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

Abstract

Provided is an image acquisition method including: a step of repeatedly generating an excitation light pulse group including a plurality of excitation light pulses; a step for irradiating an object containing the fluorescent dye with the excitation light pulse group; a step for detecting the intensity of fluorescence generated at a plurality of sites of the object by irradiation with the excitation light pulse group; and a step for generating a fluorescence image on the basis of the intensities of the fluorescence at the plurality of sites of 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 less than or equal to the relaxation time between excitation states in the excitation triplet state of the fluorescent dye or to be shorter than 10 picoseconds.

Description

Technical Field

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

[0002] Patent Document 1 and Non-Patent Document 1 disclose that the pulse interval of the excitation light pulse is set to 10 picoseconds to 50 picoseconds or more in order to reduce the fading of the fluorescence when the fluorescent pigment is excited. Non-Patent Document 2 discloses that the pulse interval of the excitation light pulse is set to be greater than 1 microsecond in order to reduce the fading of the fluorescence when the fluorescent pigment is excited. Patent Document 2 discloses a fluorescence microscope that controls the number of pulses and the pulse interval of the excitation light.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: International Publication No. 2009 / 035768

[0006] Patent Document 2: U.S. Patent Application Publication No. 2010 / 0187208

[0007] Non-patent literature

[0008] Non-patent document 1: Na Ji et al., "High-speed, low-photodamage nonlinear imaging using passive pulse splitters", Nature Methods, Volume 5, No. 2, pp. 197-202 (2008)

[0009] Non-patent document 2: Gerald Donnert et al., "Major signal increase in fluorescence microscopy through dark-state relaxation", Nature Methods, Volume 4, No. 1, pp. 81-86 (2007) Summary of the invention

[0010] Problems to be solved by the invention

[0011] A fluorescence microscope irradiates multiple parts of an object containing a fluorescent pigment with excitation light, detects the fluorescence generated by the fluorescent pigment, and outputs a fluorescence image. In such a fluorescence microscope, pulsed excitation light is sometimes irradiated on the object. For example, in a multiphoton excitation fluorescence microscope, in order to increase the photon density of the excitation light and generate multiphoton absorption, an excitation light pulse with an extremely short pulse width, such as a picosecond or femtosecond level, is irradiated on the object. However, if fluorescence is continuously detected while irradiating the excitation light pulse, the fluorescence intensity gradually decreases. This phenomenon is called photofading. Photofading limits the observation time of the object, and therefore, it is desirable to reduce photofading in a fluorescence microscope.

[0012] An object of the present disclosure is to provide an image acquisition method, a fluorescence microscope, an excitation light irradiation unit, and a waveform control unit that can reduce photofading.

[0013] Technical solutions to solve problems

[0014] [1] One embodiment provides an image acquisition method, comprising: repeatedly generating an excitation light pulse group including a plurality of excitation light pulses; irradiating an object including a fluorescent pigment with the excitation light pulse group; detecting the intensity of fluorescence generated at a plurality of locations of the object by irradiation with the excitation light pulse group; and generating a fluorescent image based on the intensity of the fluorescence at the plurality of locations of 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 less than a relaxation time between excitation states in an excited triplet state of the fluorescent pigment or shorter than 10 picoseconds.

[0015] [2] One embodiment provides a fluorescence microscope comprising: a pulse group generating unit that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses; an optical system that irradiates an object including a fluorescent pigment with the excitation light pulse group; a photodetector that detects the intensity of fluorescence generated at a plurality of locations of the object by irradiation with the excitation light pulse group; and a processing unit that generates a fluorescence image based on the intensity of the fluorescence at the plurality of locations of the object. The time interval between the plurality of excitation light pulses is less than or equal to a relaxation time between excitation states in an excited triplet state of the fluorescent pigment or is shorter than 10 picoseconds.

[0016] Photofading is caused by the following mechanism. First, an excitation light pulse is incident on the object and absorbed by the fluorescent pigment. At this time, the fluorescent pigment is excited from the base state S0 to the excited singlet state (for example, excited state S1). Then, a large number of molecules return to the base state S0 again, thereby generating fluorescence. However, a part of the molecules do not return to the base state S0, but are transformed into an excited triplet state (for example, excited state T1). This transition is called intersystem crossing. Moreover, if the molecule is in the excited triplet state, the next excitation light pulse is incident on the object and is absorbed by the fluorescent pigment, and the molecule is transformed into a higher-order excited triplet state (for example, excited state T2). When the molecule is in the excited triplet state, it reacts with oxygen to produce active oxygen, thereby destroying the molecule and generating photofading. Especially in the case of multi-photon excitation microscopy using near-infrared light, molecules in the higher-order excited triplet state (for example, excited state T2) contribute greatly to photofading.

[0017] In the image acquisition method of [1] and the fluorescence microscope of [2], (a) the time interval between multiple excitation light pulses is set to be less than the relaxation time between the excited states in the triplet excited state of the fluorescent pigment or (b) shorter than 10 picoseconds. In the case of (a), when the molecules of the fluorescent pigment are in a higher-order triplet excited state (for example, excited state T2), the next excitation light pulse is incident on the object and absorbed by the fluorescent pigment, thereby converting the molecules of the fluorescent pigment to a higher-order triplet excited state (for example, excited state T3). As a result, the potential energy difference between the triplet excited state and the singlet excited state (for example, excited state S1) of the molecule becomes larger, and the molecule is easily converted to the singlet excited state before reacting with oxygen. Therefore, the destruction of the molecule is prevented, and as a result, photofading can be reduced. There are various fluorescent pigments among fluorescent pigments, among which there are also fluorescent pigments whose relaxation time between the excited states in the triplet excited state is more than 10 picoseconds. As in (b), by shortening the time interval between multiple excitation light pulses to be shorter than 10 picoseconds, the photofading of such fluorescent pigments can be reduced.

[0018] [3] In the image acquisition method of [1] or the fluorescence microscope of [2], the relaxation time between the excited states in the triplet excited state of the fluorescent dye may be the relaxation time from the excited state T2 to the excited state T1, so-called T2 lifetime. In this case, in a fluorescent dye having the characteristic of transitioning from the excited state T1 to the excited state T2 by an excitation light pulse, photofading can be effectively reduced.

[0019] [4] The image acquisition method of [1] or [3] may also include a step of inputting information related to the type of fluorescent pigment 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 to be less than the relaxation time between the excitation states in the triplet excited state of the fluorescent pigment based on the information. The fluorescence microscope of [2] or [3] may also have an information input unit for inputting information related to the type of fluorescent pigment. The pulse group generating unit may also set the time interval between the multiple excitation light pulses to be less than the relaxation time between the excitation states in the triplet excited state of the fluorescent pigment based on the information. According to these image acquisition methods and fluorescence microscopes, the time interval between the multiple excitation light pulses can be set according to the relaxation time of the fluorescent pigment used. Therefore, the photofading of the fluorescent pigment can be reduced more effectively.

[0020] [5] In the step of generating an excitation light pulse group in the image acquisition method of any one of [1], [3] or [4], when 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 the fluorescence microscope of any one of [2] to [4], when 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, the photofading of the fluorescent pigment having a relaxation time of 1 picosecond or more can be reduced.

[0021] [6] In the step of generating an excitation light pulse group in the image acquisition method of any one of [1], [3] to [5], the peak intensity of multiple excitation light pulses can also be made uniform in each excitation light pulse group. Similarly, in the fluorescence microscope of any one of [2] to [5], the peak intensity of multiple excitation light pulses can also be made uniform in each excitation light pulse group. In this case, the peak intensity of the excitation light pulse when it is transformed into a higher-order excited triplet state (for example, excited state T2) is approximately equal to the peak intensity of the excitation light pulse when it is transformed into a higher-order excited triplet state (for example, excited state T3). Therefore, the transition from a high-order excited triplet state to a higher-order excited triplet state is effectively performed. Therefore, the photofading of the fluorescent pigment can be more effectively reduced.

[0022] [7] In the step of generating an excitation light pulse group in the image acquisition method of any one of [1], [3] to [6], the repetition frequency when repeatedly generating the excitation light pulse group may also be greater than 1 MHz. Similarly, in the fluorescence microscope of any one of [2] to [6], the repetition frequency when repeatedly generating the excitation light pulse group may also be greater than 1 MHz. The relaxation time of the excited triplet state (e.g., T1 lifetime) is less than a few microseconds in many fluorescent pigments. Therefore, when the repetition frequency of the excitation light pulse group is greater than 1 MHz, in other words, when the time interval between the excitation light pulse groups is less than 1 microsecond, photofading based on the above mechanism is likely to occur. Therefore, any of the image acquisition methods and fluorescence microscopes described above are useful.

[0023] [8] In the fluorescence microscope of any one of [2] to [7], the pulse group generating unit may include: an excitation light source that repeatedly outputs a single light pulse; and a waveform control unit that is optically coupled to the excitation light source and modulates the single light pulse output from the excitation light source to generate a plurality of excitation light pulses. In this case, the pulse group generating unit that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses can be simply configured.

[0024] [9] One embodiment provides an excitation light irradiation unit for a fluorescence microscope, wherein the unit comprises a pulse group generating unit that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses for irradiating an object including a fluorescent pigment. The time interval between the plurality of excitation light pulses is less than or shorter than 10 picoseconds of a relaxation time between excitation states in an excited triplet state of the fluorescent pigment. According to the excitation light irradiation unit, photofading can be reduced.

[0025]

[10] The excitation light irradiation unit of [9] may also include an information input unit for inputting information related to the type of fluorescent pigment. The pulse group generation unit may also set the time interval between the multiple excitation light pulses to be less than the relaxation time between the excitation states in the triplet excited state of the fluorescent pigment based on the information. According to the excitation light irradiation unit, the time interval between the multiple excitation light pulses can be set according to the relaxation time of the fluorescent pigment used. Therefore, the photofading of the fluorescent pigment can be reduced more effectively.

[0026]

[11] A waveform control unit in one embodiment is a waveform control unit for a fluorescence microscope that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses for irradiating an object including a fluorescent pigment. The waveform control unit includes a waveform control unit. The waveform control unit is optically coupled to an excitation light source that repeatedly outputs a single light pulse, modulates the single light pulse output from the excitation light source, and generates a plurality of excitation light pulses. The time interval between the plurality of excitation light pulses is less than or shorter than 10 picoseconds of the relaxation time between the excitation states in the triplet excited state of the fluorescent pigment. According to the waveform control unit, photofading can be reduced.

[0027]

[12] The waveform control unit of

[11] may also include an information input unit for inputting information related to the type of fluorescent pigment. The waveform control unit may also set the time interval between the multiple excitation light pulses to be less than the relaxation time between the excitation states in the triplet excited state of the fluorescent pigment based on the information. According to the waveform control unit, the time interval between the multiple excitation light pulses can be set according to the relaxation time of the fluorescent pigment used. Therefore, the photofading of the fluorescent pigment can be reduced more effectively.

[0028] Effects of the Invention

[0029] 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 photofading. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram showing the structure of a fluorescence microscope according to one embodiment.

[0031] Figure 2 1 and 2 are diagrams schematically showing the temporal waveform of the excitation light. Part (a) schematically shows the temporal waveform of the excitation light output from the excitation light source, and part (b) schematically shows the temporal waveform of the excitation light output from the waveform control unit.

[0032] Figure 3 It is a diagram showing a specific configuration example of a waveform control unit.

[0033] Figure 4 A diagram showing a modulation surface of a spatial light modulator (SLM).

[0034] Figure 5 1 is a graph showing an example of excitation light, wherein part (a) shows a spectrum waveform of a single pulse excitation light, and part (b) shows a temporal intensity waveform of the excitation light.

[0035] Figure 6 1 is a graph showing an example of excitation light, wherein part (a) shows the spectrum waveform of the excitation light when rectangular wave-shaped phase spectrum modulation is given to the SLM, and part (b) shows the time intensity waveform of the excitation light.

[0036] Figure 7 It is a diagram showing a specific configuration example of a fluorescence microscope.

[0037] Figure 8 Detailed description of the invention The invention is a flowchart showing an image acquisition method according to one embodiment.

[0038] Fig. 9 It is a graph showing the time waveforms of light pulses and light pulse groups, wherein parts (a) to (d) respectively 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 sixteen light pulses.

[0039] Fig.10 The graphs show the dependence of the photofading speed on the excitation light intensity, wherein (a) shows a graph related to Eosin Y, (b) shows a graph related to fluorescein, and (c) shows a graph related to C-Naphox-TEG.

[0040] Fig.11 The graphs show the dependence of the photofading speed on the number of pulses (N), wherein (a) shows a graph related to Eosin Y, (b) shows a graph related to fluorescein, and (c) shows a graph related to C-Naphox-TEG.

[0041] Fig.12 is a graph showing the relationship between the excitation light intensity and the light fading speed, wherein (a) represents the average intensity of the excitation light (I N / √N) and light fading speed P N The relationship between the light fading speed P and the light fading speed P is shown in the graph shown in part (a). N The case where the light fading speed P1 is normalized when N=1.

[0042] Fig.13 It is a diagram showing the mechanism of photofading.

[0043] Fig.14 is a graph showing the dependence of the light fading speed on the number of pulses (N), wherein parts (a) to (c) respectively show the pulses including Fig.11 The same graphs as those shown in parts (a) to (c) of FIG. Fig.11 The curves are different, indicating that Fig.13 Theoretical values ​​calculated using the mechanism shown.

[0044] Fig.15 Is based on Fig.13 Theoretically, the average intensity of the excitation light (I N / √N) and the light fading speed PN The value normalized by the light fading speed P1 when N=1 (P N / P1) relationship diagram.

[0045] Fig.16 This is a graph showing the result of calculating the relationship between the time interval of light pulses and the light fading speed.

[0046] Fig.17 This is a graph showing the time waveforms of five types of optical pulse groups having mutually different peak intensity uniformities.

[0047] Fig.18 It is a measure of the irradiation of fluorescent dyes Fig.17 The graphs of the color fading speed results for the five light pulse groups shown in parts (a) to (e) are as follows. Fig.18 It shows the relationship between the ratio (σ / μ) and the color fading speed.

[0048] Fig.19 This is a diagram schematically showing the structure of a modulation pattern calculation device.

[0049] Fig. 20 This is a diagram schematically showing a hardware configuration example of a modulation pattern calculation device.

[0050] Fig.21 This is a diagram showing the calculation procedure of the phase spectrum based on the iterative Fourier method.

[0051] Fig. 22 This is a diagram showing the calculation procedure of the phase spectrum based on the iterative Fourier method.

[0052] Fig.23 This is a diagram showing the calculation procedure of the phase spectrum.

[0053] Fig.24 This is a diagram showing the calculation procedure of the phase spectrum based on the iterative Fourier method.

[0054] Fig.25 This is a diagram showing an example of the weighting function We(t) when Target0(t) is a multi-pulse.

[0055] Fig.26 It is a diagram showing the calculation procedure in the iterative Fourier transform unit of the intensity spectrum design unit.

[0056] Fig. 27 is a flow chart showing a method for calculating a modulation pattern.

[0057] Fig.28 This is a diagram showing the calculation procedure of the phase spectrum based on the iterative Fourier method.

[0058] Fig.29This is a diagram showing the calculation procedure of the phase spectrum based on the iterative Fourier method.

[0059] Fig.30 This is a diagram showing an excitation light irradiation unit used in a fluorescence microscope.

[0060] Fig.31 is a diagram showing a waveform control unit used for a fluorescence microscope. DETAILED DESCRIPTION

[0061] 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 denoted by the same reference numerals, and repeated descriptions are omitted.

[0062] Figure 1 1 is a diagram showing the structure of a fluorescence microscope 1 according to an embodiment. The fluorescence microscope 1 is a device that irradiates an object B as an observation target with excitation light Ld and detects fluorescence Le generated in the object B thereby to obtain a fluorescence image. Figure 1 As shown, the fluorescence microscope 1 of this embodiment includes a pulse group generator 2, an optical system 3, a photodetector 4, a processor 5, a display 6, and an information input unit 11. The pulse group generator 2 includes an excitation light source 8 and a waveform controller 10.

[0063] The excitation light source 8 is optically coupled to the waveform control section 10 , and provides the excitation light La to the waveform control section 10 . Figure 2 Part (a) of FIG. 8 schematically shows a time waveform of the excitation light La output from the excitation light source 8. Figure 2 As shown in part (a) of , the excitation light La includes repetitions of a single light pulse PL1. The repetition period t1 of the light pulse PL1 is, for example, greater than 1 nanosecond and less than 10 microseconds, or greater than 10 nanoseconds and less than 100 nanoseconds. In other words, the repetition frequency of the light pulse PL1 is, for example, greater than 0.1 MHz and less than 1 GHz, or greater than 10 MHz and less than 100 MHz. In one example, the repetition frequency of the light pulse PL1 is 80 MHz. The repetition period t1 of the light pulse PL1 may also be constant. The repetition period t1 of the light pulse PL1 may be defined as the peak interval of the light pulse PL1. The full width at half maximum (FWHM) of the light pulse PL1 is, for example, greater than 5 femtoseconds and less than 200 femtoseconds, or greater than 30 femtoseconds and less than 200 femtoseconds. The excitation light source 8 repeatedly outputs this single light pulse PL1. The excitation light source 8 is, for example, a laser light source such as a solid 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.

[0064] The waveform control section 10 converts the excitation light La supplied from the excitation light source 8 into the excitation light Ld. The excitation light Ld is output from the pulse group generation section 2 . Figure 2Part (b) of FIG. 1 is a diagram schematically showing the time waveform of the excitation light Ld output from the waveform control unit 10. Figure 2 As shown in part (b) of , the excitation light Ld includes repetitions 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, for example, greater than 1 nanosecond and less than 10 microseconds, or greater than 10 nanoseconds and less than 100 nanoseconds. In other words, the repetition frequency of the light pulse group PG is, for example, greater than 0.1 MHz and less than 1 GHz, or greater than 10 MHz and less than 100 MHz. The light pulse group PG includes a plurality of excitation light pulses PL2 (hereinafter referred to as the light pulse PL2) arranged at intervals of time t2. In one example, the time interval t2 between the plurality of light pulses PL2 is constant in each light pulse group PG. The peak intensity of the light pulse PL2 is uniform in each light pulse group PG. The repetition period t1 of the light pulse group PG can be defined as the peak interval of the leading light pulse PL2 among the plurality of light pulses PL2 constituting each light pulse group PG. The time interval t2 can be defined as the peak interval of the light pulse PL2. The full width at half maximum (FWHM) of the light pulse 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.

[0065] Figure 3 1 is a diagram showing a specific configuration example of the waveform control unit 10. Figure 3 In the example shown, the waveform control unit 10 has 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 spectrally separates the excitation light La according to each wavelength component. As a spectroscopic element, other optical components such as a prism can also be used instead of the diffraction grating 12. The spectroscopic element can be a reflective type or a transmissive type. The excitation light La is incident on the diffraction grating 12 at an angle and is split into a plurality of wavelength components. The light Lb containing the plurality of wavelength components is focused by the lens 13 according to each wavelength component and is imaged on the modulation surface of the SLM 14. The lens 13 can be a convex lens composed of a light-transmitting component, or a concave mirror having a concave light-reflecting surface. In addition, the lens 15 can also be a cylindrical lens.

[0066] In order to generate an excitation light Ld having an arbitrary time intensity waveform different from the excitation light La, SLM14 simultaneously performs phase modulation and intensity modulation of the light Lb. SLM14 may also perform intensity modulation only. SLM14 is, for example, a phase modulation type. In one embodiment, SLM14 is an LCOS (Liquid crystal on silicon) type. Alternatively, SLM14 may also be an intensity modulation type SLM such as a digital micromirror device (DMD). SLM14 may be a reflective type or a transmissive type. Figure 4 1 is a diagram showing the modulation surface 17 of the SLM 14. Figure 4 As shown, on the modulation surface 17, a plurality of modulation areas 17a are arranged along a certain direction D1, and each modulation area 17a extends in a direction D2 intersecting the direction D1. The direction D1 is the spectral direction based on the diffraction grating 12. The modulation surface 17 functions as a Fourier transform surface, and the corresponding wavelength components after spectral separation are incident on the plurality of modulation areas 17a. The SLM 14 modulates the phase and intensity of each incident wavelength component in each modulation area 17a independently from other wavelength components. In the case where the SLM 14 is a phase modulation type, the intensity modulation is realized by the phase pattern (phase image) presented on the modulation surface 17.

[0067] The SLM 14 is electrically connected to the modulation pattern calculation device 20. The modulation pattern calculation device 20 calculates the modulation pattern to be presented in the SLM 14, and provides data Da representing the modulation pattern to the SLM 14. The modulation pattern is, for example, a Computer-Generated Hologram (CGH).

[0068] Each wavelength component of the modulated light Lc modulated by the SLM14 is concentrated on a point on the diffraction grating 16 by the lens 15. At this time, the lens 15 acts as a focusing optical system for focusing the modulated light Lc. The lens 15 can be a convex lens composed of a light-transmitting component, or a concave mirror with a concave light-reflecting surface. In addition, the lens 15 can also be a cylindrical lens. The diffraction grating 16 acts as a combining optical system to combine the modulated multiple wavelength components. That is, by using these lenses 15 and the diffraction grating 16, the multiple wavelength components of the modulated light Lc are focused and combined to become the excitation light Ld.

[0069] The region before the lens 15 (spectral region) and the region after the diffraction grating 16 (temporal region) are in a Fourier transform relationship. Therefore, the phase modulation and intensity modulation in the spectral region affect the temporal intensity waveform in the temporal region. Therefore, the excitation light Ld has a desired temporal intensity waveform that is different from the excitation light La and corresponds to the modulation pattern of the SLM 14. Here, Figure 5Part (a) shows the spectrum waveform (spectral phase G11 and spectral intensity G12) of the single pulse excitation light La as an example. Figure 5 Part (b) shows the temporal intensity waveform of the excitation light La. Figure 6 Part (a) shows, as an example, the spectrum waveform (spectral phase G21 and spectral intensity G22) of the excitation light Ld when rectangular wave-shaped phase spectrum modulation is applied to the SLM 14. Figure 6 Part (b) shows the temporal intensity waveform of the excitation light Ld. Figure 5 Part (a) and Figure 6 In part (a), the horizontal axis represents the wavelength (nm), the left vertical axis represents the intensity value of the intensity spectrum (arbitrary unit), and the right vertical axis represents the phase value of the phase spectrum (rad). Figure 5 Part (b) and Figure 6 In part (b), the horizontal axis represents time (femtoseconds) and the vertical axis represents light intensity (arbitrary units). In this example, a single light pulse PL1 of the excitation light La is converted into a light pulse group PG including a plurality of light pulses PL2 by giving the excitation light Ld a rectangular wave-shaped phase spectrum waveform. Figure 5 and Figure 6 The spectra and waveforms shown are examples, and the number, pulse width, peak intensity, and time interval t2 of the optical pulses PL2 of the optical pulse group PG can be variously controlled by combining various spectral phases and spectral intensities.

[0070] Refer again Figure 1 . The excitation light Ld including the light pulse group PG output from the pulse group generating unit 2 is input to the optical system 3. The optical system 3 irradiates the excitation light Ld to the object B as the observation object. The object B is previously dyed with a fluorescent dye. The fluorescent dye includes, for example, at least one material selected from a methanol solution of eosin Y, an aqueous solution of eosin Y, a methanol solution of rose bengal, an ethanol solution of rhodamine 6G, rose bengal, an aqueous solution of rhodamine 6G, and anthracene. The object B may also be a biological molecule or biological tissue that has been recombined with a gene to emit fluorescence.

[0071] The fluorescent pigment of the object B is excited by the irradiation of the excitation light Ld including the light pulse group PG, and generates fluorescence Le at multiple locations of the object B. At this time, the fluorescent pigment of the object B can also generate fluorescence Le by multiphoton absorption (for example, two-photon absorption). For example, by irradiating the object B with a light pulse PL2 having an extremely short pulse width such as picosecond or femtosecond, the photon density of the excitation light Ld can be increased and multiphoton absorption can be generated. The fluorescence Le is input to the photodetector 4. The photodetector 4 detects the intensity of the fluorescence Le at each location of 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 the processing unit 5.

[0072] The processing unit 5 is electrically connected to the photodetector 4 and receives the electrical signal Sa from the photodetector 4. The processing unit 5 generates data Sb related to the fluorescent image of the object B based on the intensity of the fluorescence Le at multiple parts of the object B. The processing unit 5 provides the data Sb to the display unit 6. The display unit 6 displays the fluorescent image of the object B based on the data Sb. The processing unit 5 is a smart device such as a personal computer, a smart phone or a tablet terminal, or a computer such as a cloud server. The computer serving as the processing unit 5 has a storage device such as an HDD, a flash memory or a RAM, and a processor (CPU). The processing unit 5 can also be composed of a microcomputer or an FPGA (Field-Programmable Gate Array).

[0073] Figure 2 The time interval t2 of the light pulse PL2 shown in part (b) is set in the modulation pattern calculation device 20 to be less than the relaxation time between the excited states in the triplet excited state of the fluorescent dye (for example, the relaxation time from the excited state T2 to the excited state T1, that is, the T2 lifetime) or shorter than 10 picoseconds as described in detail later. When 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. The T2 lifetime of the fluorescent dye cited above is as follows.

[0074] Eosin Y in methanol: 1 picosecond

[0075] · Aqueous solution of Eosin Y: 1 picosecond

[0076] Rose Bengal methanol solution: 2.2 picoseconds

[0077] Rhodamine 6G ethanol solution: 2 picoseconds

[0078] Rose red: 5.8 picoseconds

[0079] · Aqueous solution of Rhodamine 6G: 0.2 picoseconds

[0080] Anthracene: 11 picoseconds

[0081] The information input unit 11 inputs information related to the type of the fluorescent pigment of the object B. The information input unit 11 inputs information related to the type of the fluorescent pigment of the object B, for example, through an input operation of 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 the information Db related to the type of the fluorescent pigment of the object B to the modulation pattern calculation device 20. The modulation pattern calculation device 20 sets the time interval t2 of the light pulse PL2 to be less than the relaxation time between the excited states in the triplet excited state of the fluorescent pigment based on the information Db.

[0082] Figure 7 1 is a diagram showing a specific configuration example of the fluorescence microscope 1. Figure 7 In the example shown, the fluorescence microscope 1 further includes an intensity controller 7 and a waveform measuring device 9. The optical system 3 includes a light branching element 31, galvanometer mirrors 32 and 33, and coupling lenses 34 and 35.

[0083] The intensity controller 7 is disposed on the optical path of the excitation light La between the excitation light source 8 and the waveform control unit 10. The intensity controller 7 adjusts the light intensity of the excitation light La by attenuating the excitation light La. The intensity controller 7 includes, for example, an acousto-optic modulator (AOM), an electro-optic modulator (EOM), and at least one optical element selected from a combination of a half-wavelength plate and a polarizer.

[0084] The optical branching element 31 is arranged on the optical axis of the excitation light Ld output from the waveform control unit 10. The optical branching element 31 separates the excitation light Lf, which is a part of the excitation light Ld, from the excitation light Ld. The excitation light Lf is input to the waveform measuring device 9. The waveform measuring device 9 measures the time waveform of the excitation light Ld by measuring the time waveform of the excitation light Lf. The waveform measuring device 9 may also include a phase measuring device composed of a nonlinear crystal, a delay stage, and a spectrometer. Alternatively, the waveform measuring device 9 may also include an interference measuring device composed of a spectrometer. The measurement result of the waveform measuring device 9 is provided to the modulation pattern calculation device 20 of the waveform control unit 10. The modulation pattern calculation device 20 controls the modulation pattern presented in the SLM14 so that the time waveform of the excitation light Ld measured by the waveform measuring device 9 is close to the desired time waveform (specifically, the number of light pulses PL2 of the light pulse group PG, the pulse width, the peak intensity, and the time interval t2).

[0085] The galvanometer mirrors 32 and 33 are optical elements for scanning (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, so that the optical axis of the excitation light Ld moves in a direction orthogonal to the optical axis of the excitation light Ld. The galvanometer mirror 33 is optically coupled to the galvanometer mirror 32, so that the optical axis of the excitation light Ld moves in another direction orthogonal to both the optical axis of the excitation light Ld and the above-mentioned one direction. The coupling lenses 34 and 35 are optical elements for optically coupling the excitation light Ld whose optical axis moves 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.

[0086] The excitation light Ld is input to the microscope body 40. The microscope body 40 has a stage for placing the object B, and has the above-mentioned photodetector 4, processing unit 5, and display unit 6 built therein. The excitation light Ld is irradiated from below to the object B placed on the stage. Fluorescence Le generated in the object B is incident on the photodetector 4 via an objective lens (not shown) arranged below the object B.

[0087] Figure 8 : is a flowchart showing the image acquisition method of the present embodiment. The image acquisition method can also be judged as the operation method of the above-mentioned fluorescence microscope 1. First, in step S11, the information input unit 11 inputs information Db related to the type of fluorescent pigment. Then, 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 to the object B, and the fluorescence intensity is detected by the light detector 4. Based on the detection result, the sensitivity of the light detector 4 and the detection limit based on S / N are confirmed, and the intensity I1 of the excitation light is determined (step S122). Next, the waveform control unit 10 modulates the light pulse PL1 output from the excitation light source 8, and repeatedly outputs a light pulse group PG consisting of N (N is an integer greater than 2) light pulses PL2 (step S123). At this time, the waveform control unit 10 may also set the time interval t2 of the light pulse PL2 to be less than the relaxation time between the excited states in the triplet excited state of the fluorescent dye of the object B (for example, the relaxation time from the excited state T2 to the excited state T1, that is, the T2 life) or shorter than 10 picoseconds. When the time interval t2 is shorter than 10 picoseconds, the time interval t2 may be shorter than 5 picoseconds, shorter than 3 picoseconds, or shorter than 1 picosecond. The waveform control unit 10 sets the time interval t2 of the light pulse PL2 to be less than the relaxation time between the excited states in the triplet excited state of the fluorescent dye based on the information Db related to the type of the fluorescent dye obtained in step S11.

[0088] Next, based on the time waveform of the excitation light Ld (i.e., the light pulse group PG) measured by the waveform measuring device 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 is uniform in each light pulse group PG (step S124). Next, the intensity controller 7 is used to make the average power of the excitation light Ld close to (√N)·I1 (step S125).

[0089] Next, in step S13, the optical system 3 irradiates the object B containing the fluorescent pigment with the excitation light Ld including the light pulse group PG. Then, in step S14, the light detector 4 detects the intensity of the fluorescence Le generated by the fluorescent pigment of the object B by the irradiation of the excitation light Ld. Here, it is determined whether the excitation light Ld is irradiated to all the irradiation positions (step S15). When there are irradiation positions that are not 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 operation starting from step S13. When the irradiation of all the irradiation positions with the excitation light Ld is completed (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 the fluorescence Le at multiple parts of the object B, that is, at all the irradiation positions. Then, the display unit 6 receives the data Sb related to the fluorescence image from the processing unit 5 and displays the fluorescence image.

[0090] The operations and effects obtained by the fluorescence microscope 1 and the image acquisition method according to the present embodiment described above will be described.

[0091] For example, in a multiphoton excitation fluorescence microscope, a pulsed excitation light is irradiated onto an object. However, if fluorescence is continuously detected while irradiating the pulsed excitation light, the fluorescence intensity gradually decreases. This phenomenon is called photofading. Photofading limits the observation time of an object, and therefore, it is desirable to reduce photofading in a fluorescence microscope.

[0092] The above-mentioned non-patent document 1 discloses that by irradiating a light pulse group including a plurality of light pulses, the light fading speed is reduced compared to the case of irradiating a single light pulse. Furthermore, non-patent document 1 discloses that, based on theoretical investigation, for an observation object whose light fading speed is proportional to the cube of the peak energy of the light pulse, when a light pulse group including N light pulses is irradiated, the light fading speed may be reduced to (1 / √N) times. In order to confirm the effect of the method described in non-patent document 1, the present inventors conducted the following experiments. First, as Fig. 9 As shown, a single optical pulse is generated (ie, N = 1, refer to Fig. 9 (a) of FIG. 1 ), a light pulse group consisting of four light pulses (i.e., N=4, see Fig. 9(b)), a light pulse group consisting of nine light pulses (i.e., N = 9, see Fig. 9 (c) of FIG. 1 ), and a light pulse group consisting of sixteen light pulses (ie, N = 16, see Fig. 9 Then, three kinds of fluorescent dyes (eosin Y, fluorescein, and C-Naphox-TEG) ​​were irradiated with these light pulses and light pulse groups to investigate the dependence of the photofading speed on the excitation light intensity and the pulse number (N).

[0093] Fig.10 This is a graph showing the dependence of the photofading speed on the excitation light intensity. Fig.10 Part (a) shows a graph related to Eosin Y, Part (b) shows a graph related to Fluorescein, and Part (c) shows a graph related to C-Naphox-TEG. Straight lines L11 to L13 are approximate straight lines of these graphs. It can be seen from these approximate straight lines L11 to L13 that in Eosin Y, the photofading speed is proportional to the 2.93 power of the excitation light intensity, in Fluorescein, the photofading speed is proportional to the 2.66 power of the excitation light intensity, and in C-Naphox-TEG, the photofading speed is proportional to the 3.08 power of the excitation light intensity.

[0094] Fig.11 This is a graph showing the dependence of the photofading speed on the number of pulses (N). Fig.11 Part (a) shows a graph related to Eosin Y, Part (b) shows a graph related to Fluorescein, and Part (c) shows a graph related to C-Naphox-TEG. Curves C11 to C13 show the graphs based on Fig.10 The theoretical values ​​of the above power exponents are calculated from the graph. Fig.11 In the case of (a), the pulse number (N) dependence of the photofading speed associated with Eosin Y is roughly consistent with the theoretical value. Fig.11 In part (b), the pulse number (N) dependence of the photofading rate associated with fluorescein deviates from the theoretical value. Fig.11 As can be seen from part (c), the pulse number (N) dependence of the photofading speed associated with C-Naphox-TEG is more different from the theoretical value. Thus, the inventors' experiments have shown that the theory described in Non-Patent Document 1 is not valid depending on the type of fluorescent pigment.

[0095] The inventors have studied the single optical pulse (N=1, referring to Fig. 9 (a)), and a light pulse group consisting of nine light pulses (N = 9, see Fig. 9 (c) of the present invention), the changes in the photofading rate caused by changes in the excitation light intensity were investigated. Fig.12 The portion (a) represents the average intensity of the excitation light (I N / √N) and light fading speed P N In the same figure, plot P11 represents the case where N=1, and plot P12 represents the case where N=9. Fig.12 Part (b) of Fig.12 In the graph shown in part (a) of FIG. 1 , the light fading speed P is N The case where the light fading speed P1 is normalized when N = 1. In the same figure, plot P21 represents the case of N = 1, and plot P22 represents the case of N = 9.

[0096] According to the theory described in Non-Patent Document 1, Fig.12 In the graph shown in part (b) of FIG. 1 , the standard value of the light fading speed when N=9 (P N / P1) should be constant regardless of the average intensity of the excitation light. However, the standard value of the photofading speed when N=9 (P N / P1) gradually decreases as the average intensity of the excitation light increases. That is, it can be said that the greater the intensity of the excitation light, the greater the effect of reducing the light fading speed. This phenomenon cannot be explained by the theory recorded in non-patent document 1.

[0097] Fig.13 : is a diagram showing the mechanism of photofading. Photofading occurs by the following mechanism. First, an excitation light pulse is incident on an object and absorbed by a fluorescent dye. At this time, the fluorescent dye is excited from a base state S0 to an excited singlet state (e.g., an excited state S1) (arrows Aa1 and Aa2 in the figure). Fig.13An example of two-photon absorption. In more detail, the fluorescent dye is first excited to a state S1' with a higher potential than the excited state S1. Then, the fluorescent dye quickly changes to an excited state S1 with a zero vibration energy level through vibration energy relaxation (arrow Ab1 in the figure). Then, a large number of molecules return to the base state S0 again (arrow Ab2 in the figure), thereby generating fluorescence Le. However, a part of the molecules does not return to the base state S0, but changes to an excited triplet state (for example, an excited state T1), i.e., intersystem crossing (arrow Ae1 in the figure). Then, if the time interval of the excitation light pulse is longer than the relaxation time (T1 life) from the excited triplet state, the part of the molecules returns to the base state S0 (arrow Ae2 in the figure). However, in the case where the time interval of the excitation light pulse is shorter than the relaxation time (T1 life) from the excited triplet state, the next excitation light pulse is incident on the object when it is in the excited triplet state and is absorbed by the fluorescent dye. As a result, the molecule changes to a higher-order excited triplet state (for example, an excited state T2) (arrow Ac1 in the figure). In more detail, the fluorescent dye is first excited to a state T2' with a higher potential than the excited state T2. Then, the fluorescent dye quickly changes to an excited state T2 with a zero vibration energy level (arrow Ad1 in the figure) through vibration energy relaxation. Then, when in this high-order excited triplet state, the molecule reacts with oxygen to produce active oxygen, thereby destroying the molecule and causing photofading (arrow Lg in the figure). The molecules that have not reacted with oxygen return to the excited state T1 again (arrow Ad2 in the figure).

[0098] In the image acquisition method and fluorescence microscope 1 of the present embodiment, the time interval t2 of the plurality of light pulses PL2 is set to be less than the relaxation time between the excited states in the triplet excited state of the fluorescent pigment, or is set to be shorter than 10 picoseconds. When the time interval t2 is set to be less than the relaxation time (e.g., T2 lifetime) between the excited states in the triplet excited state of the fluorescent pigment, when the molecules of the fluorescent pigment are in a higher order triplet excited state (e.g., excited state T2), the next light pulse PL2 is incident on the object B and absorbed by the fluorescent pigment. As a result, the molecules of the fluorescent pigment are transformed into a higher order triplet excited state (e.g., excited state T3) (arrow Ac2 in the figure). As a result, the potential energy difference between the triplet excited state and the singlet excited state (e.g., excited state S1) of the molecule becomes larger, and the molecule is easily transformed into the singlet excited state (arrow Ae3 in the figure) before reacting with oxygen. Therefore, the destruction of the molecule can be prevented, and as a result, photofading is reduced.

[0099] Fig.14 Parts (a) to (c) of Fig.11 However, curves C21 to C23 are plotted in the same manner as the graphs shown in parts (a) to (c) of FIG. Fig.11The curves C11 to C13 are different, indicating the theoretical values ​​calculated based on the above mechanism. Fig.14 It can be seen that for any fluorescent dye, the pulse number (N) dependence of the photofading speed is close to the theoretical value. Therefore, it can be said that the above mechanism is correct.

[0100] Fig.15 is the theoretical average intensity of the excitation light (I N / √N) and the light fading speed P N The value normalized by the light fading speed P1 when N=1 (P N In the same figure, straight line L31 represents the case of N=1, and curve C31 represents the case of N=9. Fig.15 and Fig.12 As can be seen from part (b), at least when the average intensity of the excitation light (I N / √N) is in the range of 0.4 to 1.6, the experimental value ( Fig.12 (b)) and the theoretical value ( Fig.15 Therefore, it can be said that the above mechanism is correct.

[0101] Fig.16 2 is a graph showing the result of calculating the relationship between the time interval t2 of the light pulse PL2 and the light fading speed. Fig.16 In the figure, straight line L41 represents the case where the excitation light is a single light pulse, and curve C41 represents the case where the excitation light is a light pulse group composed of multiple light pulses. In this calculation, the relaxation time between the excited states in the excited triplet state is set to 5 picoseconds. When referring to this chart, at a time interval t2 of 6.2 picoseconds, which is slightly longer than the relaxation time (5 picoseconds) between the excited states in the excited triplet state, curve C41 intersects with straight line L41, and when the time interval t2 is shorter than the intersection, the light fading speed represented by curve C41 is smaller than the light fading speed represented by straight line L41. It can be seen from this that if the time interval t2 of the light pulse PL2 is less than the relaxation time between the excited states in the excited triplet state, the light fading speed can be effectively reduced.

[0102] There are various fluorescent pigments among fluorescent pigments, and there are also fluorescent pigments whose relaxation time between excited states in the triplet excited state is more than 10 picoseconds. Among the above types of fluorescent pigments, anthracene meets the requirements. By making the time interval t2 of the light pulse PL2 shorter than 10 picoseconds, the photofading of such fluorescent pigments can be effectively reduced. By making the time interval t2 of the light pulse PL2 less than 6 picoseconds, the photofading of fluorescent pigments whose relaxation time between excited states in the triplet excited state is longer than 6 picoseconds can be effectively reduced. Fig.16The calculation results shown in the figure show that by setting the time interval t2 of the light pulse PL2 to 6 picoseconds or less, the photofading of fluorescent dyes such as Rose Bengal that are longer than 5 picoseconds can also be effectively reduced. By setting the time interval t2 of the light pulse PL2 to be shorter than 2 picoseconds, the photofading of fluorescent dyes whose relaxation time between excited states in the triplet excited state is 2 picoseconds or longer (for example, the above-mentioned methanol solution of Rose Bengal, ethanol solution of Rhodamine 6G, Rose Bengal, and anthracene) can be effectively reduced. By setting the time interval t2 of the light pulse PL2 to be shorter than 1 picosecond, the photofading of fluorescent dyes whose relaxation time between excited states in the triplet excited state is 1 picosecond or longer (for example, 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) can be effectively reduced.

[0103] When the time interval t2 of the light pulse PL2 is set to be less than the relaxation time between the excited states in the triplet excited state, the relaxation time may be the relaxation time from the excited state T2 to the excited state T1 (so-called T2 life). In this case, in the fluorescent dye having the characteristic of transitioning from the excited state T1 to the excited state T2 by the light pulse PL2, photofading can be effectively reduced.

[0104] As in the present embodiment, the image acquisition method may include a step S11 of inputting information related to the type of fluorescent pigment before the step S12 of generating the light pulse group PG. Moreover, in the step S12 of generating the light pulse group PG, the time interval t2 of the plurality of light pulses PL2 may be set to be less than the relaxation time between the excited states in the triplet excited state of the fluorescent pigment based on the information. Similarly, the fluorescence microscope 1 may also include an information input unit 11 for inputting information related to the type of fluorescent pigment. The pulse group generating unit 2 may also set the time interval t2 of the light pulses PL2 to be less than the relaxation time between the excited states in the triplet excited state of the fluorescent pigment based on the information. 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 pigment used. Therefore, the photofading of the fluorescent pigment can be reduced more effectively.

[0105] As in the present embodiment, in step S12 of generating the light pulse group PG, the peak intensity of the plurality of light pulses PL2 can also be made uniform in each light pulse group PG. Similarly, in the fluorescence microscope 1, the peak intensity of the plurality of light pulses PL2 can also be made uniform in each light pulse group PG. In this case, the peak intensity of the light pulse PL2 when it is transformed into a higher-order excited triplet state (e.g., excited state T2) is substantially equal to the peak intensity of the light pulse PL2 when it is transformed into a higher-order excited triplet state (e.g., excited state T3). Therefore, since the transition from the higher-order excited triplet state to the higher-order excited triplet state is effectively performed, the photofading of the fluorescent dye can be more effectively reduced.

[0106] Regarding this situation, the inventors conducted the following experiments. Fig.17 As shown in parts (a) to (e) of FIG. 5 , five light pulse groups with different peak intensity uniformities were generated. Specifically, five light pulse groups were generated in which the ratio (σ / μ) of the standard deviation σ of the peak intensity of each light pulse to the average peak intensity μ was 0.02, 0.15, 0.26, 0.40, and 0.76, respectively. Then, the color fading speed when the fluorescent dye was irradiated with these light pulse groups was measured. Fig.18 The following is a graph showing the results, which shows the relationship between the ratio (σ / μ) and the color fading speed. Fig.18 It can be seen that the smaller the ratio (σ / μ), that is, the closer the peak intensity of the light pulse is to being uniform, the slower the color fading speed is. It is believed that this result is because the more non-uniform the peak intensity of the light pulse is, the closer it is to the case of irradiating a single light pulse. That is, by making the peak intensity of the plurality of light pulses PL2 uniform in each light pulse group PG, the light fading of the fluorescent dye can be more effectively reduced.

[0107] As described above, the repetition frequency of the light pulse group PG may be 1 MHz or more. The relaxation time of the excited triplet state (e.g., T1 lifetime) is several microseconds or less in many fluorescent pigments. 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, photofading based on the above-mentioned mechanism is likely to occur, and therefore, the image acquisition method and fluorescence microscope 1 of this embodiment are useful.

[0108] As in the present embodiment, the pulse group generating unit 2 may also include an excitation light source 8 that repeatedly outputs a single light pulse PL1, and a waveform control unit 10 that modulates the single light pulse PL1 output from the excitation light source 8 and generates a plurality of light pulses PL2. In this case, the pulse group generating unit 2 that repeatedly generates a light pulse group PG including a plurality of light pulses PL2 can be simply configured.

[0109] Here, the configuration and operation of the modulation pattern calculation device 20 will be described in detail. At the same time, a method of calculating a modulation pattern to be displayed in 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 will be described.

[0110] Fig.191 is a diagram schematically showing the structure of the modulation pattern calculation device 20. The modulation pattern calculation device 20 is, for example, a smart device such as a personal computer, a smart phone, and a tablet terminal, or a computer having a processor such as a cloud server. The modulation pattern calculation device 20 is electrically connected to the SLM14, calculates a phase modulation pattern for making the temporal intensity waveform of the excitation light Ld close to the waveform of the light pulse group PG composed of a plurality of light pulses PL2, and provides the data Da including the phase modulation pattern to the SLM14. The modulation pattern calculation device 20 of the present embodiment makes the SLM14 present a phase pattern including a phase pattern for phase modulation for obtaining a phase spectrum of the waveform including the light pulse group PG by giving the excitation light Ld, and a phase pattern for intensity modulation for obtaining an intensity spectrum of the waveform including the light pulse group PG by giving the excitation light Ld. Therefore, the modulation pattern calculation device 20 has 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 functions of 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. Each function may be realized by the same processor or by different processors.

[0111] Fig. 20 2 is a diagram schematically showing a hardware configuration example of the modulation pattern calculation device 20. Fig. 20 As shown, the modulation pattern calculation device 20 can be constructed as an ordinary computer, which physically includes a processor (CPU) 201, main storage devices such as ROM202 and RAM203, input devices 204 such as keyboard, mouse and touch screen, output devices 205 such as display (including touch screen), communication modules 206 such as network cards for sending and receiving data with other devices, auxiliary storage devices 207 such as hard disks, etc.

[0112] The processor 201 of the computer can realize the above-mentioned functions (arbitrary waveform input unit 21, phase spectrum design unit 22, intensity spectrum design unit 23, and modulation pattern generation unit 24) through the modulation pattern calculation program. Therefore, the modulation pattern calculation program enables the processor 201 of the computer to act as the arbitrary waveform input unit 21, phase spectrum design unit 22, intensity spectrum design unit 23, and 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) such as an auxiliary storage device 207, which is internal or external to the computer. The storage device can also be a non-transitory recording medium. As the recording medium, examples include recording media such as floppy disks, CDs, DVDs, ROMs, semiconductor memories, cloud servers, etc.

[0113] The arbitrary waveform input unit 21 receives input of information related to the optical pulse group PG from the operator. The operator inputs information related to the optical pulse group PG (for example, the repetition period t1 of the optical pulse group PG, the pulse width of the optical pulse PL2, the number of pulses of the optical pulse PL2, the time interval t2 of the optical pulse PL2, etc.) to the arbitrary waveform input unit 21. The information related to the optical pulse group PG is given to the phase spectrum design unit 22 and the intensity spectrum design unit 23. The phase spectrum design unit 22 calculates the phase spectrum of the excitation light Ld suitable for realizing the waveform of the given optical pulse group PG. The intensity spectrum design unit 23 calculates the intensity spectrum of the excitation light Ld suitable for realizing the waveform of the given optical pulse group PG. The modulation pattern generation unit 24 calculates a phase modulation pattern (for example, a computer-generated hologram) for giving the excitation light Ld the phase spectrum required in the phase spectrum design unit 22 and the intensity spectrum required in the intensity spectrum design unit 23. Then, the data Da containing the calculated phase modulation pattern is provided to the SLM14, and the SLM14 is controlled based on the data Da.

[0114] Here, a method for calculating the phase spectrum and intensity spectrum corresponding to the time waveform of the light pulse group PG is described in detail. The time waveform of the light pulse group PG is expressed as a function of the time domain, and the phase spectrum and intensity spectrum are expressed as functions of the frequency domain. Therefore, the phase spectrum and intensity spectrum corresponding to the time waveform of the light pulse group PG are obtained by iterative Fourier transform based on the time waveform of the light pulse group PG. In the method described below, the iterative Fourier transform method is used to calculate the phase spectrum and intensity spectrum. Therefore, Fig.19 As shown, the phase spectrum design unit 22 includes an iterative Fourier transform unit 22a, and the intensity spectrum design unit 23 includes an iterative Fourier transform unit 23a.

[0115] Fig.21 The calculation procedure of the phase spectrum based on the iterative Fourier method is shown as follows. First, the initial intensity spectrum function A0(ω) and the phase spectrum function Ψ as a function of the frequency ω are prepared. n＝0 (ω) (processing number (1) in the figure). In one example, these intensity spectrum function A0(ω) and phase spectrum function Ψ n＝0 (ω) respectively represent the intensity spectrum and phase spectrum of the excitation light La. Next, prepare a function including the intensity spectrum function A0(ω) and the phase spectrum function Ψ n The waveform function (a) in the frequency region of (ω) (processing number (2) in the figure).

[0116] [Mathematical formula 1]

[0117]

[0118] The subscript n indicates the nth Fourier transform. Before the initial (first) Fourier transform, as the phase spectrum function Ψ n (ω), using the initial phase spectrum function Ψ mentioned above n＝0 (ω). i is an imaginary unit.

[0119] Next, the function (a) is Fourier transformed from the frequency domain to the time domain (arrow A1 in the figure). Thus, the time-intensity waveform function b is obtained. n The waveform function (b) in the frequency region of (t) (processing number (3) in the figure).

[0120] [Mathematical formula 2]

[0121]

[0122] Next, the time intensity waveform function b contained in the above function (b) is n (t) is replaced by Target0(t) based on the desired waveform (processing numbers (4) and (5) in the figure).

[0123] [Mathematical formula 3]

[0124] b n (t): = Target0(t)...(c)

[0125] [Formula 4]

[0126]

[0127] Next, the above function (d) is subjected to an inverse Fourier transform from the time domain to the frequency domain (arrow A2 in the figure). Thus, the intensity spectrum function B is obtained. n (ω) and phase spectrum function Ψ n The waveform function (e) in the frequency region of (ω) (processing number (6) in the figure).

[0128] [Formula 5]

[0129]

[0130] Next, in order to constrain the intensity spectrum function B contained in the above function (e) n (ω), and replace it with the initial intensity spectrum function A0(ω) (processing number (7) in the figure).

[0131] [Mathematical formula 6]

[0132] B n (ω): =A0(ω)...(f)

[0133] Then, by repeating the above-mentioned processes (1) to (7) for a plurality of times, the phase spectrum function Ψ in the waveform function can be made n The phase spectrum shape represented by (ω) is close to the phase spectrum shape corresponding to the time waveform of the desired optical pulse group PG. IFTA (ω) is used for the calculation of the modulation pattern.

[0134] The above-mentioned iterative Fourier method may include processing for suppressing the introduction of local solutions. Fig. 22 The calculation sequence of the phase spectrum based on this iterative Fourier method (hereinafter referred to as IFTA-Fienup) is shown. Fig. 22 In the process (1) to (3) and (6) to (7), the processes are the same as those in the above-mentioned method, and therefore, the description thereof is omitted.

[0135] In this IFTA-Fienup, the time intensity waveform function b contained in the Fourier transformed function (b) after processing (4) and (5) is n (t) When performing substitution based on the desired waveform, use the Target calculated by the following formula (g) n (t) instead of Target0(t) (processing numbers (4) and (5) in the figure).

[0136] [Mathematical formula 7]

[0137]

[0138] [Mathematical formula 8]

[0139]

[0140] In the above equation (g), the function Target0(t) representing the desired waveform and the waveform function b after Fourier transformation are expressed as n The difference between Target0(t) and b n (t)) is multiplied by a predetermined coefficient β, and the result is added to the desired waveform Target0(t), thereby calculating Target n (t). If this value is less than 0, Target n (t)=0.

[0141] However, even in this IFTA-Fienup, for example, the function Target0(t) representing the desired waveform is different from the waveform function b after Fourier transformation. n (t) When the difference is large, it is sometimes still introduced into the local solution.

[0142] Therefore, as described below, the iterative Fourier method can also be improved. Fig.23The calculation procedure of the phase spectrum is shown in FIG. First, the initial intensity spectrum function A0(ω) and the phase spectrum function Ψ as a function of the frequency ω are prepared. n＝0 (ω) (processing number (1) in the figure). In one example, these intensity spectrum function A0(ω) and phase spectrum function Ψ n＝0 (ω) represent the intensity spectrum and phase spectrum of the input light respectively.

[0143] Next, prepare a spectrum including the intensity spectrum function A0(ω) and the phase spectrum function Ψ n The waveform function (i) in the frequency region of (ω) (processing number (2) in the figure).

[0144] [Mathematical formula 9]

[0145]

[0146] The subscript n indicates the nth Fourier transform process. Before the initial (first) Fourier transform process, the initial phase spectrum function Ψ is used. n＝0 (ω) as a phase spectrum function Ψ n (ω). i is an imaginary unit.

[0147] Next, the above function (i) is Fourier transformed from the frequency domain to the time domain. Thus, the time intensity waveform function b is obtained. n The waveform function (j) in the frequency region of (t) (processing number (3) in the figure).

[0148] [Formula 10]

[0149]

[0150] Next, the waveform function b after Fourier transformation is obtained n The difference between (t) and the result of multiplying the function Target0(t) by the coefficient α (α×Target0(t)) is the waveform function b n In one example, the coefficient α is such that the difference between Target0(t) and the function Target0(t) is small (processing number (4) in the figure). In one example, the evaluation function represented by the following equation (k) is used to exploratoryally derive the value of α×Target0(t) relative to the waveform function b after Fourier transformation. n The standard deviation σ of (t) becomes the minimum (σ min ) coefficient α. In formula (k), D represents the number of data points, t e ,t s Respectively represent the start and end point of the time axis.

[0151] [Mathematical formula 11]

[0152]

[0153] Next, the time intensity waveform function b contained in the Fourier transformed function (j) is n (t) is replaced based on the desired waveform (first replacement). At this time, the function Target0(t) representing the desired waveform is multiplied by the coefficient α (α×Target0(t)). In one example, Target0(t) in the formula (g) in the above IFTA-Fienup is replaced by Target0(t) calculated by replacing it with the formula (m) of α×Target0(t). n (t) (processing numbers (5) and (6) in the figure). β in the formula is an arbitrary coefficient. By appropriately selecting the coefficient β, it is expected that a better solution can be found with a smaller number of iterations n and the effect of preventing falling into a local solution can be achieved.

[0154] [Mathematical formula 12]

[0155]

[0156] [Mathematical formula 13]

[0157]

[0158] Next, the above function (n) is subjected to an inverse Fourier transform from the time domain to the frequency domain (arrow A2 in the figure). Thus, the intensity spectrum function B is obtained. n (ω) and phase spectrum function Ψ n The waveform function (o) in the frequency region of (ω) (processing number (7) in the figure).

[0159] [Formula 14]

[0160]

[0161] Next, in order to constrain the intensity spectrum function B contained in the above function (o) n (ω), and replace it with the initial intensity spectrum function A0(ω) (the second replacement, processing number (8) in the figure).

[0162] [Mathematical formula 15]

[0163] B n (ω): =A0(ω)...(p)

[0164] Then, by repeating the above-mentioned processes (1) to (8) for a plurality of times, the phase spectrum function Ψ in the waveform function can be made n The phase spectrum shape represented by (ω) is close to the phase spectrum shape corresponding to the expected time intensity waveform. IFTA(ω) is provided for the calculation of the modulation pattern.

[0165] In the iterative Fourier transform unit 22 a of the phase spectrum design unit 22 , the iterative Fourier method may be further improved as described below. Fig.24 The calculation sequence of the phase spectrum based on the improved iterative Fourier method is shown in Figure 2. Fig.23 The calculation order shown is the same, so the description is omitted appropriately.

[0166] First, the iterative Fourier transform unit 22a performs Fig.23 The calculation order shown is the same as that of the processing numbers (1) to (3). Next, the iterative Fourier transform unit 22a obtains the coefficient α having the following characteristics (A) and (B) (processing number (4) in the figure).

[0167] (A) Waveform function b after Fourier transformation n The difference between (t) and the function Target0(t) multiplied by the coefficient α (α×Target0(t)-b n (t)) ratio waveform function b n The difference between (t) and the function Target0(t) (Target0(t)-b n Specifically, the difference (α×Target0(t)-b n (t)) time integral value is greater than the difference (Target0(t)-b n The time integral value of (t)) becomes smaller.

[0168] (B) At each time of the function Target0(t), the above difference (α×Target0(t)-b n (t)), that is, the difference (α×Target0(t)-b n The ratio of (t)) decreases as the intensity increases.

[0169] In one example, the evaluation function represented by the following equation (q) is used to exploratory derive the relationship between α×Target0(t) and the waveform function b after Fourier transformation: n The suspected standard deviation σ of (t) becomes the minimum (σ min ) coefficient α. In formula (q), D represents the number of data points, t e ,t s They represent the starting point and the end point of the time axis respectively. We(t) is the first weighting function.

[0170] [Formula 16]

[0171]

[0172] As shown in formula (q), the evaluation function includes the waveform function b after Fourier transformation. n The difference between (t) and the multiplied function α×Target0(t) (α×Target0(t)-b n (t)), specifically (α×Target0(t)-b n (t)) 2 The evaluation function also includes the weighted function We(t) multiplied by the function, and includes the time integral obtained by multiplying the function by the weighted function We(t). Then, the evaluation function is exploratory, that is, the time integral becomes the minimum (σ min ). The weighting function We(t) is a function that has a weighting value that increases as the intensity of the function Target0(t) before multiplication increases. In one example, the weighting function We(t) includes a function obtained by multiplying the function Target0(t) by another coefficient C1, for example, represented by the following formula (r). In other words, the weighting function We(t) can also be determined based on the function Target0(t).

[0173] [Mathematical formula 17]

[0174] We(t)=C1·Target0(t)...(r)

[0175] As described above, by including the weighting function We(t) in the evaluation function shown in the formula (q), the above-mentioned feature (B) can be given to the coefficient α. Fig.25 ] shows an example of the weighting function We(t) when Target0(t) is a light pulse group composed of a plurality of light pulses. Fig.25 Curve C51 represents the case where the coefficient C of the formula (r) is 1, and curve C52 represents the case where the coefficient C of the formula (r) is 2.

[0176] Then, the iterative Fourier transform unit 22a performs the Fig.23 The calculation sequence is the same as that of the processing numbers (5) to (8) shown in the figure. After that, by repeating the processing numbers (1) to (8) multiple times, the phase spectrum function Ψ in the waveform function can be made n The phase spectrum shape represented by (ω) is close to the phase spectrum shape corresponding to the expected time intensity waveform. IFTA (ω) is supplied to the modulation pattern generation unit 24.

[0177] Fig.261 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 by the same method as the calculation method by the iterative Fourier transform unit 22a described above.

[0178] First, the iterative Fourier transform unit 23a prepares an initial intensity spectrum function A as in the case of calculating the phase spectrum. k＝0 (ω) and phase spectrum function Ψ0(ω) (processing number (1) in the figure). Next, the iterative Fourier transform unit 23a prepares a system including the intensity spectrum function A k (ω) and the waveform function (s) of the frequency domain of the phase spectrum function Ψ0(ω) (processing number (2) in the figure).

[0179] [Mathematical formula 18]

[0180]

[0181] The subscript k indicates the kth Fourier transform process. Before the initial (first) Fourier transform process, the initial intensity spectrum function A is used. k＝0 (ω) as a function of the intensity spectrum A k (ω). i is an imaginary unit.

[0182] Next, the iterative Fourier transform unit 23a performs Fourier transform on the above function (s) from the frequency domain to the time domain. Thus, the time intensity waveform function b is obtained. k The waveform function (t) in the frequency region of (t) (processing number (3) in the figure).

[0183] [Mathematical formula 19]

[0184]

[0185] Next, the iterative Fourier transform unit 23a obtains a coefficient α having the following characteristics (C) and (D) (processing number (4) in the figure).

[0186] (C) Waveform function b after Fourier transformation k The difference between (t) and the function Target0(t) multiplied by the coefficient α (α×Target0(t)-b k (t)) ratio waveform function b k The difference between (t) and the function Target0(t) (Target0(t)-b k Specifically, the difference (α×Target0(t)-b k (t)) time integral value is greater than the difference (Target0(t)-b k The time integral value of (t)) becomes smaller.

[0187] (D) At each time of the function Target0(t), the above difference (α×Target0(t)-b k (t)), that is, the difference (α×Target0(t)-b k The ratio of (t)) decreases as the intensity increases.

[0188] In one example, the evaluation function represented by the following equation (u) is used to exploratory derive the relationship between α×Target0(t) and the waveform function b after Fourier transformation: k The suspected standard deviation σ of (t) becomes the minimum (σ min ) coefficient α. In formula (u), D represents the number of data points, t e ,t s They represent the starting point and the end point of the time axis respectively. We(t) is the first weighting function.

[0189] [Mathematical formula 20]

[0190]

[0191] As shown in formula (u), the evaluation function includes the waveform function b after Fourier transformation. k The difference between (t) and the multiplied function α×Target0(t) (α×Target0(t)-b k (t)), specifically (α×Target0(t)-b k (t)) 2 The evaluation function also includes a weighting function We(t) multiplied by the function, and includes a time integral obtained by multiplying the function by the weighting function We(t). Then, the evaluation function is exploratory, i.e., the time integral is minimized (σ min ) coefficient α. The characteristics and specific examples of the weighting function We(t) are the same as those in the case of calculating the above-mentioned phase spectrum function (refer to equations (q) and (r)). However, the following equation (v) is used instead of the above-mentioned equation (q).

[0192] [Mathematical formula 21]

[0193]

[0194] Next, the iterative Fourier transform unit 23a performs Fourier transformation on the time intensity waveform function b contained in the function (v) after the Fourier transformation. k(t) performs a substitution based on the desired waveform (first substitution). At this time, the iterative Fourier transform unit 23a performs substitution using the result obtained by multiplying the function Target0(t) representing the desired waveform by the coefficient α (α×Target0(t)). In one example, the substitution is performed by Target0(t) calculated by the formula (w). k (t) (processing numbers (5), (6) in the figure).

[0195] [Mathematical formula 22]

[0196]

[0197] [Mathematical formula 23]

[0198]

[0199] Next, the iterative Fourier transform unit 23a performs an inverse Fourier transform on the function (w) from the time domain to the frequency domain. k (ω) and phase spectrum function Ψ k The waveform function (y) in the frequency region of (ω) (processing number (7) in the figure).

[0200] [Mathematical formula 24]

[0201]

[0202] Next, the iterative Fourier transform unit 23a constrains the phase spectrum function Ψ contained in the above function (y) k (ω), and replace it with the initial phase spectrum function Ψ0(ω) (second replacement, processing number (8) in the figure).

[0203] [Mathematical formula 25]

[0204] Ψ k (ω):=Ψ0(ω)...(z)

[0205] The iterative Fourier transform unit 23a performs Fourier inverse transform on the intensity spectrum function C in the frequency region. k (ω) performs filtering based on the intensity spectrum of the excitation light La. Specifically, the intensity spectrum function C is cut k The portion of the intensity spectrum represented by (ω) that exceeds the cutoff intensity for each wavelength determined based on the intensity spectrum of the excitation light La. In one example, the cutoff intensity for each wavelength is set to correspond to the intensity spectrum of the excitation light La (in this embodiment, the initial intensity spectrum function A k＝0 (ω)) is consistent. In this case, as shown in the following formula (z1), in the intensity spectrum function C k (ω) Ratio to initial intensity spectral function Ak＝0 (ω) large frequency, as the intensity spectrum function A k (ω) is taken into the initial intensity spectrum function A k＝0 (ω) value. In the intensity spectrum function C k (ω) is the initial intensity spectrum function A k＝0 (ω) or less, as the intensity spectrum function A k (ω) is taken into the intensity spectrum function C k The value of (ω).

[0206] [Mathematical formula 26]

[0207]

[0208] The intensity spectrum function C contained in the above function (y) k (ω) is replaced by the intensity spectrum function A after filtering based on the above formula (z1): k (ω). It is also possible to k (ω) Define a function C' multiplied by an arbitrary coefficient k (ω), and a method of relatively changing the cutoff strength was used (processing number (9) in the figure).

[0209] Thereafter, the iterative Fourier transform unit 23a can make the intensity spectrum function A in the waveform function k The intensity spectrum shape represented by (ω) is close to the intensity spectrum shape corresponding to the expected time intensity waveform. IFTA (ω) is supplied to the modulation pattern generation unit 24.

[0210] Fig. 27 The modulation pattern calculation program is a flowchart showing a modulation pattern calculation method implemented by the modulation pattern calculation device 20 described above. Fig. 20 ) executes each step contained in the flowchart. Fig. 27 As shown, first, the operator inputs information related to the desired time waveform of the optical pulse group PG into the arbitrary waveform input unit 21 (input step S20). Then, the phase spectrum design unit 22 and the intensity spectrum design unit 23 respectively calculate the phase spectrum and the intensity spectrum for making the time intensity waveform close to the desired waveform (phase spectrum calculation step S21, intensity spectrum calculation step S23).

[0211] The phase spectrum calculation step S21 includes an iterative Fourier transform step S22 by the iterative Fourier transform unit 22a. The details of the iterative Fourier transform step S22 are the same as the operation of the iterative Fourier transform unit 22a described above. The phase spectrum function Ψ is finally obtained. IFTA (ω) is then provided to the 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 intensity spectrum function A finally obtained is IFTA (ω) is then provided to the modulation pattern calculation step S25.

[0212] In the modulation pattern calculation step S25, based on the phase spectrum function Ψ IFTA (ω) and intensity spectrum function A IFTA (ω), calculate the modulation pattern. The modulation pattern is presented in SLM14.

[0213] In the above description, in order to make the time intensity waveform close to the desired waveform, the phase spectrum function Ψ is calculated IFTA (ω) and intensity spectrum function A IFTA (ω) and the modulation pattern based on these functions is presented in SLM14. This is not limited to this form. For example, only the phase spectrum function Ψ for making the time intensity waveform close to the desired waveform can be calculated. IFTA (ω) and intensity spectrum function A IFTA In this case, a pre-prepared (or selected) spectrum may be used as another spectrum, or the other spectrum may not be modulated in the state of the excitation light La.

[0214] Fig.28 and Fig.29 1 is a diagram showing a modified example of the calculation sequence of the phase spectrum based on the iterative Fourier method. Fig.24 and Fig.26 ) is different in that the coefficient β in processing number (5) is replaced by the weighting function Wr(t). In this modification, the above-mentioned equations (m) and (w) are replaced by the following equations (z2) and (z3), respectively.

[0215] [Mathematical formula 27]

[0216]

[0217] That is, in this modification, the time intensity waveform function b obtained by subtracting Fourier transformation from the function {α×Target0(t)} is used. n (t)(or b kThe first permutation is performed by multiplying the result of the function after α (t)) by the weighting function Wr(t) and the sum of the function {α×Target0(t)}.

[0218] The weighting function Wr(t) is a function that has a weighting value that increases as the intensity increases at each time of the function Target0(t). In one example, the weighting function Wr(t) includes the function Target0(t) multiplied by another coefficient C2, and is represented by the following formula, for example. In other words, the weighting function Wr(t) can also be determined based on the function Target0(t).

[0219] [Mathematical formula 28]

[0220] Wr(t)=C2·Target0(t)...(z 4)

[0221] By replacing the coefficient β with the weighting function Wr(t), the size of the difference is enhanced in the interval with high intensity in Target0(t) compared with other intervals. Therefore, when iterative Fourier operation is performed, the result of particularly reducing the difference in this interval is calculated. Therefore, the time waveform of the excitation light Ld, especially the time waveform of the interval with high light intensity, can be made closer to the desired waveform with higher accuracy.

[0222] The present invention is not limited to the above-described embodiment, and various other modifications are possible. For example, in the above-described embodiment, the fluorescence microscope 1 including the pulse group generation unit 2 , the optical system 3 , the photodetector 4 , and the processing unit 5 is described. Fig.30 1 is a diagram showing an excitation light irradiation unit 100 for a fluorescence microscope. The excitation light irradiation unit 100 includes a pulse group generation unit 2. The structure of the pulse group generation unit 2 is the same as that of the fluorescence microscope 1. The effects of the fluorescence microscope 1 of the above-mentioned embodiment are also achieved in the excitation light irradiation unit 100. In addition to the pulse group generation unit 2, the excitation light irradiation unit 100 may also include an information input unit 11. Fig.31 2 is a diagram showing a waveform control unit 200 for a fluorescence microscope. The waveform control unit 200 repeatedly generates a light pulse group PG including a plurality of light pulses PL2 for irradiating an object B including a fluorescent pigment. Therefore, the waveform control unit 200 includes a waveform control unit 10, and the waveform control unit 10 is optically coupled to an excitation light source 8 provided outside the waveform control unit 200. The waveform control unit 10 modulates a single light pulse PL1 output from the excitation light source 8 to generate a plurality of light pulses PL2. The effects achieved by the fluorescence microscope 1 of the above-mentioned embodiment are also achieved in the waveform control unit 200. In addition to the waveform control unit 10, the waveform control unit 200 may also include an information input unit 11.

[0223] Description of Reference Numerals

[0224] 1...fluorescence microscope, 2...pulse group generator, 3...optical system, 4...photodetector, 5...processor, 6...display, 7...intensity controller, 8...excitation light source, 9...waveform measuring device, 10...waveform controller, 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 calculator, 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 generator, 31...optical spectrometer Branch element, 32, 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...electric signal, Sb...data, t1...repetition period, t2...time interval.

Claims

1. A method for acquiring an image, wherein: include: Repeating the step of generating an excitation light pulse group including a plurality of excitation light pulses; a step of irradiating an object containing a fluorescent pigment with the group of excitation light pulses; a step of detecting the intensity of fluorescence generated at a plurality of locations of the object by irradiation with the excitation light pulse group; and generating a fluorescence image based on the intensity of the fluorescence at the plurality of locations of 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 a relaxation time between excitation states in the triplet excited state of the fluorescent dye or shorter than 10 picoseconds.

2. The image acquisition method according to claim 1, wherein: The relaxation time between excited states in the triplet excited state of the fluorescent dye is the relaxation time from the excited state T2 to the excited state T1.

3. The image acquisition method according to claim 1 or 2, wherein: Before the step of generating the excitation light pulse group, the method further includes the step of inputting information related to the type of the fluorescent pigment. In the step of generating the excitation light pulse group, the time intervals between the plurality of excitation light pulses are set to be equal to or shorter than a relaxation time between excitation states in the triplet excited state of the fluorescent dye based on the information.

4. The image acquisition method according to any one of claims 1 to 3, wherein: In the step of generating the excitation light pulse group, when the time interval between the plurality of excitation light pulses is made shorter than 10 picoseconds, the time interval between the plurality of excitation light pulses is made shorter than 1 picosecond.

5. The image acquisition method according to any one of claims 1 to 4, wherein: In the step of generating the excitation light pulse group, peak intensities of the plurality of excitation light pulses are made uniform in each of the excitation light pulse groups.

6. The image acquisition method according to any one of claims 1 to 5, wherein: In the step of generating the excitation light pulse group, a repetition frequency when repeatedly generating the excitation light pulse group is 1 MHz or more.

7. A fluorescence microscope, wherein: have: 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 the object including the fluorescent pigment with the group of excitation light pulses; a photodetector for detecting the intensity of fluorescence generated at a plurality of locations of the object by irradiation with the excitation light pulse group; as well as a processing unit that generates a fluorescence image based on the intensity of the fluorescence at the plurality of sites of the object, The time interval between the plurality of excitation light pulses is equal to or shorter than a relaxation time between excited states in the triplet excited state of the fluorescent dye or is shorter than 10 picoseconds.

8. The fluorescence microscope according to claim 7, wherein: The relaxation time between excited states in the triplet excited state of the fluorescent dye is the relaxation time from the excited state T2 to the excited state T1.

9. The fluorescence microscope according to claim 7 or 8, wherein: It also includes: an information input unit that inputs information related to the type of the fluorescent dye, The pulse group generating unit sets the time intervals between the plurality of excitation light pulses to be equal to or shorter than a relaxation time between excitation states in the triplet excited state of the fluorescent dye based on the information.

10. The fluorescence microscope according to any one of claims 7 to 9, wherein When the time interval between the plurality of excitation light pulses is shorter than 10 picoseconds, the time interval between the plurality of excitation light pulses is shorter than 1 picosecond.

11. The fluorescence microscope according to any one of claims 7 to 10, wherein The peak intensities of the plurality of excitation light pulses are uniform in each of the excitation light pulse groups.

12. The fluorescence microscope according to any one of claims 7 to 11, wherein The pulse group generating unit comprises: an excitation light source that repeatedly outputs a single light pulse; as well as The waveform control unit is optically coupled to the excitation light source, and modulates the single light pulse output from the excitation light source to generate the plurality of excitation light pulses.

13. The fluorescence microscope according to any one of claims 7 to 12, wherein: The pulse group generating unit repeatedly generates the excitation light pulse group at a repetition frequency of 1 MHz or more.

14. An excitation light irradiation unit, wherein: It is an excitation light irradiation unit for fluorescence microscopy. The excitation light irradiation unit includes: a pulse group generation unit that repeatedly generates an excitation light pulse group including a plurality of excitation light pulses for irradiating an object including a fluorescent dye; The time interval between the plurality of excitation light pulses is equal to or shorter than a relaxation time between excited states in the triplet excited state of the fluorescent dye or is shorter than 10 picoseconds.

15. The excitation light irradiation unit according to claim 14, wherein: It also includes: an information input unit that inputs information related to the type of the fluorescent dye, The pulse group generating unit sets the time intervals between the plurality of excitation light pulses to be equal to or shorter than a relaxation time between excitation states in the triplet excited state of the fluorescent dye based on the information.

16. A waveform control unit, wherein: It is a waveform control unit for fluorescence microscope. The waveform control unit repeatedly generates an excitation light pulse group including a plurality of excitation light pulses for irradiating an object including a fluorescent dye, The waveform control unit comprises: a waveform control unit optically coupled to an excitation light source that repeatedly outputs a single light pulse, modulating the single light pulse output from the excitation light source to generate the plurality of excitation light pulses; The time interval between the plurality of excitation light pulses is equal to or shorter than a relaxation time between excited states in the triplet excited state of the fluorescent dye or is shorter than 10 picoseconds.

17. The waveform control unit according to claim 16, wherein: It also includes: an information input unit that inputs information related to the type of the fluorescent dye, The waveform control unit sets the time intervals between the plurality of excitation light pulses to be equal to or shorter than a relaxation time between excitation states in the triplet excited state of the fluorescent dye based on the information.

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