Image acquisition method, fluorescence microscope, excitation light irradiation unit and waveform control unit

Abstract

This image acquisition method includes: a step for repeatedly generating a group of excitation light pulses including a plurality of excitation light pulses; a step for irradiating the target object containing a fluorescent dye with the group of excitation light pulses; a step for detecting the intensity of fluorescence generated at a plurality of locations of the target object by the irradiation with the group of excitation light pulses; and a step for generating a fluorescence image on the basis of the intensity of fluorescence at the plurality of locations of the target object. In the step for generating a group of excitation light pulses, the time interval between the plurality of excitation light pulses is set to be equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or shorter than 10 picoseconds.

Description

Image acquisition method, fluorescence microscope, excitation light irradiation unit, and waveform control unit

[0001] The present disclosure relates to an image acquisition method, a fluorescence microscope, an excitation light irradiation unit, and a waveform control unit.

[0002] Patent Document 1 and Non-Patent Document 1 disclose that the pulse interval of excitation light pulses is set to 10 picoseconds to 50 picoseconds or more to reduce bleaching of fluorescence when a fluorescent dye is excited. Non-Patent Document 2 discloses that the pulse interval of excitation light pulses is set to more than 1 microsecond to reduce bleaching of fluorescence when a fluorescent dye is excited. Patent Document 2 discloses a fluorescence microscope in which the number of pulses and pulse interval of excitation light are controlled.

[0003] International Publication No. 2009 / 035768 U.S. Patent Application Publication No. 2010 / 0187208

[0004] Na Ji et al., "High-speed, low-photodamage nonlinear imaging using passive pulse splitters", Nature Methods, Volume 5, No. 2, pp. 197-202 (2008)Gerald Donnert et al., "Major signal increase in fluorescence microscopy through dark-state relaxation", Nature Methods, Volume 4, No. 1, pp. 81-86 (2007)

[0005] Fluorescence microscopes irradiate multiple locations on an object containing a fluorescent dye with excitation light, detect the fluorescence emitted from the fluorescent dye, and output a fluorescence image. In some cases, pulsed excitation light is used to irradiate the object. For example, in multiphoton excitation fluorescence microscopes, excitation light pulses with extremely short pulse widths, such as on the order of picoseconds or femtoseconds, are used to irradiate the object to increase the photon density of the excitation light and induce multiphoton absorption. However, if fluorescence is continuously detected while irradiating the object with excitation light pulses, the fluorescence intensity gradually decreases. This phenomenon is called photobleaching. Because photobleaching limits the observation time of an object, reducing photobleaching is desirable in fluorescence microscopes.

[0006] The present disclosure aims to provide an image acquisition method, a fluorescence microscope, an excitation light irradiation unit, and a waveform control unit that can reduce photobleaching.

[0007] [1] An image acquisition method according to one embodiment includes the steps of repeatedly generating an excitation light pulse group including a plurality of excitation light pulses, irradiating an object containing a fluorescent dye with the excitation light pulse group, detecting the intensities of fluorescence generated at a plurality of locations on the object as a result of irradiation with the excitation light pulse group, and generating a fluorescence image based on the intensities of fluorescence at the plurality of locations on the object. In the step of generating the excitation light pulse group, the time interval between the plurality of excitation light pulses is set to be equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or shorter than 10 picoseconds.

[0008] [2] A fluorescence microscope according to one embodiment includes a pulse group generator that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses, an optical system that irradiates an object containing a fluorescent dye with the excitation light pulse group, a photodetector that detects the intensities of fluorescence generated at a plurality of locations on the object as a result of irradiation with the excitation light pulse group, and a processing unit that generates a fluorescence image based on the intensities of fluorescence at the plurality of locations on the object, wherein the time intervals between the plurality of excitation light pulses are equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye or are shorter than 10 picoseconds.

[0009] Photobleaching occurs through the following mechanism: First, an excitation light pulse is incident on the object and absorbed by the fluorescent dye. At this time, the fluorescent dye is in the ground state S ０ to an excited singlet state (e.g., excited state S １ ) after which most molecules return to the ground state S ０ However, some molecules return to the ground state S ０ does not return to an excited triplet state (e.g., excited state T １ ) This transition is called intersystem crossing. When the molecule is in the excited triplet state and the next excitation light pulse is incident on the target object and absorbed by the fluorescent dye, the molecule transitions to a higher excited triplet state (for example, excited state T ２ ) transition to the excited triplet state. Photobleaching occurs when the molecule reacts with oxygen in the excited triplet state to generate active oxygen, which destroys the molecule. In particular, in the case of multiphoton excitation microscopes using near-infrared light, the molecule transitions to a higher excited triplet state (for example, excited state T ２ ) molecules contribute significantly to photobleaching.

[0010] In the image acquisition method of [1] above and the fluorescence microscope of [2] above, (a) the time interval between the multiple excitation light pulses is set to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or (b) is shorter than 10 picoseconds. In the case of (a), the fluorescent dye molecule is in a higher excited triplet state (for example, excited state T ２ ), the next excitation light pulse is incident on the object and is absorbed by the fluorescent dye, so the fluorescent dye molecule moves to a higher excited triplet state (for example, excited state T ３ ) transition. Then, the molecule transitions from an excited triplet state to an excited singlet state (for example, an excited state S １) increases the potential energy difference between the excited triplet state and the excited triplet state, making it easier for the molecule to transition to the excited singlet state before reacting with oxygen. This prevents the molecule from being destroyed, thereby reducing photobleaching. There are a variety of fluorescent dyes, including some with a relaxation time between excited triplet states of 10 picoseconds or longer. By setting the time interval between multiple excitation light pulses to be shorter than 10 picoseconds, as in (b), photobleaching of such fluorescent dyes can be reduced.

[0011] [3] In the image acquisition method of the above [1] or the fluorescence microscope of the above [2], the relaxation time between excited states in the excited triplet state of the fluorescent dye is ２ to excited state T １ The relaxation time to ２ In this case, the excitation light pulse causes the excited state T １ to excited state T ２ In fluorescent dyes with a transition property, photobleaching can be effectively reduced.

[0012] [4] The image acquisition method of [1] or [3] above may further include a step of inputting information about the type of fluorescent dye before the step of generating the excitation light pulse group. In the step of generating the excitation light pulse group, the time interval between the multiple excitation light pulses may be set based on the information to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye. The fluorescence microscope of [2] or [3] above may further include an information input unit for inputting information about the type of fluorescent dye. Based on the information, the pulse group generator may set the time interval between the multiple excitation light pulses to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye. These image acquisition methods and fluorescence microscopes allow the time interval between the multiple excitation light pulses to be set according to the relaxation time of the fluorescent dye used. Therefore, photobleaching of the fluorescent dye can be more effectively reduced.

[0013] [5] In the step of generating an excitation light pulse group in any one of the image acquisition methods [1], [3], or [4] above, if the time interval between the multiple excitation light pulses is shorter than 10 picoseconds, the time interval between the multiple excitation light pulses may be shorter than 1 picosecond. Similarly, in any one of the fluorescence microscopes [2] to [4] above, if the time interval between the multiple excitation light pulses is shorter than 10 picoseconds, the time interval between the multiple excitation light pulses may be shorter than 1 picosecond. In this case, photobleaching of a fluorescent dye with a relaxation time of 1 picosecond or longer can be reduced.

[0014] [6] In the step of generating an excitation light pulse group in any one of the image acquisition methods [1], [3] to [5] above, the peak intensities of the multiple excitation light pulses may be uniform for each excitation light pulse group. Similarly, in any one of the fluorescence microscopes [2] to [5] above, the peak intensities of the multiple excitation light pulses may be uniform for each excitation light pulse group. In this case, a higher-order excited triplet state (e.g., excited state T ２ ) the peak intensity of the excitation light pulse when the excitation light pulse transitions to a higher excited triplet state (e.g., excited state T ３ ) is approximately equal to the peak intensity of the excitation light pulse when the triplet state transitions from the excited triplet state to the excited triplet state of the fluorophore. Therefore, the transition from a higher excited triplet state to an even higher excited triplet state is efficiently carried out. This makes it possible to more effectively reduce photobleaching of the fluorescent dye.

[0015] [7] In the step of generating an excitation light pulse group in any one of the image acquisition methods [1] and [3] to [6] above, the repetition frequency when the excitation light pulse group is repeatedly generated may be 1 MHz or more. Similarly, in any one of the fluorescence microscopes [2] to [6] above, the repetition frequency when the excitation light pulse group is repeatedly generated may be 1 MHz or more. The relaxation time (e.g., T １ The lifetime (lifetime) of many fluorescent dyes is several microseconds or less. Therefore, photobleaching due to the above-mentioned mechanism is likely to occur when the repetition frequency of the excitation light pulse group is 1 MHz or higher, in other words, when the time interval between excitation light pulse groups is 1 microsecond or less. Therefore, any of the image acquisition methods and fluorescence microscopes described above is useful.

[0016] [8] In the fluorescence microscope of any of [2] to [7] above, the pulse group generator may include an excitation light source that repeatedly outputs a single light pulse, and a waveform controller that is optically coupled to the excitation light source and modulates the single light pulse output from the excitation light source to generate multiple excitation light pulses. In this case, it is possible to easily configure the pulse group generator that repeatedly generates an excitation light pulse group including multiple excitation light pulses.

[0017] [9] An excitation light irradiation unit according to one embodiment is an excitation light irradiation unit for use in a fluorescence microscope, and includes a pulse group generator that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses to be irradiated onto an object containing a fluorescent dye. The time interval between the plurality of excitation light pulses is equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or is shorter than 10 picoseconds. This excitation light irradiation unit can reduce photobleaching.

[0018]

[10] The excitation light irradiation unit of [9] above may further include an information input unit for inputting information about the type of fluorescent dye. Based on the information, the pulse group generator may set the time interval between the multiple excitation light pulses to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye. This excitation light irradiation unit allows the time interval between the multiple excitation light pulses to be set according to the relaxation time of the fluorescent dye used. Therefore, photobleaching of the fluorescent dye can be more effectively reduced.

[0019]

[11] A waveform control unit according to one embodiment is a waveform control unit for a fluorescence microscope that repeatedly generates an excitation light pulse group including multiple excitation light pulses to be irradiated onto an object containing a fluorescent dye. The waveform control unit includes a waveform controller. The waveform controller is optically coupled to an excitation light source that repeatedly outputs a single light pulse, and modulates the single light pulse output from the excitation light source to generate multiple excitation light pulses. The time interval between the multiple excitation light pulses is equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or is shorter than 10 picoseconds. This waveform control unit can reduce photobleaching.

[0020]

[12] The waveform control unit of

[11] above may further include an information input section for inputting information about the type of fluorescent dye. Based on that information, the waveform control section may set the time interval between the multiple excitation light pulses to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye. This waveform control unit allows the time interval between the multiple excitation light pulses to be set according to the relaxation time of the fluorescent dye used. Therefore, photobleaching of the fluorescent dye can be more effectively reduced.

[0021] According to the present disclosure, it is possible to provide an image acquisition method, a fluorescence microscope, an excitation light irradiation unit, and a waveform control unit that can reduce photobleaching.

[0022] FIG. 1 is a diagram showing the configuration of a fluorescence microscope according to one embodiment. FIG. 2 is a diagram showing a schematic diagram of the time waveform of excitation light. Part (a) is a schematic diagram showing the time waveform of excitation light output from an excitation light source. Part (b) is a schematic diagram showing the time waveform of excitation light output from a waveform control unit. FIG. 3 is a diagram showing a specific configuration example of a waveform control unit. FIG. 4 is a diagram showing a modulation surface of a spatial light modulator (SLM). FIG. 5 is a graph showing an example of excitation light. Part (a) shows the spectral waveform of single-pulse excitation light. Part (b) shows the time intensity waveform of the excitation light. FIG. 6 is a graph showing an example of excitation light. Part (a) shows the spectral waveform of excitation light when a rectangular wave-shaped phase spectrum modulation is applied in an SLM. Part (b) shows the time intensity waveform of the excitation light. FIG. 7 is a diagram showing a specific configuration example of a fluorescence microscope. FIG. 8 is a flowchart showing an image acquisition method according to one embodiment. FIG. 9 is a graph showing the time waveforms of a light pulse and a light pulse group. Parts (a) to (d) show the time waveforms of a single light pulse, a light pulse group consisting of four light pulses, a light pulse group consisting of nine light pulses, and a light pulse group consisting of 16 light pulses, respectively. Fig. 10 is a graph showing the dependence of photobleaching rate on excitation light intensity. Part (a) shows a graph for eosin Y, part (b) shows a graph for fluorescein, and part (c) shows a graph for C-Naphox-TEG. Fig. 11 is a graph showing the dependence of photobleaching rate on the number of pulses (N). Part (a) shows a graph for eosin Y, part (b) shows a graph for fluorescein, and part (c) shows a graph for C-Naphox-TEG. Fig. 12 is a graph showing the relationship between excitation light intensity and photobleaching rate. Part (a) shows the dependence of photobleaching rate on the average intensity (I Ｎ / √N) and the photobleaching rate P Ｎ Part (b) shows the relationship between the photobleaching rate P Ｎ The photobleaching rate P when N = 1 １11. The graphs show the results normalized by the excitation light intensity (I ). FIG. 13 is a diagram showing the mechanism by which photobleaching occurs. FIG. 14 is a graph showing the pulse number (N) dependency of the photobleaching rate. Parts (a) to (c) show graphs containing the same plots as the graphs shown in parts (a) to (c) of FIG. 11, respectively. However, unlike the curves in FIG. 11, the curves show theoretical values ​​calculated based on the mechanism shown in FIG. 13. FIG. 15 shows the theoretical average intensity (I ) of excitation light based on the mechanism shown in FIG. 13. Ｎ / √N) and the photobleaching rate P Ｎ The photobleaching rate P when N = 1 １ The value normalized by (P Ｎ / P １ ) is a graph showing the relationship between the time interval of a light pulse and the photobleaching rate. FIG. 16 is a graph showing the results of calculating the relationship between the time interval of a light pulse and the photobleaching rate. FIG. 17 is a graph showing the time waveforms of five types of light pulse groups with different uniformities of peak intensity. FIG. 18 is a graph showing the results of measuring the color fading rate when a fluorescent dye is irradiated with the five types of light pulse groups shown in parts (a) to (e) of FIG. 17. FIG. 18 shows the relationship between the ratio (σ / μ) and the color fading rate. FIG. 19 is a diagram showing a schematic configuration of a modulation pattern calculation device. FIG. 20 is a diagram showing a schematic configuration example of the hardware of a modulation pattern calculation device. FIG. 21 is a diagram showing the procedure for calculating a phase spectrum using the iterative Fourier method. FIG. 22 is a diagram showing the procedure for calculating a phase spectrum using the iterative Fourier method. FIG. 23 is a diagram showing the procedure for calculating a phase spectrum. FIG. 24 is a diagram showing the procedure for calculating a phase spectrum using the iterative Fourier method. FIG. 25 is a diagram showing an example of the weighting function We(t) when Target0(t) is a multipulse. Fig. 26 is a diagram showing the calculation procedure in the iterative Fourier transform unit of the intensity spectrum design unit. Fig. 27 is a flowchart showing a modulation pattern calculation method. Fig. 28 is a diagram showing the calculation procedure for a phase spectrum using the iterative Fourier method. Fig. 29 is a diagram showing the calculation procedure for a phase spectrum using the iterative Fourier method. Fig. 30 is a diagram showing an excitation light irradiation unit used in a fluorescence microscope. Fig. 31 is a diagram showing a waveform control unit used in a fluorescence microscope.

[0023] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are designated by the same reference numerals, and duplicated explanations will be omitted.

[0024] Fig. 1 is a diagram showing the configuration of a fluorescence microscope 1 according to one embodiment. The fluorescence microscope 1 is a device that obtains a fluorescence image by irradiating an object B, which is an observation target, with excitation light Ld and detecting fluorescence Le generated in the object B. As shown in Fig. 1, the fluorescence microscope 1 of this embodiment includes a pulse group generator 2, an optical system 3, a photodetector 4, a processing unit 5, a display unit 6, and an information input unit 11. The pulse group generator 2 includes an excitation light source 8 and a waveform controller 10.

[0025] The excitation light source 8 is optically coupled to the waveform control unit 10 and provides excitation light La to the waveform control unit 10. Part (a) of FIG. 2 is a diagram schematically illustrating the time waveform of the excitation light La output from the excitation light source 8. As shown in part (a) of FIG. 2, the excitation light La includes repetitions of a single optical pulse PL1. The repetition period t1 of the optical pulse PL1 is, for example, 1 nanosecond to 10 microseconds, or 10 nanoseconds to 100 nanoseconds. In other words, the repetition frequency of the optical pulse PL1 is, for example, 0.1 MHz to 1 GHz, or 10 MHz to 100 MHz. In one example, the repetition frequency of the optical pulse PL1 is 80 MHz. The repetition period t1 of the optical pulse PL1 may be constant. The repetition period t1 of the optical pulse PL1 may be defined as the peak interval of the optical pulse PL1. The full width at half maximum (FWHM) of the optical pulse PL1 is, for example, 5 femtoseconds or more and 200 femtoseconds or less, or 30 femtoseconds or more and 200 femtoseconds or less. The excitation light source 8 repeatedly outputs such a single optical pulse PL1. The excitation light source 8 is, for example, a laser light source such as a solid-state laser light source, a gas laser light source, a semiconductor laser light source, or a fiber laser light source. The excitation light La is, for example, coherent light.

[0026] The waveform controller 10 converts the excitation light La provided from the excitation light source 8 into excitation light Ld. The excitation light Ld is output from the pulse group generator 2. Part (b) of FIG. 2 is a diagram schematically illustrating the time waveform of the excitation light Ld output from the waveform controller 10. As shown in part (b) of FIG. 2, the excitation light Ld includes a repetition of an excitation light pulse group PG (hereinafter referred to as the light pulse group PG). The repetition period t1 of the light pulse group PG is the same as the repetition period t1 of the light pulse PL1, and is, for example, from 1 nanosecond to 10 microseconds, or from 10 nanoseconds to 100 nanoseconds. In other words, the repetition frequency of the light pulse group PG is, for example, from 0.1 MHz to 1 GHz, or from 10 MHz to 100 MHz. The light pulse group PG includes a plurality of excitation light pulses PL2 (hereinafter referred to as the light pulses PL2) arranged at a time interval t2. In one example, the time interval t2 between the multiple light pulses PL2 is constant for each light pulse group PG. The peak intensity of the light pulses PL2 is uniform for each light pulse group PG. The repetition period t1 of the light pulse group PG may be defined as the peak interval between the leading light pulses PL2 among the multiple light pulses PL2 that make up each light pulse group PG. The time interval t2 may be defined as the peak interval between the light pulses PL2. The full width at half maximum (FWHM) of the light pulses PL2 is, for example, not less than 5 femtoseconds and not more than 200 femtoseconds, or not less than 30 femtoseconds and not more than 200 femtoseconds.

[0027] FIG. 3 is a diagram showing a specific configuration example of the waveform control unit 10. In the example shown in FIG. 3, the waveform control unit 10 includes a diffraction grating 12, a lens 13, a spatial light modulator (SLM) 14, a lens 15, a diffraction grating 16, and a modulation pattern calculation device 20. The diffraction grating 12 is an example of a spectroscopic element and is optically coupled to the excitation light source 8. The SLM 14 is optically coupled to the diffraction grating 12 via the lens 13. The diffraction grating 12 disperses the excitation light La into individual wavelength components. Instead of the diffraction grating 12, other optical components such as a prism may be used as the spectroscopic element. The spectroscopic element may be either a reflective or transmissive type. The excitation light La is incident obliquely on the diffraction grating 12 and dispersed into multiple wavelength components. The light Lb containing the multiple wavelength components is condensed by the lens 13 into each wavelength component and formed into an image on the modulation surface of the SLM 14. The lens 13 may be a convex lens made of a light-transmitting member or a concave mirror having a concave light-reflecting surface. The lens 15 may also be a cylindrical lens.

[0028] The SLM 14 simultaneously performs phase modulation and intensity modulation of the light Lb to generate excitation light Ld having an arbitrary time intensity waveform different from that of the excitation light La. The SLM 14 may also perform intensity modulation only. The SLM 14 is, for example, a phase modulation type. In one embodiment, the SLM 14 is an LCOS (Liquid Crystal on Silicon) type. Alternatively, the SLM 14 may be an intensity modulation type SLM such as a digital micromirror device (DMD). The SLM 14 may be a reflective type or a transmissive type. FIG. 4 is a diagram showing the modulation surface 17 of the SLM 14. As shown in FIG. 4, the modulation surface 17 has multiple modulation regions 17a arranged along a direction D1, and each modulation region 17a extends in a direction D2 intersecting the direction D1. The direction D1 is the direction of light separation by the diffraction grating 12. The modulation surface 17 functions as a Fourier transform plane, and each of the multiple modulation regions 17a is incident on each of the corresponding wavelength components after separation. The SLM 14 modulates the phase and intensity of each incident wavelength component in each modulation region 17 a independently of other wavelength components. When the SLM 14 is a phase modulation type, the intensity modulation is realized by a phase pattern (phase image) presented on the modulation surface 17.

[0029] The SLM 14 is electrically connected to a modulation pattern calculation device 20. The modulation pattern calculation device 20 calculates a modulation pattern to be presented by the SLM 14 and provides data Da indicating the modulation pattern to the SLM 14. The modulation pattern is, for example, a computer-generated hologram (CGH).

[0030] Each wavelength component of the modulated light Lc modulated by the SLM 14 is focused to a single point on the diffraction grating 16 by the lens 15. In this case, the lens 15 functions as a focusing optical system that focuses the modulated light Lc. The lens 15 may be a convex lens made of a light-transmitting member, or a concave mirror having a concave light-reflecting surface. The lens 15 may also be a cylindrical lens. The diffraction grating 16 functions as a combining optical system that combines the multiple wavelength components after modulation. In other words, the multiple wavelength components of the modulated light Lc are focused and combined by the lens 15 and the diffraction grating 16 to become the excitation light Ld.

[0031] The region in front of the lens 15 (spectral region) has a Fourier transform relationship with the region behind the diffraction grating 16 (time domain). Therefore, phase modulation and intensity modulation in the spectral domain affect the time-intensity waveform in the time domain. Therefore, the excitation light Ld has a desired time-intensity waveform different from that of the excitation light La, depending on the modulation pattern of the SLM 14. Here, part (a) of FIG. 5 shows, as an example, the spectral waveform (spectral phase G11 and spectral intensity G12) of the single-pulse excitation light La, and part (b) of FIG. 5 shows the time-intensity waveform of the excitation light La. Part (a) of FIG. 6 shows, as an example, the spectral waveform (spectral phase G21 and spectral intensity G22) of the excitation light Ld when a rectangular-wave phase spectral modulation is applied in the SLM 14, and part (b) of FIG. 6 shows the time-intensity waveform of the excitation light Ld. In part (a) of Figure 5 and part (a) of Figure 6, the horizontal axis represents wavelength (nm), the left vertical axis represents intensity values ​​(arbitrary units) of the intensity spectrum, and the right vertical axis represents phase values ​​(rad) of the phase spectrum. In part (b) of Figure 5 and part (b) of Figure 6, the horizontal axis represents time (femtoseconds), and the vertical axis represents light intensity (arbitrary units). In this example, by providing a rectangular wave-shaped phase spectrum waveform to the excitation light Ld, a single light pulse PL1 of the excitation light La is converted into a light pulse group PG including multiple light pulses PL2. The spectra and waveforms shown in Figures 5 and 6 are exemplary, and the number, pulse width, peak intensity, and time interval t2 of the light pulses PL2 of the light pulse group PG can be variously controlled by combining various spectral phases and spectral intensities.

[0032] Referring again to FIG. 1 , the excitation light Ld including the light pulse group PG output from the pulse group generator 2 is input to the optical system 3. The optical system 3 irradiates the excitation light Ld onto the object B to be observed. The object B has been stained in advance with a fluorescent dye. The fluorescent dye includes at least one material selected from the group consisting of, for example, a methanol solution of eosin Y, an aqueous solution of eosin Y, a methanol solution of rose bengal, an ethanol solution of rhodamine 6G, an aqueous solution of rose bengal and rhodamine 6G, and anthracene. The object B may be a biomolecule or biological tissue genetically modified to emit fluorescence.

[0033] The fluorescent dye in the object B is excited by irradiation with excitation light Ld containing a light pulse group PG, generating fluorescence Le at multiple locations in the object B. At this time, the fluorescent dye in the object B may generate fluorescence Le through multiphoton absorption (e.g., two-photon absorption). By irradiating the object B with light pulses PL2 having an extremely short pulse width, for example, on the order of picoseconds or femtoseconds, the photon density of the excitation light Ld can be increased to cause multiphoton absorption. The fluorescence Le is input to a photodetector 4. The photodetector 4 detects the intensity of the fluorescence Le at each location in the object B. The photodetector 4 is, for example, a semiconductor light-receiving element such as a photodiode, an avalanche photodiode, or a single-photon avalanche diode, or a photomultiplier tube. The photodetector 4 generates an electrical signal Sa corresponding to the intensity of the fluorescence Le. The photodetector 4 provides the generated electrical signal Sa to a processing unit 5.

[0034] The processing unit 5 is electrically connected to the photodetector 4 and receives an electrical signal Sa from the photodetector 4. The processing unit 5 generates data Sb relating to a fluorescence image of the object B based on the intensity of the fluorescence Le at multiple locations on the object B. The processing unit 5 provides the data Sb to the display unit 6. The display unit 6 displays the fluorescence image of the object B based on the data Sb. The processing unit 5 is a computer such as a personal computer, a smart device such as a smartphone or a tablet terminal, or a cloud server. The computer serving as the processing unit 5 includes a HDD, a storage device such as a flash memory or RAM, and a processor (CPU). The processing unit 5 may be configured using a microcomputer or an FPGA (Field-Programmable Gate Array).

[0035] The time interval t2 of the light pulse PL2 shown in part (b) of FIG. 2 is the relaxation time between excited states in the excited triplet state of the fluorescent dye (for example, the time between excited states T ２ to excited state T １ The relaxation time to ２The time interval t2 is set in the modulation pattern calculation device 20 so that it is equal to or shorter than the lifetime) or shorter than 10 picoseconds. If the time interval t2 is shorter than 10 picoseconds, the time interval t2 may be shorter than 6 picoseconds, shorter than 3 picoseconds, or shorter than 1 picosecond. ２ The lifetimes are as follows: Eosin Y in methanol: 1 picosecond Eosin Y in water: 1 picosecond Rose Bengal in methanol: 2.2 picoseconds Rhodamine 6G in ethanol: 2 picoseconds Rose Bengal: 5.8 picoseconds Rhodamine 6G in water: 0.2 picoseconds Anthracene: 11 picoseconds

[0036] The information input unit 11 inputs information about the type of fluorescent dye in the object B. The information input unit 11 inputs information about the type of fluorescent dye in the object B, for example, through an input operation by a user of the fluorescence microscope 1. The information input unit 11 is, for example, an input device such as a keyboard or a touch panel. The information input unit 11 provides information Db about the type of fluorescent dye in the object B to the modulation pattern calculation device 20. Based on the information Db, the modulation pattern calculation device 20 sets the time interval t2 of the light pulse PL2 to be equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye.

[0037] Fig. 7 is a diagram showing a specific configuration example of the fluorescence microscope 1. In the example shown in Fig. 7, the fluorescence microscope 1 further includes an intensity controller 7 and a waveform measuring device 9. The optical system 3 has a light branching element 31, galvanometer mirrors 32 and 33, and coupling lenses 34 and 35.

[0038] The intensity controller 7 is disposed on the optical path of the excitation light La between the excitation light source 8 and the waveform controller 10. The intensity controller 7 attenuates the excitation light La to adjust the optical intensity of the excitation light La. The intensity controller 7 includes at least one optical element selected from the group consisting of an acousto-optic modulator (AOM), an electro-optic modulator (EOM), and a combination of a half-wave plate and a polarizer, for example.

[0039] The optical branching element 31 is disposed on the optical axis of the pump light Ld output from the waveform control unit 10. The optical branching element 31 separates the pump light Lf, which is a part of the pump light Ld, from the pump light Ld. The pump light Lf is input to the waveform measurement unit 9. The waveform measurement unit 9 measures the time waveform of the pump light Lf to measure the time waveform of the pump light Ld. The waveform measurement unit 9 may include a correlation measurement unit composed of a nonlinear crystal, a delay stage, and a spectrometer. Alternatively, the waveform measurement unit 9 may include an interferometer composed of a spectrometer. The measurement results by the waveform measurement unit 9 are provided to the modulation pattern calculation unit 20 of the waveform control unit 10. The modulation pattern calculation unit 20 controls the modulation pattern presented by the SLM 14 so that the time waveform of the pump light Ld measured by the waveform measurement unit 9 approaches a desired time waveform (specifically, the number, pulse width, peak intensity, and time interval t2 of the optical pulse group PG).

[0040] The galvanometer mirrors 32 and 33 are optical elements for scanning the optical axis of the excitation light Ld. The galvanometer mirror 32 is optically coupled to the waveform control unit 10 via the optical branching element 31, and moves the optical axis of the excitation light Ld in one direction perpendicular to the optical axis of the excitation light Ld. The galvanometer mirror 33 is optically coupled to the galvanometer mirror 32, and moves the optical axis of the excitation light Ld in another direction perpendicular to both the optical axis of the excitation light Ld and the one direction. The coupling lenses 34 and 35 are optical elements for optically coupling the excitation light Ld, whose optical axis is moving, with the object B. The coupling lens 34 is optically coupled to the galvanometer mirror 33, and the coupling lens 35 is optically coupled to the coupling lens 34.

[0041] The excitation light Ld is input to the microscope main body 40. The microscope main body 40 has a mounting stage on which the object B is placed, and also incorporates the above-mentioned photodetector 4, processing unit 5, and display unit 6. The object B placed on the mounting stage is irradiated with the excitation light Ld from below. Fluorescence Le generated in the object B is incident on the photodetector 4 via an objective lens (not shown) arranged below the object B.

[0042] 8 is a flowchart showing an image acquisition method according to this embodiment. This image acquisition method can also be considered as the operating method of the fluorescence microscope 1 described above. First, in step S11, the information input unit 11 inputs information Db relating to the type of fluorescent dye. Next, in step S12, the pulse group generation unit 2 repeatedly generates a light pulse group PG including a plurality of light pulses PL2. In this step S12, first, the excitation light source 8 repeatedly outputs a single light pulse PL1 (step S121). Then, the light pulse PL1 is directly irradiated onto the object B, and the fluorescence intensity is detected by the photodetector 4. Based on the detection result, the sensitivity of the photodetector 4 and the detection limit based on the S / N ratio are confirmed, and the intensity I of the excitation light is calculated. １ (Step S122). Subsequently, the waveform controller 10 modulates the light pulse PL1 output from the excitation light source 8 to repeatedly output a light pulse group PG consisting of N (N is an integer of 2 or more) light pulses PL2 (Step S123). At this time, the waveform controller 10 determines the time interval t2 of the light pulses PL2 based on the relaxation time between excited states in the excited triplet state of the fluorescent dye of the object B (for example, the excited state T ２ to excited state T １ The relaxation time to ２ The time interval t2 is set to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye, based on the information Db regarding the type of fluorescent dye obtained in step S11.

[0043] Next, based on the time waveform of the excitation light Ld (i.e., the light pulse group PG) measured by the waveform measuring instrument 9, the modulation pattern calculation device 20 of the waveform control unit 10 controls the modulation pattern of the SLM 14 so that the peak intensity of the light pulse PL2 becomes uniform for each light pulse group PG (step S124). Next, using the intensity controller 7, the average power of the excitation light Ld is calculated as (√N)·I １ (step S125).

[0044] Next, in step S13, the optical system 3 irradiates the object B containing the fluorescent dye with excitation light Ld including the light pulse group PG. Then, in step S14, the photodetector 4 detects the intensity of fluorescence Le generated by the fluorescent dye in the object B in response to irradiation with the excitation light Ld. Here, it is determined whether the excitation light Ld has been irradiated to all irradiation positions (step S15). If there are any irradiation positions that have not been irradiated with the excitation light Ld (step S15: NO), the galvanometer mirrors 32 and 33 move the optical axis of the excitation light Ld (step S16), and the fluorescence microscope 1 repeats the operations from step S13. If all irradiation positions have been irradiated with the excitation light Ld (step S15: YES), the fluorescence microscope 1 performs the operation of step S17. In step S17, the processing unit 5 generates a fluorescence image based on the intensity of fluorescence Le at multiple locations on the object B, i.e., at all irradiation positions. Thereafter, the display unit 6 receives data Sb related to the fluorescence image from the processing unit 5 and displays the fluorescence image.

[0045] The functions and effects obtained by the fluorescence microscope 1 and image acquisition method of this embodiment described above will now be described.

[0046] For example, in multiphoton excitation fluorescence microscopes, pulsed excitation light is irradiated onto an object. However, if fluorescence is continuously detected while irradiating the object with pulsed excitation light, the fluorescence intensity gradually decreases. This phenomenon is called photobleaching. Since photobleaching limits the observation time of an object, it is desirable to reduce photobleaching in fluorescence microscopes.

[0047] The aforementioned Non-Patent Document 1 discloses that irradiation with a light pulse group including multiple light pulses reduces the photobleaching rate compared to irradiation with a single light pulse. Furthermore, Non-Patent Document 1 discloses, from theoretical considerations, that for an object of observation whose photobleaching rate is proportional to the cube of the peak energy of the light pulse, irradiation with a light pulse group including N light pulses may reduce the photobleaching rate by a factor of (1 / √N). The inventors conducted the following experiment to confirm the effectiveness of the method described in Non-Patent Document 1. First, as shown in FIG. 9 , a single light pulse (i.e., N = 1, see part (a) of FIG. 9 ), a light pulse group consisting of four light pulses (i.e., N = 4, see part (b) of FIG. 9 ), a light pulse group consisting of nine light pulses (i.e., N = 9, see part (c) of FIG. 9 ), and a light pulse group consisting of 16 light pulses (i.e., N = 16, see part (d) of FIG. 9 ) were generated. These light pulses and groups of light pulses were then irradiated onto three types of fluorescent dyes (eosin Y, fluorescein, and C-Naphox-TEG) ​​to examine the dependence of the photobleaching rate on excitation light intensity and pulse number (N).

[0048] FIG. 10 is a graph showing the dependence of photobleaching rate on excitation light intensity. Part (a) of FIG. 10 shows a graph for eosin Y, part (b) shows a graph for fluorescein, and part (c) shows a graph for C-Naphox-TEG. Lines L11 to L13 are approximate straight lines for these graphs. These approximate straight lines L11 to L13 show that the photobleaching rate for eosin Y is proportional to the 2.93 power of the excitation light intensity, the photobleaching rate for fluorescein is proportional to the 2.66 power of the excitation light intensity, and the photobleaching rate for C-Naphox-TEG is proportional to the 3.08 power of the excitation light intensity.

[0049] FIG. 11 is a graph showing the pulse number (N) dependence of photobleaching rate. Part (a) of FIG. 11 shows a graph for eosin Y, part (b) shows a graph for fluorescein, and part (c) shows a graph for C-Naphox-TEG. Curves C11 to C13 show theoretical values ​​based on the above-mentioned exponents calculated from the graph in FIG. 10. Referring to part (a) of FIG. 11, the pulse number (N) dependence of the photobleaching rate for eosin Y is nearly consistent with the theoretical value. However, referring to part (b) of FIG. 11, it can be seen that the pulse number (N) dependence of the photobleaching rate for fluorescein deviates from the theoretical value. Referring to part (c) of FIG. 11, it can be seen that the pulse number (N) dependence of the photobleaching rate for C-Naphox-TEG deviates even more significantly from the theoretical value. Thus, the inventor's experiments have revealed that the theory described in Non-Patent Document 1 does not hold true depending on the type of fluorescent dye.

[0050] The inventors investigated the change in photobleaching rate due to the change in excitation light intensity for a single light pulse (N=1, see part (a) of Figure 9) and a light pulse group consisting of nine light pulses (N=9, see part (c) of Figure 9). Part (a) of Figure 12 shows the change in photobleaching rate due to the change in excitation light intensity. Ｎ / √N) and the photobleaching rate P Ｎ In the figure, plot P11 shows the case where N=1, and plot P12 shows the case where N=9. Part (b) of FIG. 12 shows the relationship between the photobleaching rate P Ｎ The photobleaching rate P when N = 1 １ In the figure, plot P21 shows the case where N=1, and plot P22 shows the case where N=9.

[0051] According to the theory described in Non-Patent Document 1, in the graph shown in part (b) of FIG. 12, the normalized value of the photofading rate (P Ｎ / P １ ) should be constant regardless of the average intensity of the excitation light. However, the standard value of the photobleaching rate when N = 9 (P Ｎ / P １) gradually decreases as the average intensity of the excitation light increases. In other words, the higher the excitation light intensity, the greater the effect of reducing the photobleaching rate. This phenomenon cannot be explained by the theory described in Non-Patent Document 1.

[0052] FIG. 13 is a diagram showing the mechanism by which photobleaching occurs. Photobleaching occurs through the following mechanism. First, an excitation light pulse is incident on the object and absorbed by the fluorescent dye. At this time, the fluorescent dye is in the ground state S ０ to an excited singlet state (e.g., excited state S １ ) (arrows Aa1 and Aa2 in the figure). Figure 13 illustrates the case of two-photon absorption. More specifically, the fluorescent dye is first excited to the excited state S １ State S with higher potential than １ The fluorescent dye then rapidly relaxes to the excited state S ′, where the vibrational level is zero. １ (arrow Ab1 in the figure). After that, most molecules return to the ground state S ０ (arrow Ab2 in the figure), which generates fluorescence Le. However, some molecules remain in the ground state S ０ does not return to an excited triplet state (e.g., excited state T １ ), that is, an intersystem crossing occurs (arrow Ae1 in the figure). Then, the relaxation time from the excited triplet state (T １ If the time interval between excitation light pulses is longer than the lifetime, some of the molecules will be in the ground state S ０ However, the relaxation time from the excited triplet state (T １ If the time interval between excitation light pulses is shorter than the lifetime of the molecule, the next excitation light pulse is incident on the target substance when the molecule is in the excited triplet state and is absorbed by the fluorescent dye. This causes the molecule to transition to a higher excited triplet state (for example, excited state T ２ ) (arrow Ac1 in the figure). More specifically, the fluorescent dye first transitions to the excited state T ２ The state T has a higher potential than ２ The fluorescent dye then rapidly relaxes to the excited state T ２The molecule then transitions to the excited state T (arrow Ad1 in the figure). When the molecule is in this higher excited triplet state, it reacts with oxygen to generate active oxygen, which destroys the molecule and causes photobleaching (arrow Lg in the figure). The molecule that did not react with oxygen transitions back to the excited state T １ (arrow Ad2 in the figure).

[0053] In the image acquisition method and the fluorescence microscope 1 of this embodiment, the time interval t2 of the multiple light pulses PL2 is set to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or shorter than 10 picoseconds. ２ lifetime), the fluorescent dye molecule is in a higher excited triplet state (e.g., excited state T ２ ), the next light pulse PL2 is incident on the object B and is absorbed by the fluorescent dye. This causes the fluorescent dye molecules to enter a higher excited triplet state (for example, the excited state T ３ ) (arrow Ac2 in the figure). Then, the molecule transitions from an excited triplet state to an excited singlet state (for example, an excited state S １ ), the potential energy difference between the two molecules becomes large, making it easier for the molecule to transition to an excited singlet state before reacting with oxygen (arrow Ae3 in the figure). This prevents the molecule from being destroyed, and as a result, photobleaching can be reduced.

[0054] Parts (a) to (c) of Figure 14 show graphs containing the same plots as those shown in parts (a) to (c) of Figure 11, respectively. However, unlike curves C11 to C13 in Figure 11, curves C21 to C23 show theoretical values ​​calculated based on the above mechanism. Referring to Figure 14, it can be seen that the pulse number (N) dependence of the photobleaching rate for each fluorescent dye is close to the theoretical value. This indicates that the above mechanism is correct.

[0055] FIG. 15 shows the theoretical average intensity of the excitation light (I Ｎ / √N) and the photobleaching rate P Ｎ The photobleaching rate P when N = 1 １ The value normalized by (P Ｎ / P１ ) in the figure. In the figure, the line L31 shows the case where N=1, and the curve C31 shows the case where N=9. Comparing FIG. 15 with FIG. 12(b), it is clear that at least the average intensity (I Ｎ It can be seen that the experimental values ​​(part (b) of FIG. 12) agree well with the theoretical values ​​(FIG. 15) when √N / √N is in the range of 0.4 to 1.6. This also proves that the above mechanism is correct.

[0056] FIG. 16 is a graph showing the results of calculations of the relationship between the time interval t2 of the light pulse PL2 and the photobleaching rate. In FIG. 16, the line L41 represents the case where the excitation light is a single light pulse, and the curve C41 represents the case where the excitation light is a light pulse group consisting of multiple light pulses. In this calculation, the relaxation time between excited states in the excited triplet state is set to 5 picoseconds. Referring to this graph, the curve C41 intersects with the line L41 at a time interval t2 of 6.2 picoseconds, which is slightly longer than the relaxation time between excited states in the excited triplet state (5 picoseconds). When the time interval t2 is shorter than the intersection point, the photobleaching rate indicated by the curve C41 is lower than the photobleaching rate indicated by the line L41. This indicates that the photobleaching rate can be effectively reduced if the time interval t2 of the light pulse PL2 is equal to or shorter than the relaxation time between excited states in the excited triplet state.

[0057] There are a variety of fluorescent dyes, including some with a triplet relaxation time of 10 picoseconds or longer. An example of such a fluorescent dye is anthracene. Setting the time interval t2 of the light pulses PL2 to less than 10 picoseconds effectively reduces photobleaching of such fluorescent dyes. Setting the time interval t2 of the light pulses PL2 to 6 picoseconds or shorter effectively reduces photobleaching of fluorescent dyes with triplet relaxation times of longer than 6 picoseconds. The calculation results shown in FIG. 16 indicate that setting the time interval t2 of the light pulses PL2 to 6 picoseconds or shorter effectively reduces photobleaching of fluorescent dyes such as rose bengal with a triplet relaxation time of longer than 5 picoseconds. By making the time interval t2 of the light pulses PL2 shorter than 2 picoseconds, it is possible to effectively reduce photobleaching of fluorescent dyes whose relaxation time between excited states in the excited triplet state is 2 picoseconds or more (e.g., the above-mentioned methanol solution of rose bengal, ethanol solution of rhodamine 6G, rose bengal, and anthracene).By making the time interval t2 of the light pulses PL2 shorter than 1 picosecond, it is possible to effectively reduce photobleaching of fluorescent dyes whose relaxation time between excited states in the excited triplet state is 1 picosecond or more (e.g., the above-mentioned methanol solution of eosin Y, aqueous solution of eosin Y, methanol solution of rose bengal, ethanol solution of rhodamine 6G, rose bengal, and anthracene).

[0058] When the time interval t2 of the light pulse PL2 is set to be equal to or less than the relaxation time between excited states in the excited triplet state, the relaxation time is ２ to excited state T １ the relaxation time to ２ In this case, the excited state T １ to excited state T ２ In fluorescent dyes with a transition property, photobleaching can be effectively reduced.

[0059] As in this embodiment, the image acquisition method may include step S11 of inputting information about the type of fluorescent dye before step S12 of generating the light pulse group PG. Then, in step S12 of generating the light pulse group PG, the time interval t2 of the multiple light pulses PL2 may be set based on the information to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye. Similarly, the fluorescence microscope 1 may include an information input unit 11 that inputs information about the type of fluorescent dye. Based on the information, the pulse group generation unit 2 may set the time interval t2 of the light pulses PL2 to be equal to or shorter than the relaxation time between excited states in the excited triplet state of the fluorescent dye. According to these image acquisition methods and the fluorescence microscope 1, the time interval t2 of the light pulses PL2 can be set according to the relaxation time of the fluorescent dye used. Therefore, photobleaching of the fluorescent dye can be more effectively reduced.

[0060] As in the present embodiment, in step S12 of generating the light pulse group PG, the peak intensities of the plurality of light pulses PL2 may be uniform for each light pulse group PG. Similarly, in the fluorescence microscope 1, the peak intensities of the plurality of light pulses PL2 may be uniform for each light pulse group PG. In that case, a higher-order excited triplet state (for example, an excited state T ２ ) the peak intensity of the light pulse PL2 when it transitions to a higher excited triplet state (for example, excited state T ３ ) is approximately equal to the peak intensity of the light pulse PL2 when the excited triplet state transitions from the higher excited triplet state to the even higher excited triplet state. Therefore, the transition from the higher excited triplet state to the even higher excited triplet state is efficiently carried out, and photobleaching of the fluorescent dye can be more effectively reduced.

[0061] In relation to this, the inventor conducted the following experiment. First, as shown in parts (a) to (e) of Figure 17, five types of light pulse groups were generated, each with a different uniformity of peak intensity. Specifically, five types of light pulse groups were generated, with the ratio (σ / μ) of the standard deviation σ of the peak intensity of each light pulse to the average peak intensity μ being 0.02, 0.15, 0.26, 0.40, and 0.76, respectively. The fading rate was then measured when a fluorescent dye was irradiated with these light pulse groups. Figure 18 is a graph showing the results, illustrating the relationship between the ratio (σ / μ) and the fading rate. As is clear from Figure 18, the smaller the ratio (σ / μ), i.e., the more uniform the peak intensities of the light pulses, the lower the fading rate. This result is thought to be due to the fact that the less uniform the peak intensities of the light pulses, the closer they are to irradiation with a single light pulse. In other words, by ensuring that the peak intensities of the multiple light pulses PL2 are uniform for each light pulse group PG, photobleaching of the fluorescent dye can be more effectively reduced.

[0062] As described above, the repetition frequency of the optical pulse group PG may be 1 MHz or more. １ The lifetime (t1) of most fluorescent dyes is several microseconds or less. Therefore, when the repetition frequency of the light pulse group PG is 1 MHz or more, in other words, when the repetition period t1 of the light pulse group PG is 1 microsecond or less, photobleaching due to the above-mentioned mechanism is likely to occur, making the image acquisition method and fluorescence microscope 1 of this embodiment useful.

[0063] As in the present embodiment, the pulse group generating unit 2 may include an excitation light source 8 that repeatedly outputs a single optical pulse PL1, and a waveform control unit 10 that generates a plurality of optical pulses PL2 by modulating the single optical pulse PL1 output from the excitation light source 8. In this case, it is possible to easily configure the pulse group generating unit 2 that repeatedly generates an optical pulse group PG including a plurality of optical pulses PL2.

[0064] Here, we will explain in detail the configuration and operation of the modulation pattern calculation device 20. At the same time, we will explain a method for calculating a modulation pattern presented to the SLM 14 in order to generate a light pulse group PG including a plurality of light pulses PL2 from a single light pulse PL1.

[0065] FIG. 19 is a diagram schematically illustrating the configuration of the modulation pattern calculation device 20. The modulation pattern calculation device 20 is a computer having a processor, such as a smart device such as a personal computer, smartphone, or tablet terminal, or a cloud server. The modulation pattern calculation device 20 is electrically connected to the SLM 14 and calculates a phase modulation pattern for approximating the time-intensity waveform of the excitation light Ld to a waveform including a light pulse group PG consisting of a plurality of light pulses PL2, and provides data Da including the phase modulation pattern to the SLM 14. The modulation pattern calculation device 20 of this embodiment causes the SLM 14 to present a phase pattern including a phase pattern for phase modulation that imparts a phase spectrum to the excitation light Ld to obtain a waveform including the light pulse group PG, and a phase pattern for intensity modulation that imparts an intensity spectrum to the excitation light Ld to obtain a waveform including the light pulse group PG. To this end, the modulation pattern calculation device 20 includes an arbitrary waveform input unit 21, a phase spectrum design unit 22, an intensity spectrum design unit 23, and a modulation pattern generation unit 24. That is, the processor of the computer provided in the modulation pattern calculation device 20 realizes the function of the arbitrary waveform input unit 21, the function of the phase spectrum design unit 22, the function of the intensity spectrum design unit 23, and the function of the modulation pattern generation unit 24. Each function may be realized by the same processor or by different processors.

[0066] Fig. 20 is a diagram schematically illustrating an example of the hardware configuration of the modulation pattern calculation device 20. As shown in Fig. 20, the modulation pattern calculation device 20 can be physically configured as a normal computer including a processor (CPU) 201, main storage devices such as ROM 202 and RAM 203, input devices 204 such as a keyboard, mouse, and touch screen, output devices 205 such as a display (including a touch screen), a communication module 206 such as a network card for transmitting and receiving data to and from other devices, and an auxiliary storage device 207 such as a hard disk.

[0067] The processor 201 of the computer can realize the above-mentioned functions (the arbitrary waveform input unit 21, the phase spectrum design unit 22, the intensity spectrum design unit 23, and the modulation pattern generation unit 24) by the modulation pattern calculation program. Therefore, the modulation pattern calculation program causes the processor 201 of the computer to operate as the arbitrary waveform input unit 21, the phase spectrum design unit 22, the intensity spectrum design unit 23, and the modulation pattern generation unit 24 in the modulation pattern calculation device 20. The modulation pattern calculation program is stored in a storage device (storage medium) inside or outside the computer, such as the auxiliary storage device 207. The storage device may be a non-transitory recording medium. Examples of the recording medium include recording media such as a flexible disk, CD, and DVD, recording media such as ROM, semiconductor memory, and cloud server.

[0068] The arbitrary waveform input unit 21 accepts input of information related to the light pulse group PG from an operator. The operator inputs information related to the light pulse group PG (e.g., the repetition period t1 of the light pulse group PG, the pulse width of the light pulses PL2, the number of pulses of the light pulses PL2, the time interval t2 of the light pulses PL2, etc.) to the arbitrary waveform input unit 21. The information related to the light pulse group PG is provided to the phase spectrum design unit 22 and the intensity spectrum design unit 23. The phase spectrum design unit 22 calculates a phase spectrum of the excitation light Ld suitable for realizing the waveform of the given light pulse group PG. The intensity spectrum design unit 23 calculates an intensity spectrum of the excitation light Ld suitable for realizing the waveform of the given light pulse group PG. The modulation pattern generation unit 24 calculates a phase modulation pattern (e.g., a computer-generated hologram) for applying the phase spectrum determined by the phase spectrum design unit 22 and the intensity spectrum determined by the intensity spectrum design unit 23 to the excitation light Ld. Then, data Da including the calculated phase modulation pattern is provided to the SLM 14, and the SLM 14 is controlled based on the data Da.

[0069] Here, a method for calculating the phase spectrum and intensity spectrum corresponding to the time waveform of the light pulse group PG will be described in detail. The time waveform of the light pulse group PG is expressed as a function in the time domain, and the phase spectrum and intensity spectrum are expressed as functions in the frequency domain. Therefore, the phase spectrum and intensity spectrum corresponding to the time waveform of the light pulse group PG can be obtained by an iterative Fourier transform based on the time waveform of the light pulse group PG. In the method described below, the phase spectrum and intensity spectrum are calculated using an iterative Fourier transform method. Therefore, as shown in FIG. 19 , the phase spectrum design unit 22 has an iterative Fourier transform unit 22a. The intensity spectrum design unit 23 has an iterative Fourier transform unit 23a.

[0070] 21 shows the procedure for calculating the phase spectrum using the iterative Fourier method. First, the initial intensity spectrum function A(ω) and the phase spectrum function Ψ, which are functions of frequency ω, are calculated. n=0 (ω) are prepared (processing number (1) in the figure). In one example, the intensity spectrum function A(ω) and the phase spectrum function Ψ n=0(ω) respectively represent the intensity spectrum and phase spectrum of the excitation light La. Next, the intensity spectrum function A(ω) and the phase spectrum function Ψ n A waveform function (a) in the frequency domain including (ω) is prepared (processing number (2) in the figure). The subscript n indicates the time after the n-th Fourier transform process. Before the first (first) Fourier transform process, the phase spectrum function Ψ n (ω) as the initial phase spectrum function Ψ n=0 (ω) is used, where i is the imaginary unit.

[0071] Next, the function (a) is subjected to a Fourier transform from the frequency domain to the time domain (arrow A1 in the figure). This results in a time intensity waveform function b n A waveform function (b) in the frequency domain including (t) is obtained (processing number (3) in the figure).

[0072] Next, the time intensity waveform function b included in the function (b) n (t) is replaced with Target0(t) based on the desired waveform (processing numbers (4) and (5) in the figure).

[0073] Next, the function (d) is subjected to an inverse Fourier transform from the time domain to the frequency domain (arrow A2 in the figure). This results in the intensity spectrum function B n (ω) and the phase spectrum function Ψ n A waveform function (e) in the frequency domain including (ω) is obtained (processing number (6) in the figure).

[0074] Next, the intensity spectrum function B included in the above function (e) n In order to constrain (ω), it is replaced with the initial intensity spectrum function A 0 (ω) (process number (7) in the figure).

[0075] Thereafter, the above processes (1) to (7) are repeated multiple times to obtain the phase spectrum function Ψ nThe phase spectrum shape represented by (ω) can be made to approach the phase spectrum shape corresponding to the time waveform of the desired optical pulse group PG. IFTA (ω) is used to calculate the modulation pattern.

[0076] The iterative Fourier transform algorithm described above may include a process for suppressing the process from being guided to a local solution. Figure 22 shows the procedure for calculating a phase spectrum using such an iterative Fourier transform algorithm (hereinafter referred to as IFTA-Fienup). In Figure 22, steps (1) to (3) and (6) to (7) are the same as those in the previously described method, and therefore their explanations are omitted.

[0077] In this IFTA-Fienup, the time-intensity waveform function b included in the process (4) and (5), i.e., the function (b) after the Fourier transform n When replacing Target0(t) based on the desired waveform, Target0(t) is replaced by Target0(t) calculated by the following formula (g): n (t) is used (processing numbers (4) and (5) in the figure).

[0078] In the above formula (g), the function Target0(t) representing the desired waveform and the waveform function b after Fourier transform are n (t) (Target0(t)-b n (t)) is multiplied by a predetermined coefficient β and added to the desired waveform Target0(t) to obtain Target n If this value is less than 0, the Target n (t)=0.

[0079] However, even with this IFTA-Fienup, for example, if the function Target0(t) representing the desired waveform is a waveform function b n If it differs significantly from (t), it may still lead to a local minimum.

[0080] Therefore, the iterative Fourier method may be further improved as described below. Fig. 23 shows the procedure for calculating the phase spectrum. First, the initial intensity spectrum function A(ω) and the phase spectrum function Ψ, which are functions of frequency ω, are calculated. n=0 (ω) are prepared (processing number (1) in the figure). In one example, the intensity spectrum function A(ω) and the phase spectrum function Ψ n=0 (ω) respectively represent the intensity spectrum and phase spectrum of the input light.

[0081] Next, the intensity spectrum function A(ω) and the phase spectrum function Ψ n A waveform function (i) in the frequency domain including (ω) is prepared (processing number (2) in the figure). The subscript n indicates the time after the n-th Fourier transform process. Before the first (first) Fourier transform process, the phase spectrum function Ψ n (ω) as the initial phase spectrum function Ψ n=0 (ω) is used, where i is the imaginary unit.

[0082] Next, the function (i) is subjected to a Fourier transform from the frequency domain to the time domain, thereby obtaining a time intensity waveform function b n A waveform function (j) in the frequency domain including (t) is obtained (processing number (3) in the figure).

[0083] Next, the waveform function b after Fourier transformation n The difference between the waveform function b(t) and the function Target0(t) multiplied by the coefficient α (α×Target0(t)) is n A coefficient α is calculated so that it is smaller than the difference between (t) and the function Target0(t) (processing number (4) in the figure). In one example, the waveform function b after Fourier transform is calculated using the evaluation function shown in the following formula (k). n The standard deviation σ of α×Target0(t) for (t) is minimum (σ min In equation (k), D represents the number of data points, and t e , t s represent the start and end points of the time axis, respectively.

[0084] Next, the time-intensity waveform function b included in the function (j) after the Fourier transform n (t) is replaced based on the desired waveform (first replacement). At this time, the replacement is performed using a function Target0(t) representing the desired waveform multiplied by a coefficient α (α × Target0(t)). In one example, Target0(t) calculated by equation (m) in the above-mentioned IFTA-Fienup is replaced with α × Target0(t). n (t) (process numbers (5) and (6) in the figure). β in the formula is an arbitrary coefficient, and by appropriately selecting this coefficient β, it is possible to search for a better solution with a small number of iterations n and to prevent falling into a local solution.

[0085] Next, the function (n) is subjected to an inverse Fourier transform from the time domain to the frequency domain (arrow A2 in the figure). This results in the intensity spectrum function B n (ω) and the phase spectrum function Ψ n A waveform function (o) in the frequency domain including (ω) is obtained (processing number (7) in the figure).

[0086] Next, the intensity spectrum function B included in the above function (o) n In order to constrain (ω), it is replaced with the initial intensity spectrum function A 0 (ω) (second replacement, process number (8) in the figure).

[0087] Thereafter, the above processes (1) to (8) are repeated multiple times to obtain the phase spectrum function Ψ in the waveform function. n The phase spectrum shape represented by (ω) can be made to approach the phase spectrum shape corresponding to the desired time-intensity waveform. IFTA (ω) is used to calculate the modulation pattern.

[0088] In the iterative Fourier transform unit 22a of the phase spectrum design unit 22, the iterative Fourier method may be further improved as described below. Fig. 24 shows the procedure for calculating a phase spectrum using the improved iterative Fourier method. This calculation procedure is similar in many respects to the calculation procedure shown in Fig. 23, and therefore, a description thereof will be omitted where appropriate.

[0089] First, the iterative Fourier transform unit 22a performs the processes indicated by the process numbers (1) to (3) in the same manner as the calculation procedure shown in Fig. 23. Next, the iterative Fourier transform unit 22a obtains a coefficient α having the following characteristics (A) and (B) (process number (4) in the figure). (A) Waveform function b after Fourier transform n (t) and the function Target0(t) multiplied by a coefficient α (α × Target0(t) - b n (t)) is the waveform function b n (t) and the function Target0(t) (Target0(t)-b n Specifically, the difference (α×Target0(t)−b n (t)) is the time integral of the difference (Target0(t)-b n (B) At each time of the function Target0(t), the difference (α×Target0(t)−b n (t)), that is, the difference (α×Target0(t)−b n The ratio of (t) decreases as the strength increases.

[0090] In one example, the waveform function b after Fourier transform is calculated using the evaluation function shown in the following formula (q): n The pseudo standard deviation σ of α×Target0(t) for (t) is minimum (σ min In the formula (q), D represents the number of data points, and t e , t s represent the start and end points of the time axis, respectively. We(t) is the first weighting function. As shown in the formula (q), this evaluation function is a waveform function b n(t) and the difference between the multiplied function α×Target0(t) (α×Target0(t)−b n (t)), specifically, (α×Target0(t)−b n (t) ２ Furthermore, this evaluation function includes a weighting function We(t) multiplied by this function, and includes the time integral of this function multiplied by the weighting function We(t). Then, this evaluation function, i.e., the time integral, is minimized (σ min The weighting function We(t) is a function that has a larger weight value at each time of the function Target0(t) before multiplication as the intensity increases. In one example, the weighting function We(t) includes a function obtained by multiplying the function Target0(t) by another coefficient C1, and is represented by the following mathematical formula (r), for example. In other words, the weighting function We(t) may be determined based on the function Target0(t).

[0091] As explained above, by including the weighting function We(t) in the evaluation function shown in formula (q), the above-mentioned feature (B) can be imparted to the coefficient α. Fig. 25 shows an example of the weighting function We(t) when Target0(t) is an optical pulse group consisting of a plurality of optical pulses. Curve C51 in Fig. 25 shows the case where the coefficient C of formula (r) is 1, and curve C52 shows the case where the coefficient C of formula (r) is 2.

[0092] Thereafter, the iterative Fourier transform unit 22a performs the processes (5) to (8) similar to the calculation procedure shown in Fig. 23. Thereafter, by repeating the processes (1) to (8) multiple times, the phase spectrum function Ψ in the waveform function is calculated. n The phase spectrum shape represented by (ω) can be made to approach the phase spectrum shape corresponding to the desired time-intensity waveform. IFTA (ω) is provided to the modulation pattern generator 24 .

[0093] 26 shows the calculation procedure in the iterative Fourier transform unit 23a of the intensity spectrum design unit 23. The iterative Fourier transform unit 23a calculates the intensity spectrum using a method similar to the calculation method used by the iterative Fourier transform unit 22a described above.

[0094] First, the iterative Fourier transform unit 23a calculates the initial intensity spectrum function A k=0 (ω) and the phase spectrum function Ψ(ω) are prepared (processing number (1) in the figure). Next, the iterative Fourier transform unit 23a prepares the intensity spectrum function A k A waveform function (s) in the frequency domain including the phase spectrum function Ψ(ω) is prepared (process number (2) in the figure). The subscript k indicates the time after the kth Fourier transform process. Before the first (first) Fourier transform process, the intensity spectrum function A k (ω) as the above initial intensity spectrum function A k=0 (ω) is used, where i is the imaginary unit.

[0095] Next, the iterative Fourier transform unit 23a performs a Fourier transform from the frequency domain to the time domain on the function (s). As a result, the time intensity waveform function b k A waveform function (t) in the frequency domain including (t) is obtained (processing number (3) in the figure).

[0096] Next, the iterative Fourier transform unit 23a calculates a coefficient α having the following characteristics (C) and (D) (process number (4) in the figure). (C) Waveform function b after Fourier transform ｋ (t) and the function Target0(t) multiplied by a coefficient α (α × Target0(t) - b ｋ (t)) is the waveform function b ｋ (t) and the function Target0(t) (Target0(t)-b ｋ Specifically, the difference (α×Target0(t)−b ｋ (t)) is the time integral of the difference (Target0(t)-b ｋ(D) At each time of the function Target0(t), the difference (α×Target0(t)−b ｋ (t)), that is, the difference (α×Target0(t)−b ｋ The ratio of (t) decreases as the strength increases.

[0097] In one example, the waveform function b after Fourier transform is calculated using the evaluation function shown in the following formula (u): ｋ The pseudo standard deviation σ of α×Target0(t) for (t) is minimum (σ min In the formula (u), D represents the number of data points, and t e , t s represent the start and end points of the time axis, respectively. We(t) is the first weighting function. As shown in the formula (u), this evaluation function is a waveform function b ｋ (t) and the difference between the multiplied function α×Target0(t) (α×Target0(t)−b ｋ (t)), specifically, (α×Target0(t)−b ｋ (t) ２ Furthermore, this evaluation function includes a weighting function We(t) multiplied by this function, and includes the time integral of this function multiplied by the weighting function We(t). Then, this evaluation function, i.e., the time integral, is minimized (σ min The coefficient α such that: ∑ t = ...

[0098] Next, the iterative Fourier transform unit 23a calculates the time-intensity waveform function b included in the function (v) after the Fourier transform. k(t) based on the desired waveform (first replacement). At this time, the iterative Fourier transform unit 23a performs the replacement using a function Target0(t) representing the desired waveform multiplied by a coefficient α (α×Target0(t)). In one example, Target0(t) calculated by the formula (w) k (t) (processing numbers (5) and (6) in the figure).

[0099] Next, the iterative Fourier transform unit 23a performs an inverse Fourier transform on the function (w) from the time domain to the frequency domain. As a result, the intensity spectrum function C k (ω) and the phase spectrum function Ψ k A waveform function (y) in the frequency domain including (ω) is obtained (processing number (7) in the figure). Next, the iterative Fourier transform unit 23a calculates the phase spectrum function Ψ included in the function (y). k In order to constrain (ω), it is replaced with the initial phase spectrum function Ψ 0 (ω) (second replacement, process number (8) in the figure).

[0100] The iterative Fourier transform unit 23a calculates the intensity spectrum function C in the frequency domain after the inverse Fourier transform. k (ω) is subjected to filtering based on the intensity spectrum of the excitation light La. Specifically, the intensity spectrum function C k In the intensity spectrum represented by (ω), a portion exceeding the cutoff intensity for each wavelength determined based on the intensity spectrum of the excitation light La is cut off. In one example, the cutoff intensity for each wavelength is determined based on the intensity spectrum of the excitation light La (in this embodiment, the initial intensity spectrum function A k=0 In this case, the intensity spectrum function C is set to coincide with the k (ω) is the initial intensity spectrum function A k=0 At frequencies greater than (ω), the intensity spectrum function A k (ω) as the value of the initial intensity spectrum function A k=0 The value of (ω) is taken as the intensity spectrum function C k(ω) is the initial intensity spectrum function A k=0 At frequencies less than or equal to (ω), the intensity spectrum function A k The intensity spectrum function C k The value of (ω) is taken into account. The intensity spectrum function C included in the above function (y) k (ω) is the intensity spectrum function A after filtering by the above formula (z1). k (ω) is replaced by C k A function C' obtained by multiplying (ω) by an arbitrary coefficient k Alternatively, a method may be used in which (ω) is defined and the cutoff intensity is changed relatively (process number (9) in the figure).

[0101] Thereafter, the iterative Fourier transform unit 23a repeats the above processes (1) to (8) (or (1) to (9)) multiple times to obtain the intensity spectrum function A k The intensity spectrum shape represented by (ω) can be made to approach the intensity spectrum shape corresponding to the desired time intensity waveform. IFTA (ω) is provided to the modulation pattern generator 24 .

[0102] Fig. 27 is a flowchart showing a modulation pattern calculation method realized by the modulation pattern calculation device 20 described above. The above-mentioned modulation pattern calculation program causes a computer processor 201 (see Fig. 20) to execute each step included in this flowchart. As shown in Fig. 27, first, an operator inputs information about the time waveform of a desired optical pulse group PG to an arbitrary waveform input unit 21 (input step S20). Next, a phase spectrum and an intensity spectrum are calculated in the phase spectrum design unit 22 and the intensity spectrum design unit 23, respectively, to bring the time intensity waveform closer to the desired waveform (phase spectrum calculation step S21, intensity spectrum calculation step S23).

[0103] The phase spectrum calculation step S21 includes an iterative Fourier transform step S22 by the iterative Fourier transform unit 22a. Details of the iterative Fourier transform step S22 are the same as the operation of the iterative Fourier transform unit 22a described above. The finally obtained phase spectrum function Ψ IFTA (ω) is provided to the subsequent modulation pattern calculation step S25. The intensity spectrum calculation step S23 includes an iterative Fourier transform step S24 by the iterative Fourier transform unit 23a. The details of the iterative Fourier transform step S24 are the same as the operation of the iterative Fourier transform unit 23a described above. The finally obtained intensity spectrum function A IFTA (ω) is provided to the subsequent modulation pattern calculation step S25.

[0104] In the modulation pattern calculation step S25, the phase spectrum function Ψ IFTA (ω) and the intensity spectrum function A IFTA Based on (ω), a modulation pattern is calculated, which is presented to the SLM 14.

[0105] In the above explanation, in order to make the time-intensity waveform closer to the desired waveform, the phase spectrum function Ψ IFTA (ω) and the intensity spectrum function A IFTA (ω) and a modulation pattern based on these functions is presented to the SLM 14. The present invention is not limited to this configuration, and for example, a phase spectrum function Ψ IFTA (ω) and the intensity spectrum function A IFTA In this case, a spectrum prepared (or selected) in advance may be used as the other spectrum, or the other spectrum may remain the excitation light La without being modulated.

[0106] 28 and 29 show a modified example of the calculation procedure for the phase spectrum using the iterative Fourier method. This calculation procedure differs from the above calculation procedure (see FIGS. 24 and 26) in that the coefficient β in process number (5) is replaced by a weighting function Wr(t). In this modification, the above formulas (m) and (w) are replaced by the following formulas (z2) and (z3), respectively. That is, in this modified example, the time intensity waveform function b after Fourier transform from the function {α×Target0(t)} is obtained. n (t) (or b k The first substitution is performed using the sum of a function {α×Target0(t)} minus a function multiplied by a weighting function Wr(t).

[0107] The weighting function Wr(t) is a function that has a larger weight value at each time of the function Target0(t) as the intensity increases. In one example, the weighting function Wr(t) includes a function obtained by multiplying the function Target0(t) by another coefficient C2, and is represented by, for example, the following formula. In other words, the weighting function Wr(t) may be determined based on the function Target0(t).

[0108] By replacing the coefficient β with the weighting function Wr(t), the magnitude of the difference is emphasized in the section of Target0(t) where the intensity is high compared to other sections. Therefore, when performing the iterative Fourier transformation, a result is calculated that particularly reduces the difference in this section. Therefore, the time waveform of the excitation light Ld in a section where the light intensity is particularly high can be made to more accurately approximate the desired waveform.

[0109] The present invention is not limited to the above-described embodiments and various other modifications are possible. For example, in the above-described embodiments, a fluorescence microscope 1 including a pulse group generator 2, an optical system 3, a photodetector 4, and a processing unit 5 has been described. FIG. 30 is a diagram showing an excitation light irradiation unit 100 used in a fluorescence microscope. The excitation light irradiation unit 100 includes a pulse group generator 2. The configuration of the pulse group generator 2 is similar to that of the fluorescence microscope 1. The effects of the fluorescence microscope 1 of the above-described embodiments are also achieved in this excitation light irradiation unit 100. The excitation light irradiation unit 100 may include an information input unit 11 in addition to the pulse group generator 2. FIG. 31 is a diagram showing a waveform control unit 200 used in a fluorescence microscope. The waveform control unit 200 repeatedly generates a light pulse group PG including multiple light pulses PL2 to be irradiated onto an object B containing a fluorescent dye. To this end, the waveform control unit 200 includes a waveform control unit 10 optically coupled to an excitation light source 8 provided outside the waveform control unit 200. The waveform control unit 10 generates multiple light pulses PL2 by modulating the single light pulse PL1 output from the excitation light source 8. The effects of the fluorescence microscope 1 of the above-described embodiment are similarly achieved in the waveform control unit 200. The waveform control unit 200 may include an information input unit 11 in addition to the waveform control unit 10.

[0110] 1...fluorescence microscope, 2...pulse group generation unit, 3...optical system, 4...photodetector, 5...processing unit, 6...display unit, 7...intensity controller, 8...excitation light source, 9...waveform measurement device, 10...waveform control unit, 11...information input unit, 12, 16...diffraction grating, 13, 15...lens, 14...spatial light modulator (SLM), 17...modulation surface, 17a...modulation region, 20...modulation pattern calculation device, 21...arbitrary waveform input unit, 22...phase spectrum design unit, 22a...iterative Fourier transform unit, 23...intensity spectrum design unit, 23a...iterative Fourier transform unit, 24...modulation pattern generation unit, 31...light branching element, 3 2, 33...Galvanometer mirror, 34, 35...Coupling lens, 100...Excitation light irradiation unit, 200...Waveform control unit, 201...Processor (CPU), 202...ROM, 203...RAM, 204...Input device, 205...Output device, 206...Communication module, 207...Auxiliary storage device, B...Object, D1, D2...Direction, Da...Data, Db...Information, La...Excitation light, Ld...Excitation light, Le...Fluorescence, PG...Excitation light pulse group, PL1...Light pulse, PL2...Excitation light pulse, Sa...Electrical signal, Sb...Data, t1...Repetition period, t2...Time interval.

Claims

1. Repeatedly generating an excitation light pulse group including a plurality of excitation light pulses; irradiating an object containing a fluorescent dye with the group of excitation light pulses; detecting intensities of fluorescence generated at a plurality of locations on the object by irradiation with the group of excitation light pulses; generating a fluorescence image based on the intensities of the fluorescence at the multiple locations of the object; Including, An image acquisition method, wherein in the step of generating the group of excitation light pulses, the time interval between the multiple excitation light pulses is equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or is shorter than 10 picoseconds.

2. The relaxation time between excited states in the excited triplet state of the fluorescent dye is the excited state T ２ to excited state T １ The image acquisition method according to claim 1 , wherein the relaxation time to the target position is a relaxation time to the target position.

3. The method further includes a step of inputting information regarding the type of the fluorescent dye before the step of generating the group of excitation light pulses, 3. The image acquisition method according to claim 1, wherein in the step of generating the group of excitation light pulses, a time interval between the plurality of excitation light pulses is set based on the information so as to be equal to or less than a relaxation time between excited states in an excited triplet state of the fluorescent dye.

4. 3. The image acquisition method according to claim 1, wherein in the step of generating the group of excitation light pulses, when the time interval between the plurality of excitation light pulses is set to be shorter than 10 picoseconds, the time interval between the plurality of excitation light pulses is set to be shorter than 1 picosecond.

5. 3. The image acquiring method according to claim 1, wherein in the step of generating the excitation light pulse groups, peak intensities of the plurality of excitation light pulses are made uniform for each of the excitation light pulse groups.

6. 3. The image acquiring method according to claim 1, wherein in the step of generating the group of excitation light pulses, a repetition frequency when the group of excitation light pulses is repeatedly generated is 1 MHz or more.

7. a pulse group generating unit that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses; an optical system for irradiating a target containing a fluorescent dye with the group of excitation light pulses; a photodetector that detects the intensity of fluorescence generated at a plurality of points on the object by irradiation with the group of excitation light pulses; a processing unit that generates a fluorescent image based on the intensities of the fluorescent light at the plurality of points of the object; Equipped with A fluorescence microscope, wherein the time interval between the multiple excitation light pulses is equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or shorter than 10 picoseconds.

8. The relaxation time between excited states in the excited triplet state of the fluorescent dye is the excited state T ２ to excited state T １ The fluorescence microscope according to claim 7, wherein the relaxation time to the

9. An information input unit for inputting information regarding the type of the fluorescent dye, 9. The fluorescence microscope according to claim 7, wherein the pulse group generating unit sets, based on the information, a time interval between the plurality of excitation light pulses to be equal to or less than a relaxation time between excited states in an excited triplet state of the fluorescent dye.

10. 9. The fluorescence microscope of claim 7, wherein when the time interval between the plurality of excitation light pulses is less than 10 picoseconds, the time interval between the plurality of excitation light pulses is less than 1 picosecond.

11. 9. The fluorescence microscope according to claim 7, wherein the peak intensities of the plurality of excitation light pulses are uniform for each group of excitation light pulses.

12. The pulse group generating unit An excitation light source that repeatedly outputs a single optical pulse; a waveform control unit optically coupled to the pump light source, modulating the single light pulse output from the pump light source to generate the plurality of pump light pulses; The fluorescence microscope according to claim 7 or 8, further comprising:

13. 9. The fluorescence microscope according to claim 7, wherein the pulse group generating section repeatedly generates the excitation light pulse group at a repetition frequency of 1 MHz or more.

14. An excitation light irradiation unit for use in a fluorescence microscope, comprising: a pulse group generating unit that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses to be irradiated onto an object including a fluorescent dye; An excitation light irradiation unit, wherein the time interval between the multiple excitation light pulses is equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or shorter than 10 picoseconds.

15. An information input unit for inputting information regarding the type of the fluorescent dye, 15. The excitation light irradiation unit according to claim 14, wherein the pulse group generation section sets, based on the information, a time interval between the plurality of excitation light pulses to be equal to or less than a relaxation time between excited states in an excited triplet state of the fluorescent dye.

16. A waveform control unit for a fluorescence microscope that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses to be irradiated onto an object including a fluorescent dye, the waveform control unit comprising: a waveform control unit that is optically coupled to a pump light source that repeatedly outputs a single optical pulse, and modulates the single optical pulse output from the pump light source to generate the multiple pump light pulses; A waveform control unit, wherein the time interval between the multiple excitation light pulses is equal to or less than the relaxation time between excited states in the excited triplet state of the fluorescent dye, or shorter than 10 picoseconds.

17. An information input unit for inputting information regarding the type of the fluorescent dye, The waveform control unit according to claim 16, wherein the waveform control section sets, based on the information, a time interval between the plurality of excitation light pulses to be equal to or less than a relaxation time between excited states in an excited triplet state of the fluorescent dye.