fluorescence. Thereby, the spatial resolution can be improved.
- the laser light is modulated such that the intensity changes according to time, and the intensity of the laser light is changed. Fluorescence becomes the non-linear region at the time when the modulated laser beam peaks.
- scanning is performed so as to change the relative position of the sample and the laser light in a state where the laser light is condensed and irradiated on the sample and the laser light is modulated. Thereby, the spatial resolution can be improved by a simple method.
- An observation method is an observation method in which, in the above-described observation method, a harmonic component with respect to a modulation frequency is extracted from the detected fluorescence. Thereby, spatial resolution can be improved easily.
- An observation method is the observation method described above, wherein the laser beam is a pulse laser beam and the pulse laser beam is intensity-modulated. Thereby, detection with higher sensitivity can be performed.
- the observation method according to the fourteenth aspect of the present invention is the observation method described above, wherein at least a first intensity at which fluorescence is in the nonlinear region and a second intensity different from the first intensity.
- the intensity of the laser beam is changed so that the sample is irradiated with two intensities, scanning is performed for each of the first intensity and the second intensity, and the first intensity
- the saturation component of the fluorescence is calculated based on the fluorescence intensity at the second intensity and the fluorescence intensity at the second intensity.
- An observation method according to the fifteenth aspect of the present invention is characterized in that in the above-described observation method, fluorescence is detected by a multiphoton excitation method. Thereby, detection with higher sensitivity can be performed.
- An observation method according to the sixteenth aspect of the present invention is the observation method described above, wherein the sample is labeled with quantum dots. Thereby, a low intensity laser beam can be used.
- the observation method according to the seventeenth aspect of the present invention is the above-described observation method in which the fluorescence is detected by changing the focal position of the laser light in the sample along the optical axis direction. It is. Thereby, the spatial resolution can be improved with a simple configuration.
- An observation method according to an eighteenth aspect of the present invention is the above-described observation method, wherein the fluorescence generated by irradiating the sample with the laser light is detected via a confocal optical system. . Thereby, the spatial resolution can be further improved.
- An observation method according to the nineteenth aspect of the present invention includes a laser light source that emits laser light serving as excitation light, an objective lens that collects the laser light and irradiates the sample, and the laser light.
- a detector for detecting fluorescence generated in the sample and a stirrer for performing scribing by changing a relative position between the laser beam and the sample; and when the intensity of the laser beam is maximum, The sample is irradiated with the laser light with varying intensity so that saturation occurs, the fluorescence corresponding to the intensity of the laser light is detected by the detector, and observation is performed based on the saturation component of the fluorescence. It is. Thereby, spatial resolution can be improved.
- Fig. 1 is a diagram showing a configuration of a fluorescence microscope which is effective in the present invention.
- FIG. 2 is a diagram schematically showing a spatial distribution of laser light as excitation light and fluorescence generated by the excitation light in the present invention.
- FIG. 3A is a diagram schematically showing excitation light modulated by the fluorescence microscope according to the first embodiment of the present invention and changes in fluorescence intensity generated by the excitation light.
- FIG. 3B is a diagram schematically showing excitation light modulated by the fluorescence microscope according to the first embodiment of the present invention and changes in the intensity of fluorescence generated by the excitation light.
- FIG. 4 is a diagram schematically showing the relationship between excitation light intensity and fluorescence intensity.
- FIG. 5 is a diagram schematically showing an amplitude spectrum with respect to an angular frequency of fluorescence intensity.
- FIG. 6A is a diagram schematically showing the spatial distribution of fluorescence.
- FIG. 6B is a diagram schematically showing the spatial distribution of the primary frequency component of fluorescence.
- FIG. 6C is a diagram schematically showing a spatial distribution of second-order frequency components of fluorescence.
- FIG. 7 is a diagram schematically showing an optical transfer function when a high-order frequency component is detected. Explanation of symbols
- the fluorescence microscope according to the first embodiment of the present invention is a confocal microscope, and is a laser scanning microscope.
- the spatial resolution is improved by utilizing the saturation of fluorescence.
- the spatial resolution is improved by observing the saturated component of fluorescence.
- FIG. 1 is a diagram schematically showing a configuration of a fluorescence microscope according to the present invention. 10 is a light source, 11 is a lens, 12 is a dichroic mirror, 13 is an objective lens, 14 is a sample, 15 is a stage, 16 is a modulator, 21 is a pinhole, 22 is a detector, 23 is a lock-in amplifier, 24 Is a processing device.
- the light source 10 is a laser light source that continuously oscillates excitation light.
- a semiconductor ion laser having a wavelength in the visible region or visible region can be used.
- the wavelength of the light source 10 is a wavelength necessary for excitation of the fluorescent material.
- the laser beam as the excitation light is intensity-modulated by the modulator 16 and becomes a periodic function of the frequency f.
- the modulator 16 an electro-optic modulator, an acousto-optic modulator, or the like can be used.
- the modulated excitation light is refracted by the lens 11 and enters the dichroic mirror 12.
- the dichroic mirror 12 reflects only light of a specific wavelength range and transmits light of other wavelengths.
- the light having the wavelength of the laser beam is transmitted, and the light having the wavelength of the fluorescence from the sample 14 is reflected.
- excitation light and fluorescence are separated based on the difference in wavelength.
- fluorescent light has a wavelength longer than that of excitation light due to a stochastic shift. Therefore, excitation light and fluorescence can be efficiently separated by using a dichroic mirror 12.
- the fluorescence and the excitation light may be separated by using a light separation means other than the Dyke mouth mirror. For example, by using a combination of a filter and a beam splitter, it is possible to separate the fluorescence from the excitation light.
- the excitation light transmitted through the dichroic mirror 12 is incident on the objective lens 13.
- the objective lens 13 collects the excitation light so as to form an image on or in the sample and makes it incident on the sample 14.
- a biological sample stained by an immunostaining method can be used.
- fluorescence is generated based on the excitation light.
- the intensity of the fluorescence is based on the intensity of the excitation light.
- the fluorescent light is refracted by the objective lens 13 and enters the dike mouth mirror 12. Since the dichroic mirror 12 reflects light according to the wavelength as described above, it reflects the laser light reflected by the sample 14 and reflects only fluorescence. Thereby, the excitation light reflected from the sample 14 and the fluorescence can be separated.
- the fluorescence reflected by the dichroic mirror 12 passes through the pinhole 21 and enters the detector 22.
- the fluorescence is refracted by the objective lens 13 so as to form an image at the pinhole 21.
- the fluorescence incident on the detector 22 is based on the modulated excitation light.
- Detector 2 Reference numeral 2 denotes a point sensor such as a photomultiplier tube.
- This detector 22 outputs a detection signal corresponding to the intensity of the received light to the lock-in amplifier 23.
- the lock-in amplifier 23 locks in a predetermined repetition frequency, and detects the lock-in of the signal from the detector 22.
- the reference signal from the modulator 16 is input to the lock-in amplifier 23, and a signal is detected at a frequency n times (n is an integer of 2 or more) the modulation frequency f at the modulator 16.
- n is an integer of 2 or more
- the modulation frequency is 100 kHz
- detection is performed at a repetition frequency of 200 kHz and 300 kHz '''.
- higher-order frequency components can be extracted and detected.
- the stage 15 is an XYZ stage provided with a drive mechanism and can move in a three-dimensional direction. That is, the stage 15 is provided so as to be movable in the horizontal direction (XY direction) and the vertical direction (Z direction). While moving this stage 15, fluorescence is detected. For example, the stage 15 is moved at a constant speed while irradiating the sample 14 with laser light, and the relative position between the sample 14 and the excitation light is changed. Then, the entire surface of the sample 14 is scanned.
- the processing device 24 is a processing device such as a personal computer and controls the movement of the stage 15. For example, the stage 15 is moved in the X direction, and the sample 14 is scanned from one end to the other end.
- the stage 15 moves in the Z direction, and scan in the XY direction as well. That is, the stage 15 is moved in the Z direction, and the focal position is shifted in the optical axis direction.
- the focal position in the sample moves along the optical axis direction, and three-dimensional observation of the sample becomes possible.
- the focal position can be changed along the optical axis.
- the XY direction and the Z direction are repeated, and the whole or a part of the sample 14 is scanned three-dimensionally.
- the scanning in the XY directions is not limited to driving the stage 15 but may be a beam deflecting device such as a galvanometer mirror or an acoustooptic device.
- the processing device 24 controls the stage 15 and the lock-in amplifier 23 to perform lock-in detection while the sample 14 is being scanned. Further, the processing device 24 forms a fluorescent image based on the signal output from the lock-in amplifier. That is, while sample 14 is being scanned A fluorescence image is formed based on the detected signal. By performing a predetermined operation with the processing device 24, a fluorescent image can be displayed on the screen, and fluorescent image data can be stored. As a result, images can be observed and imaged.
- the fluorescence microscope which is effective in the present embodiment constitutes a confocal microscope. That is, the light source 10 which is a point light source and the sample 14 are arranged so as to have a conjugate imaging relationship, and the sample 14 and the pinhole 21 are arranged so as to have a conjugate imaging relationship. Thereby, fluorescence can be detected through the confocal optical system. Therefore, the spatial resolution can be improved.
- FIG. 2 shows the spatial distribution of the intensity of laser light (excitation light) and fluorescence.
- the horizontal axis indicates the position, and the vertical axis indicates the light intensity.
- 3A and 3B are diagrams showing the intensity of the modulated laser beam (excitation light) and the temporal change in fluorescence.
- the horizontal axis indicates time, and the vertical axis indicates light intensity.
- the solid line indicates the excitation light intensity, and the broken line indicates the fluorescence intensity.
- Figure 4 shows the relationship between excitation light intensity and fluorescence intensity.
- the horizontal axis indicates the excitation light intensity
- the vertical axis indicates the fluorescence intensity.
- the excitation light since the excitation light is modulated, the excitation light changes according to the cosine function at any position.
- fluorescence saturation means that the relationship between the intensity of excitation light and the intensity of fluorescence deviates from a linear relationship. That is, fluorescence saturation occurs when the excitation light intensity is such that the broken line and the solid line in FIG. In other words, the saturation region where fluorescence saturation occurs is a non-linear region where the relationship between the intensity of the excitation light and the intensity of the laser light is non-linear.
- the fluorescence intensity and the excitation light intensity are proportional to each other, and they are shown in the same manner in FIG. 3B. .
- the fluorescence intensity is proportional to l + cos (co t).
- the second, third, ... harmonic components appear in the fluorescence intensity.
- the fluorescence intensity in the absence of saturation is generally proportional to the quantum yield, the detection sensitivity of the detection system, the absorption cross section, and the excitation light intensity. Therefore, the proportionality constant based on the quantum yield and the detection sensitivity of the detection system is A, the absorption cross section is ⁇ , the spontaneous emission coefficient is C, and the excitation light intensity is B.
- I is expressed by a power series. (L + cos (co t)) 2 has a cos (2 co t) term, (f
- 1 + cos ( ⁇ t)) 3 shows the term cos (3 ⁇ t). Accordingly, the term of (1 + cos ( ⁇ t)) n , that is, a harmonic component of cos (n co t) appears in the fluorescence. Therefore, it can be seen that higher-order modulation frequency components are obtained in proportion to the power of the amplitude B of the excitation light intensity.
- FIG. 5 is a diagram schematically showing an amplitude spectrum with respect to the angular frequency ⁇ of the fluorescence. Since fluorescence is a periodic function as shown in Equation 3, the spectrum is a line spectrum having a peak at a predetermined angular frequency.
- peaks appear at the positions of ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ .
- the height of the peak at the position of ⁇ ⁇ is proportional to ⁇ ⁇ .
- the peak position of 2 omega is proportional to the beta 2. The peak decreases as ⁇ increases. That is, the highest peak is at ⁇ , the second highest peak is at 2 ⁇ , the third highest, and the peak is at 3 ⁇ .
- FIG. 6A is a diagram showing the spatial distribution of fluorescence, which is the same as the spatial distribution shown by the broken line in FIG.
- FIG. 6B shows the first order ( ⁇ ) frequency component of fluorescence
- FIG. 6C shows the second order (2 ⁇ ) harmonic component of fluorescence.
- the horizontal axis indicates the position
- the vertical axis indicates the fluorescence intensity.
- the vertical axis is shown in different scales in each figure.
- the spatial distribution in Fig. 6 ⁇ is proportional to ⁇ because it is a first-order frequency component. Moreover proportional to beta 2 for the spatial distribution is second-order harmonic component illustrated in FIG. 6C. Therefore, as shown in Fig. 6 (b) and Fig. 6C, the half-width of the peak in the spatial distribution of the second harmonic component is narrower than the first frequency component. Similarly, ⁇ -order high-frequency components are proportional to ⁇ ⁇ , so the higher-order harmonic components have a narrower peak half-value width. That is, the higher order harmonic components, the narrower the peak and the steeper the peak.
- a substantial fluorescent spot can be obtained as a power of a point spread function, and spatial resolution can be improved. Spatial resolution can be improved in proportion to the order of the harmonic components to be detected.
- the The sum of the first-order frequency component and all harmonic components is shown in Fig. 6A.
- the spatial distribution shown in Fig. 6C is based on the fluorescence saturation component. That is, the spatial distribution of harmonic components changes based on the fluorescence saturation component. That is, the intensity of the harmonic component changes according to the intensity of the fluorescence saturation component.
- FIG. 7 shows an example of the optical transfer function when high-order frequency components are detected.
- the higher the order the better the spatial resolution.
- the spatial resolution can be further improved by combining with the confocal detection method.
- this embodiment can improve the spatial resolution in all three-dimensional directions. Note that the confocal detection method or the two-photon fluorescence without using the detection method of the present invention is substantially the same as the optical transfer function of 2 ⁇ .
- the second-order harmonic component by detecting the second-order harmonic component, it is possible to detect the second-order harmonic component with a spatial resolution twice that of the case where the first-order frequency component is detected.
- the spatial resolution can be doubled or tripled. Therefore, it becomes possible to detect with a spatial resolution exceeding the diffraction limit, and it is possible to detect with a higher resolution than a conventional fluorescence microscope.
- the spatial resolution can be increased by a factor of ⁇ by detecting the ⁇ -order harmonic component.
- spatial resolution can be improved by extracting and detecting harmonic components of the ⁇ order ( ⁇ is a natural number of 2 or more). Therefore, lock-in detection is performed with the lock-in amplifier 23 fixed at a predetermined frequency. At this time, the detection frequency of the lock-in amplifier 23 is ⁇ times the modulation frequency ( ⁇ is a natural number of 2 or more). In addition, detection is performed by locking in the excitation light at a peak phase. As a result, highly sensitive detection can be performed, and higher-order harmonic components can be detected. Therefore, the spatial resolution can be further improved. Thus, since it is an integral multiple of the modulation frequency of the excitation light, it is possible to extract harmonic components with very high sensitivity by electrically filtering the frequency band.
- the modulation frequency in the modulator 16 is sufficiently faster than the scanning of the sample 14. That is, the modulation frequency is increased so that a plurality of peaks are included in the time for scanning one pixel of the fluorescent image. For example, if the scanning time corresponding to one pixel of the fluorescent image is lmsec, the modulation frequency is 100 kHz. In this case, the peak power of the excitation light appears within the time required to scan one pixel. By making the modulation frequency faster than the sample, the number of occurrences of absorption saturation can be increased within the time it takes to scan one pixel, so that accurate detection can be achieved.
- the greater the degree of saturation of fluorescence the easier it is to detect saturation and the resolution can be improved.
- the intensity (density) of the laser beam is increased to increase the excitation light intensity, the sample may be damaged and the fluorescent light may be faded.
- a pulse laser light source is preferably used as the light source.
- the cosine function shown by the dotted line in FIG. 3 becomes an envelope, and the excitation light has a nore intensity corresponding to the envelope.
- each frequency is set to include multiple pulses during one modulation period.
- the modulation frequency is 100 kHz
- a repetition frequency of 80 MHz can be used.
- 800 pulses are included in one cycle. In this way, by making the repetition frequency of the pulse laser light higher than the modulation frequency, accurate detection can be performed.
- Quantum dots have a large absorption cross section that is strong in amber. Therefore, it is possible to easily prevent damage to the sample and fading of fluorescence while realizing light intensity that reaches absorption saturation.
- the function for modulating the laser beam may be a function other than the cosine function as long as it is a periodic function.
- the intensity of the laser beam is set to an intensity at which fluorescence saturation occurs.
- FIG. 1 has been described using an epi-illumination type fluorescence microscope, the present invention can also be used for a transmission illumination type fluorescence microscope.
- the fluorescence microscope according to the present embodiment can be configured not to have a confocal optical system.
- the pinhole 21 can be removed, and an optical microscope that is not a confocal microscope can be obtained.
- the fluorescence from the position other than the focal position has a low fluorescence saturation due to the low density of the laser light. That is, at a place other than the focal position, the fluorescence whose laser light intensity is low is a linear region that is not a nonlinear region. Therefore, the saturation component of fluorescence from other than the focal position is reduced. As a result, the resolution can be improved in the Z direction even in a configuration that does not use a confocal optical system.
- the fluorescence becomes a linear region at a position shifted from the focal position in the optical axis direction. Therefore, no saturation of fluorescence occurs, and information from only the focal position and its vicinity can be extracted.
- This enables three-dimensional observation with a simple configuration that does not use a confocal optical system.
- the resolution is further improved. It becomes possible to go up.
- a fluorescence microscope having the same configuration as that of Embodiment 1 is used, but a pulsed laser light source is used as the light source 10.
- the spatial resolution is improved by observing in the same manner as in Embodiment 1 using the two-photon excitation light. Note that the description of the same configuration as that of Embodiment 1 is omitted.
- a mode-locked titanium sapphire laser (wavelength 750 nm, pulse width 100 fs, repetition frequency 80 MHz) is used. Then, as in the first embodiment, intensity modulation is performed using the modulator 16.
- the modulator uses an electro-optic modulator (EO modulator) and modulates at 100kHz. As described in the first embodiment, the repetition frequency of the pulse laser beam is sufficiently higher than the modulation frequency.
- the optical excitation itself is localized inside the laser focus. Therefore, it is possible to detect absorption saturation with higher sensitivity than the need to consider fluorescence emission other than the focal plane.
- the light scattering efficiency power S1 / 16 is lower than that of single-photon excitation, so that the light intensity can be easily adjusted.
- the same effect can be obtained by using a multiphoton excitation method of two or more photons, which is not limited to the two-photon excitation method.
- quantum dots may be used as fluorescent probes as in the first embodiment.
- the fluorescence microscope that is useful in the present embodiment can be used in a fluorescence microscope other than the confocal system as in the first embodiment.
- the fluorescent microscope according to the present invention if intensity modulation and lock-in detection are not performed, it can be used as a normal fluorescent microscope. Therefore, it is possible to easily switch between high-resolution detection and normal detection.
- the fluorescence microscope which is effective in the present embodiment performs observation by irradiating laser light with different intensities without modulating laser light.
- Basics of the fluorescence microscope that can be used in this embodiment Since the general configuration is the same as that of the fluorescence microscope shown in the first embodiment, description thereof is omitted.
- the fluorescence microscope that is useful in the present embodiment has a configuration in which the modulator 16 used in the fluorescence microscope shown in FIG. 1 is not provided. Then, the sample 14 is irradiated from the light source 10 with different intensity of laser light.
- the output of the light source 10 may be changed, or the intensity of the laser light may be changed using a filter such as an ND filter. Two or more light sources may be used. Further, the intensity of the laser beam may be changed by combining these. Note that laser beams with different intensities have substantially the same wavelength.
- the different intensities of the laser light are defined as the first intensity and the second intensity. That is, the sample 14 is irradiated with the laser light from the light source 10 at the first intensity and the second intensity.
- the sample 14 may be irradiated with laser light at an intensity other than the first intensity and the second intensity.
- the sample 14 may be irradiated with laser light with an intensity of 3 or more.
- the first strength is stronger than the second strength.
- At least the first intensity is an intensity at which fluorescence saturation occurs. That is, when the first intensity is used, the sample is irradiated with laser light in a non-linear region where the relationship between excitation light and fluorescence is non-linear.
- the second intensity may be the intensity at which fluorescence saturation occurs.
- the first intensity is set so that the excitation light intensity is such that the excitation light and the fluorescence are not proportional.
- the first intensity corresponds to a non-linear region where the relationship between excitation light and fluorescence is nonlinear.
- the first intensity is set as B
- the second intensity is set as B.
- the sample 14 is scanned with respect to the first intensity B and the second intensity B.
- a fluorescence image can be taken for each of B. That is, at the first intensity B
- a fluorescence image of 2 1 and a fluorescence image at the second intensity B can be taken.
- the fluorescence at intensity B contains a saturated component of fluorescence.
- the fluorescence intensity corresponding to the first intensity B and the second intensity B at the same location of the sample 14 Based on the degree, the saturation component of the fluorescence at that location is calculated.
- the fluorescence intensity corresponding to the first intensity B is I
- the fluorescence intensity corresponding to the second intensity B is I. Therefore, as shown in FIG. 4, in the graph with the excitation light intensity on the horizontal axis and the fluorescence intensity on the vertical axis, the detection results exist at the coordinates (B, I) and (B, I). Further, when the excitation light intensity is 0, the fluorescence intensity is also 0. Therefore, in the graph in which the horizontal axis is the excitation light intensity and the vertical axis is the fluorescence intensity, (0, 0), (B, I) and (
- I pB 2 + qB + r.
- I indicated by the quadratic function is similar to the function indicated by the solid line in FIG. 4, for example.
- I is the fluorescence intensity
- B is the excitation light intensity
- the B component of I indicates a component in which fluorescence and excitation light are proportional. Specifically, qB is based on the broken line in FIG.
- the square of B in I indicates the saturation component of fluorescence.
- pB 2 indicates the difference between the broken line in FIG. 4 and the solid line that is the actual fluorescence intensity. More precisely, pB 2 is based on the difference between the broken line in FIG. 4 and the solid line that is the actual fluorescence intensity. In other words, the difference between the broken line in FIG. 4 and the solid line that is the actual fluorescence intensity includes terms with an order of 3 or more.
- p 0 is not satisfied.
- each coefficient calculated in this way is a function of (x, y, z). That is, the second-order coefficient p represents the spatial distribution of the intensity of the fluorescence saturation component. Therefore, the image of the fluorescence saturation component is denoted by p (x, y, z). In other words, an image of the fluorescence saturation component is formed by the coefficient p of the quadratic term of the fitted quadratic function.
- the image of the fluorescence saturation component is a spatial distribution of the intensity of the fluorescence saturation component when the sample 14 is scanned. In this way, an image of the fluorescence saturation component can be taken. The spatial resolution can be improved by observing the image of the saturated component of the fluorescence.
- the fluorescence may be detected for the laser light intensity of 1S3 or higher in which the fluorescence is detected only for the first intensity and the second intensity.
- the second-order and third-order terms indicate the fluorescence saturation component.
- the spatial resolution can be further improved. In other words, the spatial resolution can be tripled by focusing on the coefficient of the third-order term.
- the spatial resolution By detecting fluorescence with n laser light intensities, fitting can be performed with an n-order function. By focusing on higher-order terms, the spatial resolution can be further improved. In other words, when focusing on the coefficients of the polynomial, the spatial resolution can be improved in proportion to the order of the polynomial. Specifically, the spatial resolution can be increased by n times. In this case, the sum of the second and higher terms indicates the fluorescence saturation component. In order to perform detection more accurately, it is preferable to perform detection by changing the intensity of the laser beam in a large number.
- the force of scanning with the laser light kept at a constant intensity S is not limited to this.
- each portion on the sample 14 is irradiated with laser light with an intensity of at least 2 or more. Then, fitting is performed with a function of the order of 2 or more, and the coefficient of the term of the order of 2 or more is calculated. The spatial distribution of this coefficient becomes an image of the fluorescence saturation component.
- the sample 14 can be observed based on the fluorescence saturation component.
- the present invention focuses on the fact that the fluorescence is saturated when the intensity of the excitation light is increased, and the spatial resolution can be improved by performing the observation based on the saturation component of the fluorescence. . In this way, by improving the spatial resolution, the fluorescent substance is brought closer to the sample 14. Even when they are present in contact, they can be separated and observed.
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Abstract
Description
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JP2006547672A JP4487078B2 (ja) | 2004-12-08 | 2005-10-14 | 蛍光顕微鏡及び観察方法 |
EP05793232A EP1835323B1 (en) | 2004-12-08 | 2005-10-14 | Fluorescence microscope and observation method |
US11/792,304 US7781711B2 (en) | 2004-12-08 | 2005-10-14 | Fluorescence microscope for which a sample is observed based on the saturation components of fluorescence and fluorescence microscopy method |
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EP (1) | EP1835323B1 (ja) |
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WO2009096432A1 (ja) | 2008-01-30 | 2009-08-06 | Osaka University | 光記録材料、光記録方法、感光性材料、フォトリソグラフィー方法、光重合開始剤、及び光増感剤 |
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Also Published As
Publication number | Publication date |
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EP1835323A1 (en) | 2007-09-19 |
EP1835323A4 (en) | 2010-07-21 |
JP4487078B2 (ja) | 2010-06-23 |
EP1835323B1 (en) | 2012-08-01 |
JPWO2006061947A1 (ja) | 2008-06-05 |
US7781711B2 (en) | 2010-08-24 |
US20080215272A1 (en) | 2008-09-04 |
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