Fluorescence detection device and fluorescence detection method

Abstract

A scanning microscope disclosed herein comprises: a pulse light generation device (or a pulse light generation device) that emits excitation light toward a sample, wherein the excitation wavelength of the excitation light is switched for each pulse; and a detector that detects fluorescence from a cell mass S irradiated with the excitation light and outputs a detection signal. The sensor bandwidth of the detector is adjusted so that the detection signal attenuates within a pulse-off period of the excitation light.

Description

Fluorescence detection device and fluorescence detection method

[0001] The present disclosure relates to a fluorescence detection device and a fluorescence detection method.

[0002] Patent Document 1 discloses a multiphoton microscope that generates multiphoton excitation, in which multiple excitation photons are simultaneously absorbed when fluorescent molecules are irradiated with excitation light.

[0003] Special Publication No. 2022-524180

[0004] When observing deep inside a sample using a multiphoton microscope, the laser intensity increases as the depth increases. Therefore, the possibility of damaging the sample increases when observing deep inside. To avoid damage, the laser intensity must be kept as low as possible even when observing deep inside. However, when the laser intensity is reduced, the amount of detected signal decreases, potentially making it impossible to observe properly.

[0005] The present disclosure has been made in view of the above circumstances, and has an object to provide a fluorescence detection device and a fluorescence detection method that improve the amount of detected signals and thereby improve the accuracy of fluorescence observation.

[0006] (1) A fluorescence detection device according to one aspect of the present disclosure includes an excitation light source that emits excitation light, the excitation wavelength of which changes with each pulse, toward a sample, and a detection unit that detects fluorescence from the sample irradiated with the excitation light and outputs a detection signal, wherein the detection band of the detection unit is adjusted so that the detection signal attenuates during the pulse-off period of the excitation light.

[0007] In a fluorescence detection device according to one aspect of the present disclosure, fluorescence from a sample irradiated with excitation light whose excitation wavelength changes with each pulse is detected by a detection unit, and a detection signal is output from the detection unit. In the fluorescence detection device according to one aspect of the present disclosure, the detection band of the detection unit is adjusted so that the detection signal decays within the pulse-off period of the excitation light. By adjusting the detection band so that the detection signal decays within the pulse-off period (before the arrival of the next pulse), different pulses are temporally distinguished from one another, preventing the mixing of detection signals associated with different pulses. This eliminates the need for a configuration that filters signals according to wavelength to distinguish between different pulses, thereby suppressing the reduction in the amount of detection signal that would be a problem if such a configuration were used. As described above, the fluorescence detection device according to one aspect of the present disclosure can improve the accuracy of fluorescence observation by increasing the amount of detection signal.

[0008] (2) In the fluorescence detection device described in (1) above, the detection unit may have a detection band adjusted so that the combined time taken for the fluorescence to decay and the detection signal to decay is shorter than the repetition interval of the excitation light pulses. With this configuration, the time taken for the fluorescence and the detection signal to decay is made shorter than the repetition interval of the pulses, thereby more reliably achieving the temporal separation of the different pulses described above.

[0009] (3) In the fluorescence detection device described in (1) or (2) above, the detection unit may detect the fluorescence without passing through a filter for wavelength separation of the fluorescence, thereby avoiding a reduction in the amount of the detected signal due to passing through a filter and enabling the amount of the detected signal to be appropriately improved.

[0010] (4) In the fluorescence detection device according to any one of (1) to (3) above, the detection unit may be configured with a single optical sensor that detects all of the fluorescence corresponding to the excitation wavelengths of the pulses. This configuration allows for a simplified configuration while improving the amount of detected signal without providing a configuration for filtering signals according to wavelength.

[0011] (5) In the fluorescence detection device according to any one of (1) to (4) above, the excitation light source may emit excitation light by switching between a first excitation wavelength for stimulating the sample and a second excitation wavelength for observing the effect of the optical stimulation of the first excitation wavelength for each pulse, and the detection unit may not detect light at the timing for detecting fluorescence from the sample corresponding to the pulse of the first excitation wavelength, but may detect light at the timing for detecting fluorescence from the sample corresponding to the pulse of the second excitation wavelength. This configuration makes it possible to detect only necessary fluorescence while avoiding detection of the stimulation fluorescence (removing the stimulation fluorescence), thereby contributing to an improvement in the lower detection limit.

[0012] (6) The fluorescence detection device according to any one of (1) to (5) above may further include an A / D converter that converts the detection signal output from the detection unit into a digital signal, and the sampling frequency of the A / D converter may be synchronized with the repetition frequency of the excitation light pulses. This configuration makes it easier to obtain the necessary information from the analog signal corresponding to each pulse without providing a high-speed A / D converter, and enables high-speed signal processing at low cost.

[0013] (7) In the fluorescence detection device described in (6) above, the A / D converter may be configured to acquire the maximum value of the detection signal. As described above, by synchronizing the frequency to always acquire the same position of the signal waveform and further acquiring the maximum value of the detection signal, necessary information can be more appropriately acquired from the analog signal corresponding to each pulse.

[0014] (8) In the fluorescence detection device described in (6) above, the A / D converter may be configured to acquire an integrated value of the detection signal. When acquiring the maximum value as described above, there is a risk that the maximum value cannot be acquired successfully due to, for example, fluctuations in the fluorescence signal. In this regard, by acquiring an integrated value, it is possible to more appropriately acquire the necessary information from the analog signal corresponding to each pulse.

[0015] (9) A fluorescence detection method according to one aspect of the present disclosure includes emitting excitation light, the excitation wavelength of which changes with each pulse, toward a sample, detecting fluorescence from the sample irradiated with the excitation light, and outputting a detection signal, wherein the detection band is adjusted so that the detection signal attenuates within the pulse-off period of the excitation light.

[0016] According to one aspect of the present invention, it is possible to provide a fluorescence detection device and a fluorescence detection method that improve the amount of detected signals and thereby improve the accuracy of fluorescence observation.

[0017] FIG. 1 is a schematic diagram of a scanning microscope according to an embodiment. FIG. 2 is a diagram illustrating the challenges of deep imaging. FIG. 3 is a diagram illustrating the excitation light and fluorescence of GFP and RFP. FIG. 4(a) is a diagram illustrating an example of excitation in two wavelength bands, FIG. 4(b) is a diagram illustrating a fluorescence signal corresponding to the excitation light of FIG. 4(a), and FIG. 4(c) is a diagram illustrating the sensor band. FIG. 5 is a schematic diagram of an example of the configuration of a detector according to a comparative example. FIG. 6 is a graph illustrating the fact that a bandpass filter makes it impossible to acquire the entire fluorescence spectrum. FIG. 7(a) is a diagram illustrating an example of excitation in two wavelength bands, FIG. 7(b) is a diagram illustrating a fluorescence signal corresponding to the excitation light of FIG. 7(a), and FIG. 7(c) is a diagram illustrating the sensor band. FIG. 8 is a schematic diagram of an example of the configuration of a detector according to this embodiment. FIGS. 9(a) and 9(b) are graphs illustrating the conditions of the sensor band. FIG. 10(a) is a graph showing a case where the sensor bandwidth is sufficiently narrower than the A / D converter bandwidth. FIG. 10(b) is a graph showing a case where the sensor bandwidth is wider. FIG. 10(c) is a graph explaining a method of acquiring only the maximum value of the detection signal. FIGS. 11(a) and 11(b) are diagrams explaining the effect of synchronizing the sampling frequency of the A / D converter with the pulse repetition frequency. FIGS. 12(a) and 12(b) are graphs explaining acquisition of the integrated value of the detection signal. FIG. 13 is a diagram explaining a detection method when applied to light stimulus control. FIG. 14 is a block diagram showing a pulsed light generator. FIG. 15(a) is a graph showing the time waveform of ultrashort pulsed light output from the oscillator of FIG. 14. FIG. 15(b) is a graph showing the spectrum of ultrashort pulsed light output from the oscillator of FIG. 14. FIG. 15(c) is a graph showing the time waveform of ultrashort pulsed light output from the fiber amplifier of FIG. 14. FIG. 15(d) is a graph showing the spectrum of ultrashort pulsed light output from the fiber amplifier of FIG. 14. Fig. 16 is a graph showing a specific example of the spectrum of the ultrashort optical pulse output from the fiber amplifier of Fig. 14. Fig. 17(a) is a graph showing the time waveform of the ultrashort optical pulse output from the acousto-optic modulator of Fig. 14. Fig. 17(b) is a graph showing the spectrum of the ultrashort optical pulse output from the acousto-optic modulator of Fig. 14.FIG. 18( a) is a graph showing the time waveform of an ultrashort optical pulse output from the soliton-shifted fiber of FIG. 14 . FIG. 18( b) is a graph showing the spectrum of an ultrashort optical pulse output from the soliton-shifted fiber of FIG. 14 . FIG. 18( c) is a graph showing the time waveform of an ultrashort optical pulse output from the filter of FIG. 14 . FIG. 18( d) is a graph showing the spectrum of an ultrashort optical pulse output from the filter of FIG. 14 . FIG. 19 is a diagram explaining chirped pulse amplification. FIG. 20( a) is a diagram explaining a method of generating a difference in optical path length by using a difference in refractive index when passing through a substance, and FIG. 20( b) is a diagram explaining a method of generating a difference in optical path length by using diffraction. FIG. 21 is a schematic diagram of a configuration related to chirped pulse amplification. FIG. 22 is a schematic diagram of a configuration related to chirped pulse amplification that widens the time width using a single fiber. FIG. 23( a) is a graph showing the characteristics of the stretcher and the compressor in the configuration of FIG. 22 , and FIG. 23( b) is a graph showing the residual GVD in the configuration of FIG. 22 . FIG. 24 is a diagram explaining the generation of a second harmonic. FIG. 25 is a graph showing the wavelength conversion efficiency in each wavelength band for each crystal length. FIG. 26 is a diagram explaining angular phase matching. FIG. 27 is a diagram explaining an example of the configuration of a wavelength converter. FIG. 28 is a diagram explaining an example of the configuration of a wavelength converter according to a modified example. FIG. 29 is a block diagram showing another example of a pulsed light generator. A flowchart showing a wavelength tuning method. FIG. 31( a) is a graph explaining an example of amplifying short-wavelength ultrashort pulsed light. FIG. 31( b) is a graph explaining a continuation of FIG. 31( a). FIG. 31( c) is a graph explaining a continuation of FIG. 31( b). FIG. 31( d) is a graph explaining a continuation of FIG. 31( c). FIG. 32( a) is a graph explaining an example of amplifying long-wavelength ultrashort pulsed light. Fig. 32(b) is a graph for explaining a continuation of Fig. 32(a). Fig. 32(c) is a graph for explaining a continuation of Fig. 32(b). Fig. 32(d) is a graph for explaining a continuation of Fig. 32(c). Fig. 33 is a graph showing the relationship between the absorption cross section and stimulated emission cross section of a Tm fiber and the transmittance of a filter and wavelength.Fig. 34 is a diagram schematically showing a scanning microscope (fluorescence detection device) according to a modified example. Fig. 35 is a diagram showing the time waveforms of each signal. Fig. 36 is a diagram explaining a delay generator. Fig. 37 is a diagram schematically showing a pulsed light generating device (excitation light source) according to another modified example. Fig. 38 is a diagram showing the time waveforms of each signal.

[0018] Hereinafter, the embodiments will be described in detail with reference to the drawings. In each drawing, the same or corresponding parts are designated by the same reference numerals, and duplicated explanations will be omitted.

[0019] FIG. 1 is a schematic diagram showing a scanning microscope (fluorescence detection device) 1 according to an embodiment. The scanning microscope 1 is, for example, a laser scanning microscope (LSM). The scanning microscope 1 may also be a multiphoton microscope, for example, a two-photon microscope. The scanning microscope 1 irradiates a cell cluster S (sample) with excitation light. The cell cluster S has been subjected to a staining process using, for example, a fluorescent probe. Therefore, when the cell cluster S is irradiated with the excitation light, the cell cluster S that has incorporated the fluorescent probe emits fluorescence. In the staining process, for example, green fluorescent protein (GFP) and red fluorescent protein (RFP) may be used as fluorescent molecules (fluorescent dyes) to label the antibodies of the cell cluster S. The scanning microscope 1 detects the fluorescence emitted by the cell cluster S.

[0020] The scanning microscope 1 includes a pulsed light generator 10 (excitation light source), a dichroic mirror 20, an objective lens 30, a condenser lens 40, a detector (detection unit) 50, an A / D converter 60, and a computer 70.

[0021] The pulsed light generator 10 is, for example, a laser device, and in this embodiment, is a femtosecond laser device. The pulsed light generator 10 generates ultrashort pulsed light as excitation light L1. The pulsed light generator 10 performs wavelength conversion for each pulse. Note that the pulsed light generator 10 only needs to have an output mode that performs wavelength conversion for each pulse. In addition to this output mode, the pulsed light generator 10 may also have other output modes, such as continuously outputting pulsed light at a single wavelength, outputting multiple pulsed light at multiple wavelengths obtained by wavelength conversion, or outputting pulsed light at completely random wavelengths and at random times. That is, the pulsed light generator 10 emits excitation light L1, the excitation wavelength of which changes for each pulse, toward the cell mass S. Details of the pulsed light generator 10 will be described later.

[0022] The dichroic mirror 20 is a mirror that reflects light of a specific wavelength and transmits light of other wavelengths. The dichroic mirror 20 reflects light in a wavelength band that can be used as excitation light L1 toward the objective lens 30. The dichroic mirror 20 also transmits fluorescence L2 from the cell cluster S, which will be described later.

[0023] The objective lens 30 is disposed opposite the cell cluster S and is a lens that focuses the excitation light L1 incident thereon via the dichroic mirror 20 toward a focal point FP of the cell cluster S. Fluorescence L2 generated from the focal point FP passes through the objective lens 30 and then passes through the dichroic mirror 20. The fluorescence L2 that has passed through the dichroic mirror 20 passes through the condenser lens 40 and enters the detector 50. An excitation light cut filter (not shown) that suppresses the incidence of the excitation light L1 on the detector 50 may be provided between the condenser lens 40 and the detector 50.

[0024] The detector 50 is an optical sensor that detects fluorescence L2 from the cell mass S irradiated with excitation light L1 and outputs a detection signal. The detector 50 is, for example, an electron tube such as a photomultiplier tube (PMT) or a hybrid photodetector (HPD) that incorporates a photocathode and an electron multiplier unit in a vacuum housing. A control circuit (not shown) is connected to the photomultiplier tube, and the control circuit includes a high-voltage power supply and a gain adjustment circuit that adjusts the power supplied to the electron multiplier unit, etc. In this embodiment, the detector 50 is described as a photomultiplier tube, but is not limited thereto. It may also be a semiconductor light-receiving element such as a silicon photomultiplier (SiPM), an avalanche photodiode (APD), or a photodiode (PD), as well as an electron tube, image sensor, or camera using such elements. Details of the detector 50 will be described later.

[0025] The A / D converter 60 converts the detection signal (analog signal) output from the detector 50 into a digital signal and outputs the digital signal. The A / D converter 60 will be described in detail later.

[0026] The computer 70 performs predetermined control and signal processing on the digital signal (fluorescence signal) output from the A / D converter 60. The computer 70 may transfer the signal processing results to an external server or the like via serial communication, for example.

[0027] Next, problems that arise when observing deep areas of the cell mass S (deep imaging) using a multiphoton microscope such as a two-photon microscope will be described with reference to FIGS.

[0028] 2 is a diagram illustrating the issues involved in deep imaging. Typically, when performing deep imaging of a cell cluster S, the intensity of the excitation light L1 is increased as the focal point FP is set deeper into the cell cluster S. In this case, there is a concern that the sample may be damaged during deep observation, so it is necessary to reduce the laser intensity of the excitation light L1 as much as possible.

[0029] However, in a device for observing fluorescence, it is necessary to separate the fluorescence wavelength band using a wavelength-selective filter such as a band-pass filter, which tends to reduce the amount of detected signal, and in some cases it may not be possible to sufficiently reduce the laser intensity in order to ensure a sufficient amount of detected signal. The reason why the amount of detected signal is reduced will be explained below.

[0030] Figure 3 is a diagram explaining the excitation light and fluorescence of green fluorescent protein (GFP) and red fluorescent protein (RFP). Consider a case where GFP and RFP are fluorescent molecules (fluorescent dyes) and antibodies in a cell cluster S are labeled. The horizontal axis of Figure 3 represents wavelength. Figure 3 shows excitation light L11 when exciting GFP and fluorescence L21 corresponding to excitation light L11, and excitation light L12 when exciting RFP and fluorescence L22 corresponding to excitation light L12. The wavelength bands of fluorescence L21 and fluorescence L22 partially overlap.

[0031] Fig. 4(a) shows the waveforms of the excitation light when switching between the excitation light L11 and the excitation light L12 for each pulse. As shown in Fig. 4(a), a waveform 101 corresponding to the excitation light L11 and a waveform 102 corresponding to the excitation light L12 appear in sequence for each pulse. In this case, as shown in Fig. 4(b), a waveform 201 corresponding to the fluorescence L21 and a waveform 202 corresponding to the fluorescence L22 also appear in sequence.

[0032] FIG. 4C is a diagram illustrating the sensor band 301 of the detector (optical sensor), which is the detection band of the optical detection unit. In this embodiment, the detection band of the optical detection unit refers to the band of the output signal extracted as a detection signal from the optical detection unit. In other words, a wide detection band means a wide output signal band, i.e., a short time from when an output signal rises to when it decays (the time from when an output signal begins to rise to when it ends to fall). A narrow detection band means a narrow output signal band, i.e., a long time from when an output signal rises to when it decays. The width and narrowness of the detection band are determined, more specifically, by the width and narrowness of the frequency band of the amplifier unit in the circuitry of the optical detection unit, which amplifies and converts the current output from the optical sensor into a voltage output. In other words, a wide frequency band of the amplifier unit results in a wide detection band, whereas a narrow frequency band of the amplifier unit results in a narrow detection band. Therefore, in this embodiment, the description of the detection band of the photodetector can also be replaced with the frequency band of the amplifier section in the circuit section of the photodetector, which amplifies and converts the current output from the photosensor into a voltage output. Typically, the sensor band 301 of the detector (photosensor) is set much narrower than the repetition interval of the excitation light pulses (see FIGS. 4(a) to 4(c)). Therefore, the fluorescence L21 component and the fluorescence L22 component cannot be temporally distinguished from each other in the detection signal output from the photosensor (more specifically, the digital signal converted by the downstream A / D converter). Even when the excitation light L11 and the excitation light L12 are switched for each pulse, the narrow sensor band 301 of the photosensor makes it impossible to properly distinguish and acquire the fluorescence L21 corresponding to the excitation light L11 and the fluorescence L22 corresponding to the excitation light L12. Furthermore, even if the fluorescence L21 and the fluorescence L22 exhibit intensity fluctuations within a time period shorter than the sensor band 301, this fluctuation is not reflected in the detection signal, and therefore, this information cannot be acquired.

[0033] In order to appropriately distinguish and acquire the fluorescence L21 and the fluorescence L22, it is possible to employ a configuration including bandpass filters 503 and 603, as shown in FIG. 5 . In the example shown in FIG. 5 , for fluorescence, for example, light in the 500 nm wavelength band is reflected by a dichroic mirror 400, while light in the 600 nm wavelength band is transmitted. The light reflected by the dichroic mirror 400 passes through a lens 502 and a bandpass filter 503 and enters a photodetector 501. The light detected by the photodetector 501 is the fluorescence of GFP. The light transmitted through the dichroic mirror 400 passes through a lens 602 and a bandpass filter 603 and enters a photodetector 601. The light detected by the photodetector 601 is the fluorescence of RFP. As described above, when the sensor bandwidth is narrow and it is not possible to distinguish the fluorescence of each pulse in time, it is necessary to resolve the fluorescence signal by wavelength using a bandpass filter.

[0034] FIG. 6 is a graph illustrating the inability to capture the entire fluorescence spectrum due to the bandpass filters 503 and 603. In FIG. 6 , the horizontal axis represents wavelength, and the vertical axis represents intensity. As indicated by the legend in FIG. 6 , FIG. 6 also shows the spectrum of GFP fluorescence L21, the wavelength range of the bandpass filter 503 for detecting the fluorescence L21, the spectrum of RFP fluorescence L22, and the wavelength range of the bandpass filter 603 for detecting the fluorescence L22. As shown in FIG. 6 , the spectral range detected by the photodetectors 501 and 601 is limited by the bandpass filter 503 for GFP fluorescence L21 and the bandpass filter 603 for RFP fluorescence L22, respectively. Thus, the use of the bandpass filters 503 and 603 makes it impossible to detect the entire fluorescence spectrum, resulting in a reduced amount of detected fluorescence signal. Furthermore, the amount of light is reduced even when passing through the bandpass filters 503 and 603. In such a configuration, the laser intensity of the excitation light must be increased to a certain extent to ensure a sufficient amount of fluorescence signal, which results in the problem that fluorescence generation from sources other than the focal point cannot be sufficiently suppressed. Furthermore, even if it is possible to distinguish between the fluorescence L21 and the fluorescence L22, if the sensor band 301 of the optical sensor is narrow, and intensity fluctuations in the fluorescence L21 and the fluorescence L22 occur within a time period shorter than the sensor band 301, these fluctuations will not be reflected in the detection signal, and therefore this information cannot be acquired.

[0035] Therefore, the scanning microscope 1 according to this embodiment employs a method for distinguishing between pulses of fluorescence without using a bandpass filter in a configuration in which the excitation wavelength is switched for each pulse. Specifically, in the scanning microscope 1, the sensor band (detection band) of the detector 50 described above is adjusted so that the detection signal (signal output from the detector 50) attenuates during the pulse-off period of the excitation light L1. The pulse-off period is the off period of the pulsed light in which on / off is repeated. The attenuation of the detection signal may refer to, for example, a state in which the detection signal has attenuated to a signal amount of 10% or less of its maximum value.

[0036] As shown in Fig. 7(a), a waveform 101 corresponding to the excitation light L11 and a waveform 102 corresponding to the excitation light L12 appear in sequence for each pulse, and as shown in Fig. 7(b), a waveform 201 corresponding to the fluorescence L21 corresponding to the excitation light L11 and a waveform 202 corresponding to the fluorescence L22 corresponding to the excitation light L12 appear in a similar sequence. Now, as shown in Fig. 7(c), the sensor bands 351 and 352 of the detector 50 (optical sensor) are set sufficiently wide (equally wide) relative to the repetition interval of the excitation light pulses. In Fig. 7(c), the sensor band 351 is the sensor band corresponding to the fluorescence L21, and the sensor band 352 is the sensor band corresponding to the fluorescence L22. In Fig. 7(c), the sensor bands 351 and 352 are set to have a 1:1 relationship with the waveform 201 corresponding to the fluorescence L21 and the waveform 202 corresponding to the fluorescence L22 corresponding to the excitation light L12. 7C, the sensor bands 351 and 352 of the detector 50 are adjusted so that the detection signal (waveform of the sensor bands 351 and 352) attenuates during the pulse-off period of the excitation light. In this way, the detection signal of the detector 50 attenuates before the next pulse is detected.

[0037] 8 is a diagram schematically illustrating an example configuration of a detector 50 according to this embodiment. As shown in FIG. 8 , the detector 50 is configured with a single optical sensor 51 that detects all of the fluorescence corresponding to the excitation wavelengths of each pulse (e.g., the above-described fluorescence L21 and L22). The optical sensor 51 of the detector 50 detects the fluorescence without using a bandpass filter for wavelength separation of the fluorescence. This configuration without a bandpass filter can be adopted because the sensor bandwidth of the detector 50 is set so that the detection signal of the detector 50 attenuates during the pulse-off period of the excitation light, and the fluorescence corresponding to different pulses can be distinguished over time.

[0038] More specifically, the sensor bandwidth of the detector 50 is adjusted so that the combined time for the fluorescence to decay and the detection signal to decay is shorter than the repetition interval of the excitation light pulse. Figures 9(a) and 9(b) are graphs illustrating the sensor bandwidth conditions. In Figures 9(a) and 9(b), the horizontal axis represents time and the vertical axis represents intensity. Let frep be the repetition interval of the excitation light pulse (the picking frequency of the AOM), T be the fluorescence lifetime (the longest fluorescence lifetime in the case of multiple staining), FWHM be the full width at half maximum of the detection signal, and fsensor be the sensor bandwidth (sensor signal bandwidth) of the detector 50. The following equation (1) can be derived from the above conditions of the detector 50. The fluorescence lifetime here refers to the time it takes for the initial intensity of the afterglow to decay to 1 / e. Since 1 / e is approximately 37%, the time it takes for the fluorescence signal to decay is approximately four times that value. 1 / frep>4T+2FWHM 1 / frep>4T+2×1 / (2×fsensor) 1 / fsensor<1 / frep−4T (1) In this way, the sensor bandwidth of the detector 50 is set to fall within the range determined by the pulse repetition interval and the fluorescence lifetime.

[0039] Here, when the sensor bandwidth of the detector 50 is sufficiently narrower than the A / D converter bandwidth, the detection signal from the detector 50 can be sampled without any problems. That is, as shown in FIG. 10( a), when the sensor bandwidth 301 is sufficiently narrower than the A / D converter bandwidth and many sampling points SP can be set for the detection signal, the detection signal can be sampled without any problems and the analog signal can be properly reproduced from the A / D converter. In contrast, as described above, in the configuration according to this embodiment, the sensor bandwidth 351 of the detector 50 is wide as shown in FIG. 10( b), and if the A / D converter bandwidth is the same as in the conventional configuration, the detection signal from the detector 50 may not be sampled sufficiently, and the analog signal may not be properly reproduced from the A / D converter. To address this issue, for example, increasing the speed of the A / D converter is conceivable, but this would be technically difficult and would increase costs. Furthermore, since the scanning microscope 1 must perform scanning at high frequencies to acquire a large amount of signals and then construct an entire image, an increase in the number of samples due to an increase in the speed of the A / D converter means a huge increase in the amount of data, which is fatal in practical use.

[0040] In this regard, in the configuration according to the present embodiment, the sampling frequency of the A / D converter 60 is synchronized with the repetition frequency of the excitation light pulse, thereby enabling sampling at the same position in the detection signal. In this case, it is preferable to further provide an appropriate time delay to the sampling frequency of the A / D converter 60. In this case, the A / D converter 60 may be set to acquire the maximum value of the detection signal. That is, as shown in FIG. 10( c), the sampling point SPM of the A / D converter 60 may be set so that it is always near the maximum value of the detection signal. By synchronizing the frequency in this manner, sampling of the detector signal can be performed a minimum number of times without increasing the speed of the A / D converter 60 (in fact, by slowing it down compared to conventional methods), thereby contributing to data compression. Note that the maximum value of the detection signal in this embodiment is not limited to the value of the peak portion of the detection signal, which appears as a pulse, but also includes a range showing a signal value of 70% or more of the signal value indicated by the peak portion.

[0041] Furthermore, by synchronizing the sampling frequency of the A / D converter 60 with the repetition frequency of the excitation light pulse, it becomes possible to remove random noise (dark / stray light). As shown in FIG. 11A, if the above-described synchronization is not performed, the detection signal sampled at the sampling point SP will contain a random noise D component. In contrast, as shown in FIG. 11B, if the above-described synchronization is performed and, for example, only the maximum value of the detection signal is detected at the sampling point SPM, the random noise D tends to fall outside the detection range, and the random noise D can be properly removed. By removing the random noise, the lower detection limit can be expanded.

[0042] The A / D converter 60 may be configured to acquire values ​​other than the maximum value of the detection signal, such as an integrated value. Figures 12(a) and 12(b) are graphs illustrating the acquisition of the integrated value of the detection signal. As shown in Figure 12(a), when the sensor bandwidth 351 is relatively wide, the jitter (fluctuation) of the fluorescence signal represented by waveform 201 may affect the value variability when using the method of acquiring the maximum value of the detection signal described above. Therefore, for example, a method can be considered in which the detection signal is input to a charge amplifier circuit (not shown), charging is performed until the fluorescence signal decays, and the integrated value is sampled at a single point. In the example shown in Figure 12(b), a single point of the charge signal CS corresponding to the integrated value obtained by inputting the detection signal to the charge amplifier circuit (not shown) is set as the sampling point SPM. This configuration can suppress the effects of jitter (fluctuation) in the fluorescence signal. By sampling as quickly as possible after the fluorescence signal decays, the effects of stray light and other factors can be minimized.

[0043] The scanning microscope 1 may also be used for various other types of fluorescence observation, such as fluorescence observation with optical stimulus control. In this case, the pulsed light generator 10 emits excitation light by switching between a first excitation wavelength for stimulating the cell cluster S and a second excitation wavelength for observing the effects of the optical stimulus of the first excitation wavelength for each pulse. As shown in FIG. 13 , the detector 50 may not perform sampling by the A / D converter at the timing of detecting fluorescence from the cell cluster S in response to a pulse of the first excitation wavelength (detection timing corresponding to the excitation for stimulation), thereby not detecting light, but may detect light only at the timing of detecting fluorescence from the cell cluster S in response to a pulse of the second excitation wavelength (detection timing corresponding to the excitation for observation). This configuration can remove the fluorescence corresponding to the excitation for stimulation, thereby avoiding a situation in which the fluorescence corresponding to the excitation for observation is not properly detected due to being buried in the fluorescence corresponding to the excitation for stimulation. This contributes to improving the lower detection limit.

[0044] Next, the pulsed light generating device 10 that switches the excitation wavelength for each pulse will be described in detail with reference to FIGS.

[0045] 14 , the pulsed light generator 10 of this embodiment generates long-wavelength ultrashort pulsed light (pulsed light) by utilizing soliton self-frequency shift (Raman soliton shift). For example, the pulsed light generator 10 is a femtosecond laser device for a two-photon microscope. The pulsed light generator 10 includes an oscillator 702, a fiber amplifier 703, an acousto-optic modulator 704, a compressor 705, a soliton-shifted fiber 706, a stretcher fiber 707, a fiber amplifier 708, a compressor 709, and a wavelength conversion unit 710.

[0046] The oscillator 702 constitutes an oscillation unit that oscillates ultrashort pulsed light. As shown in FIG. 15( a), the oscillator 702 generates an ultrashort pulse train with a predetermined period F1. The wavelength band of the ultrashort pulsed light oscillated from the oscillator 702 may be, for example, a band including 1550 nm. As shown in FIG. 15( b), the oscillator 702 here oscillates ultrashort pulsed light having a spectrum with a first spectral width H1 and a first intensity K1. The oscillator 702 is not particularly limited, and various oscillators can be used.

[0047] The fiber amplifier 703 constitutes an amplifying section that broadens the spectrum of the ultrashort optical pulse oscillated by the oscillator 702. The fiber amplifier 703 broadens the spectrum of the ultrashort optical pulse and increases the output power of the ultrashort optical pulse by similariton amplification. The fiber amplifier 703 is disposed between the oscillator 702 and the soliton-shifted fiber 706 in the optical path of the ultrashort optical pulse.

[0048] The fiber amplifier 703 includes a fiber amplifier. The fiber amplifier of the fiber amplifier 703 is a normal dispersion fiber, which is a double-clad fiber co-doped with erbium and ytterbium. That is, the fiber amplifier 703 performs amplification while causing a nonlinear effect by the normal dispersion double-clad fiber so as not to stretch, and obtains ultrashort pulse light as amplified light over a wide bandwidth. The normal dispersion fiber is a fiber in which the dispersion parameter D (ps / nm / km) is negative. There are no particular limitations on the dopant used in the fiber amplifier 703, and various dopant types may be used.

[0049] 15(c) and 15(d), the fiber amplifier 703 broadens the spectral width of the ultrashort optical pulse to a second spectral width H2 that is wider than the first spectral width H1. The fiber amplifier 703 increases the intensity of the ultrashort optical pulse to a second intensity K2 that is higher than the first intensity K1. Specifically, as shown in Fig. 16, the fiber amplifier 703 sets the spectral width of the ultrashort optical pulse to 100 nm or more. In Fig. 16, the horizontal axis represents the wavelength of the ultrashort optical pulse, and the vertical axis represents the intensity of the ultrashort optical pulse relative to a predetermined intensity.

[0050] The acousto-optic modulator 704 constitutes a light intensity control unit that controls the intensity of the ultrashort optical pulse for each pulse. The acousto-optic modulator 704 is a device that modulates the ultrashort optical pulse by utilizing the force of acoustics (sound waves) and is called an AOM (Acousto-Optic Modulator). In this embodiment, the acousto-optic modulator 704 is disposed between the fiber amplifier 703 and the soliton-shifted fiber 706 in the optical path of the ultrashort optical pulse. The acousto-optic modulator 704 may be disposed anywhere between the oscillator 702 and the soliton-shifted fiber 706. As shown in FIGS. 17( a) and 17(b), the acousto-optic modulator 704 controls the intensity of the ultrashort optical pulse so that it varies for each pulse. For example, when intensity modulations M1 and M2 are applied as shown in FIG. 17(a), ultrashort optical pulses LM1 and LM2 corresponding to the intensities given by M1 and M2 are generated as shown in FIG. 17(b). The range and precision of intensity modulation of the ultrashort pulsed light L (LM1, LM2) depend on the performance of the acousto-optic modulator 704. The intensity of each pulsed light in the pulse train of ultrashort pulsed light can be modulated arbitrarily by the acousto-optic modulator 704.

[0051] The compressor 705 constitutes a pulse compression unit that compresses the pulse duration of the ultrashort optical pulse. In this embodiment, the compressor 705 is disposed between the acousto-optic modulator 704 and the soliton-shifted fiber 706 in the optical path of the ultrashort optical pulse. The compressor 705 may be disposed at any position between the fiber amplifier 703 and the soliton-shifted fiber 706. Even if the ultrashort optical pulse is stretched (for example, by several picoseconds) by the fiber amplifier 703, the compressor 705 compresses the duration of the ultrashort optical pulse and outputs an ultrashort optical pulse with a duration spread of less than a certain value (less than 1 picosecond). The compressor 705 is not particularly limited, and various compressors can be used.

[0052] The soliton-shifted fiber 706 constitutes a modulation section that utilizes soliton self-frequency shift to modulate the wavelength of the ultrashort optical pulse, the output of which has been increased while broadening the spectrum in the fiber amplifier 703. The soliton-shifted fiber 706 is disposed downstream of the fiber amplifier 703 in the optical path of the ultrashort optical pulse. As shown in FIGS. 18( a) and 18(b), the soliton-shifted fiber 706 lengthens the wavelength of the ultrashort optical pulse to generate soliton S1. The wavelength band of the lengthened ultrashort optical pulse L may be, for example, a band including 1800 nm to 2200 nm. The soliton-shifted fiber 706 may be, for example, a single-mode anomalous dispersion fiber that exhibits anomalous dispersion in the wavelength band of the ultrashort optical pulse generated in the fiber amplifier 703. In addition, by controlling the acousto-optic modulator 704, solitons with wavelengths different from the soliton S1 can be generated. For example, when the wavelength of the soliton S is modulated by intensities M1 and M2 as shown in FIG. 18( c), the soliton S shifts to a wavelength corresponding to the intensities given by M1 and M2 (solitons S1 and S2) as shown in FIG. 18( d). The range and precision of the wavelength shift of the soliton S depend on the performance of the acousto-optic modulator 704. The wavelength shift of each soliton S in the soliton train generated from the ultrashort optical pulse can be arbitrarily changed by applying intensity modulation to the pulse train using the acousto-optic modulator 704. In the illustrated example, the ultrashort optical pulse modulated by the soliton self-frequency shift contains a non-soliton component S0 (a component that does not become soliton S1 or S2). A filter (not shown) that cuts the non-soliton component S0 of the ultrashort optical pulse L may be provided downstream of the soliton-shifted fiber 6. Such a filter may have an OD value of 3 or greater.

[0053] The stretcher fiber 707, the fiber amplifier 708, and the compressor 709 are components related to chirped pulse amplification. The components related to chirped pulse amplification will be described with reference to FIGS.

[0054] First, chirped pulse amplification will be described. FIG. 19 is a diagram illustrating chirped pulse amplification. When strongly amplifying the output of ultrashort optical pulses, a process called chirped pulse amplification may be performed to prevent adverse effects due to excessive peak power increase, which could result in nonlinear effects. In chirped pulse amplification, the time width of ultrashort optical pulses is first expanded (stretched), the stretched ultrashort optical pulses are then amplified, and finally, a compression process is performed to restore the time width of the amplified ultrashort optical pulses (to the state before stretching). As shown in FIG. 19 , in chirped pulse amplification, the time width of pulsed light I, which is ultrashort optical pulses input into a stretcher fiber 707, is first expanded by the stretcher fiber 707. Then, pulsed light II, which is ultrashort optical pulses whose time width has been expanded by the stretcher fiber 707, is amplified by a fiber amplifier 708. Finally, pulsed light III, which is the ultrashort optical pulses amplified by the fiber amplifier 708, is compressed by a compressor 9 to restore its time width to the state before stretching.

[0055] Next, two examples of methods for expanding (or compressing) the time width in chirped pulse amplification will be described. In both examples, the time width is expanded (or compressed) by creating a difference in optical path length. FIG. 20( a) is a diagram illustrating a method for creating a difference in optical path length by using a difference in refractive index when passing through a material, and FIG. 20( b) is a diagram illustrating a method for creating a difference in optical path length by using diffraction. As shown in FIG. 20( a), when ultrashort pulse light, in which light of multiple wavelengths is superimposed with its phases aligned, passes through some material, differences in the optical path length of each wavelength occur due to differences in the refractive index of each wavelength, which changes the group delay time of each wavelength. This can be utilized to expand the time width or, conversely, compress the time width. Here, the material is, for example, fiber. Furthermore, as shown in FIG. 20( b), differences in the optical path length of each wavelength occur due to a diffraction grating pair 91, 92 (described in detail later), which changes the group delay time of each wavelength. This allows the time width to be widened or, conversely, compressed.

[0056] In such chirped pulse amplification, when returning the expanded time width of an ultrashort pulse light to its original state, it is preferable to compress the time width in the same way as when the time width was expanded. From this perspective, it is possible to perform stretching and compression using the same method. That is, for example, when using a method in which the optical path length of each wavelength is varied by passing the light through a fiber due to differences in refractive index (see FIG. 20(a)), fibers are used as both the stretcher and compressor, and when using a method in which the optical path difference of each wavelength is varied using a diffraction grating pair (see FIG. 20(b)), a diffraction grating pair is used as both the stretcher and compressor. In this way, by performing stretching and compression using the same type of optical element, the characteristics of stretching and compression (described in detail below) are consistent, and it is possible to compress the ultrashort pulse light by the same amount as the expanded time width.

[0057] On the other hand, when optimizing the stretcher and compressor from the perspective of stability and throughput, it may be advantageous to use separate stretchers and compressors. For example, when stretching and compressing a fiber, higher throughput (typically nearly zero loss) can be expected for stretching compared to a diffraction grating pair. However, when compressing, nonlinear effects may occur, resulting in light that does not have the expected characteristics. Specifically, complex dispersion may be introduced into the ultrashort pulse light, making it impossible to fully compress the ultrashort pulse light. As a result, the energy that should be concentrated in the main pulse is dispersed to other parts, creating an undesirable state for nonlinear effects. On the other hand, when stretching and compressing a fiber using a diffraction grating pair, for example, the ultrashort pulse light is propagated in a free space, which increases the beam diameter and reduces energy concentration, making it less likely to cause nonlinear effects. Regarding stretching, a diffraction grating pair typically has a diffraction efficiency of approximately 90%, so in the case of a diffraction grating pair with multiple diffraction gratings, energy is lost with each diffraction, resulting in a final throughput of approximately 60% of the original energy.

[0058] Considering these factors, a configuration using a fiber as a stretcher and a diffraction grating pair as a compressor can be considered to suppress the occurrence of nonlinear effects while maintaining throughput. In a configuration in which stretching and compression are performed using different methods (using different types of optical elements), it is more difficult to match the stretching and compression characteristics (described in detail below) compared to a configuration in which stretching and compression are performed using the same method (using the same types of optical elements). This makes it more difficult to restore the ultrashort pulse light to its pre-stretched state after compression. This problem becomes even more serious in broadband light sources and broadly wavelength-tunable light sources. To address this issue, the chirped pulse amplification configuration of the pulsed light generator 10 according to this embodiment includes two fibers (first fiber 71 and second fiber 72) as the stretcher fiber 707, as shown in FIG. 21 .

[0059] 21 is a schematic diagram of the chirped pulse amplification configuration of the pulsed light generator 10 according to this embodiment. As shown in FIG. 21 , the pulsed light generator 10 includes a stretcher fiber 707 (stretcher), a fiber amplifier 708 (amplification section), and a compressor 709 as the chirped pulse amplification configuration.

[0060] The stretcher fiber 707 is a stretcher that expands the time width of ultrashort pulsed light. The wavelength band of ultrashort pulsed light whose time width is expanded by the stretcher fiber 707 is, for example, 1800 nm to 2200 nm. The stretcher fiber 707 is configured by combining a first fiber 71 that expands the time width of ultrashort pulsed light with a first characteristic and a second fiber 72 that expands the time width of ultrashort pulsed light with a second characteristic different from that of the first fiber 71. The first fiber 71 and the second fiber 72 are configured to expand the time width of ultrashort pulsed light by generating differences in the optical path lengths of the wavelengths due to differences in the refractive indexes of the wavelengths when the ultrashort pulsed light passes through them. The first fiber 71 expands the time width of ultrashort pulsed light including, for example, a wavelength band of 1800 nm to 2200 nm, output from the soliton shift fiber 706, with the first characteristic, and outputs the expanded time width to the second fiber 72. The first fiber 71 may be, for example, a normal dispersion fiber. The second fiber 72 is connected to the first fiber 71, and expands the time width of the ultrashort pulse light input from the first fiber 71 using a second characteristic, and outputs the expanded light to the fiber amplifier 708. The second fiber 72 may be, for example, a normal dispersion or anomalous dispersion fiber. The first characteristic and the second characteristic will be described in detail later.

[0061] The fiber amplifier 708 amplifies (increases the output power of) the pulsed light whose time width has been expanded by the stretcher fiber 707. The fiber amplifier 708 includes a fiber amplifier. The fiber amplifier of the fiber amplifier 708 is an anomalous dispersion fiber, for example, a thulium-doped fiber. The laser medium doped in the fiber of the fiber amplifier 708 is not particularly limited, and may be a rare earth element such as ytterbium, erbium, or neodymium, or may be Bi or the like. The wavelength band of the ultrashort pulsed light L amplified by the fiber amplifier 708 is, for example, 1800 nm to 2200 nm. The fiber amplifier 708, which can reliably amplify the pulsed light in a wide wavelength band, includes, for example, a first fiber amplifier (not shown) having a high gain G1 (not shown) on the first wavelength side, which is the short wavelength side, of the ultrashort pulsed light L, and a second fiber amplifier (not shown) having a high gain G2 (not shown) on the second wavelength side, which is the long wavelength side, and is configured by combining a filter between these amplifiers that attenuates amplified light containing noise generated by ASE and soliton self-frequency shift. As an example, the short wavelength (first wavelength) is a wavelength not less than 1800 nm and less than 2000 nm, and the long wavelength (second wavelength) is a wavelength not less than 2000 nm and not more than 2200 nm. For example, light on the second wavelength side of the ultrashort pulse light L including the wavelength band of 1800 nm to 2200 nm generates amplified light of noise caused by ASE and soliton self-frequency shift during amplification in the first fiber amplifier described above, which not only prevents sufficient amplification in the second fiber amplifier described above but also causes parasitic oscillation. However, these effects can be suppressed by a filter provided between the fiber amplifiers, so the light is reliably amplified and output to the compressor 709.

[0062] The compressor 709 compresses the time width of the ultrashort pulsed light amplified by the fiber amplifier 708. The compressor 709 has a diffraction grating pair 91, 92. The diffraction grating pair 91, 92 is configured to compress the time width by changing the group delay time of each wavelength by causing a difference in the optical path length of each wavelength. The wavelength band of the ultrashort pulsed light whose time width is compressed by the diffraction grating pair 91, 92 is, for example, 1800 nm to 2200 nm.

[0063] Next, a method for determining the first characteristic of the first fiber 71, the second characteristic of the second fiber 72, and the characteristic (compression characteristic) of the diffraction grating pair 91, 92 will be described.

[0064] The first and second characteristics described above determine how the ultrashort pulsed light L expands. The compression characteristic determines how the ultrashort pulsed light L is compressed. The first, second, and compression characteristics are represented, for example, by information related to group velocity dispersion (GVD). The group velocity is the speed of a wave packet associated with the ultrashort pulsed light. The group velocity dispersion is the chromatic dispersion of the group velocity. The information related to group velocity dispersion may be the group velocity dispersion itself, group delay dispersion (GDD) obtained by multiplying the group velocity dispersion by the length of the medium (fiber length or the distance between diffraction grating pairs) in consideration of the length, or a third order group delay dispersion (TOD / GVD) ratio, which is the ratio of the group velocity dispersion to the third order group delay dispersion (TOD), which is the differentiation of the group velocity dispersion with respect to frequency.

[0065] The above-described characteristics are determined, for example, by the following procedure. First, in a configuration as shown in FIG. 22 , in which the second fiber 72 is not present, the residual group delay dispersion is derived when the time width of the ultrashort pulsed light L is expanded by the first fiber 71, the ultrashort pulsed light is amplified by the fiber amplifier 708, and the time width of the ultrashort pulsed light L is compressed by the diffraction grating pair 91, 92. In this case, the first characteristic of the first fiber 71 is the group delay dispersion G1 of the first fiber 71 (see FIG. 23( a)), which is determined by arbitrarily determining the fiber length. Furthermore, the characteristic of the diffraction grating pair 91, 92 is the group delay dispersion G2 of the diffraction grating pair 91, 92 (see FIG. 23( a)), which is determined by arbitrarily determining the distance l between the diffraction grating pair 91, 92 (see FIG. 20( b)). Specifically, the group delay dispersion G2 of the diffraction grating pair 91, 92 may be derived by the following equation (2). In the following equation (2), λ is the wavelength, c is the speed of light, d is the grating constant, l is the distance between the diffraction grating pair 91 and 92, and θ is the angle of incidence of light. Group delay dispersion G2(λ)=−λ3l / πc2d2[1−(λ / d−sinθ)2] (2)

[0066] The group delay dispersion G1 of the first fiber 71 and the group delay dispersion G2 of the second fiber 72 are then added together to derive the residual group delay dispersion G3 (see FIG. 23( b)). In FIGS. 23( a) and 23(b), the horizontal axis represents wavelength, and the vertical axis represents the group delay dispersion value. As shown in FIG. 23( b), the residual group delay dispersion G3 value for the wavelength X1 portion of the ultrashort optical pulse is zero, realizing restoration of the ultrashort optical pulse (return to the state before stretching) and appropriate compression. On the other hand, as shown in FIG. 23( b), the residual group delay dispersion G3 value for the wavelength X2 portion of the ultrashort optical pulse is a value that deviates from zero, preventing restoration of the ultrashort optical pulse (return to the state before stretching) and appropriate compression.

[0067] Then, with the group delay dispersion G1, which is the first characteristic, the group delay dispersion G2, which is the compression characteristic, and the residual group delay dispersion G3 determined, the group delay dispersion G4 of the second fiber 72, which is the second characteristic of the second fiber 72, is set so that the value of the residual group delay dispersion G3 approaches a desired value (for example, 0). Specifically, the group delay dispersion G4 may be set to a value obtained by multiplying the residual group delay dispersion G3 by −1. The group delay dispersion G4 is determined, for example, by the fiber length of the second fiber 72.

[0068] As described above, the second characteristic of the second fiber 72 is set so that when the time width is expanded by the first fiber 71 and then compressed by the diffraction grating pair 91, 92 in a configuration in which the second fiber 72 is not present, the value of the residual group delay dispersion G3 (an index related to group velocity dispersion) of each wavelength remaining in the ultrashort pulse light approaches a predetermined value, preferably approaches 0.

[0069] 14 , the wavelength conversion unit 710 is configured to perform wavelength conversion for each pulse of the ultrashort pulsed light output from the compressor 709. The wavelength conversion unit 710 may perform wavelength conversion for each pulse to a wavelength band of 900 nm to 1100 nm, for example.

[0070] The wavelength conversion unit 710 converts, for example, light of 1800 nm to 2200 nm to 900 to 1100 nm, which is usable in two-photon microscopes. The wavelength conversion unit 710 may perform the wavelength conversion by second harmonic generation, which is a phenomenon included in nonlinear effects. Second harmonic generation is a phenomenon in which the original wavelength (fundamental wave) is converted into light of half the wavelength (double wave).

[0071] Conversion of a fundamental wave to a second harmonic wave in second harmonic generation will now be described. FIG. 24 is a diagram illustrating the generation of a second harmonic wave. As shown in FIG. 24 , when high-intensity ultrashort pulsed light, which is the fundamental wave, enters a crystal (nonlinear optical medium) that is a wavelength conversion element, polarization oscillation generates a second harmonic wave, which is a harmonic component of electron oscillation. In detail, the harmonic wave (second harmonic wave) is generated at each point in the crystal through which the ultrashort pulsed light passes. The final second harmonic wave is the sum of these second harmonic waves generated at each point.

[0072] Here, in the example shown in Figure 24, the phases of the doubled waves at each point are aligned with each other, but this is not necessarily the case. Because reactions occur sequentially, such as the crystal responding to the fundamental wave and then generating a doubled wave, the difference in the crystal's response is reflected in the phase of the doubled wave, resulting in a misalignment of the phases of the doubled waves at each point. If the doubled waves at each point are added together when their phases are not aligned, the doubled waves will cancel each other out or overlap, resulting in an insufficient magnitude of the added doubled wave. For this reason, it is important to determine the crystal conditions so that the phases of the doubled waves generated at each point are aligned as closely as possible (performing phase matching). A phase-matched state is one in which the refractive index of the fundamental wave and the refractive index perceived by the harmonic wave are equal.

[0073] FIG. 25 is a graph showing the wavelength conversion efficiency in each wavelength band for a nonlinear optical crystal, such as single crystal BBO (Beta-BaB2O4), for each crystal length. In FIG. 25, the horizontal axis represents the wavelength band, and the vertical axis represents the normalized wavelength conversion efficiency in second-harmonic generation (SHG). The solid line in FIG. 25 represents the wavelength conversion efficiency in each wavelength band for a relatively thick crystal with a crystal length of 1 mm, while the dashed line represents the wavelength conversion efficiency in each wavelength band for a relatively thin crystal with a crystal length of 0.2 mm. Note that the maximum wavelength conversion efficiency is higher for thicker crystals, but because the wavelength conversion efficiency in FIG. 25 is normalized, the upper limit of the wavelength conversion efficiency is 1 for all crystals. As shown in FIG. 25, the longer (thicker) the crystal, the stricter the conditions for superimposing the doubled waves at each point, resulting in a narrower wavelength band in which wavelength conversion can be performed with high efficiency. Therefore, from the perspective of widening the wavelength band in which phase matching is achieved, it is considered to use a relatively thin crystal.

[0074] However, because the intensity of the second harmonic wave is proportional to the square of the crystal length, it is difficult to increase the wavelength conversion efficiency with relatively thin crystals. Because the intensity of the second harmonic wave also depends on the intensity of the fundamental wave, it is possible to ensure the intensity of the second harmonic wave by increasing the intensity of the fundamental wave. However, amplifying the fundamental wave to compensate for the effect of the square of the crystal length described above is not cost-effective. It is also possible to spatially increase the intensity of the fundamental wave by focusing the light onto a very small area of ​​the crystal. However, focusing the fundamental wave to a small area also generates nonlinear effects from third order onward, which inhibit second-harmonic generation and ultimately reduce wavelength conversion efficiency. Furthermore, high intensity may damage the crystal itself. For these reasons, it is difficult to sufficiently increase the wavelength conversion efficiency with the relatively thin crystals described above.

[0075] Therefore, in order to achieve broadband phase matching, which is a problem with thick crystals, while using a relatively thick crystal, a configuration can be considered in which a drive system is provided to change the angle of incidence of light on the crystal. Figure 26 is a diagram explaining angular phase matching. In the configuration shown in Figure 26, a birefringent crystal 500 is prepared, and the refractive index is adjusted by adjusting the angle of incidence of light on the crystal 500 using a drive system (not shown), thereby achieving broadband phase matching.

[0076] However, in a configuration having a driving system such as that shown in Figure 26, it is impossible to drive the driving system for each pulse of ultrashort pulsed light (for example, every several tens of MHz), and it is difficult to use this configuration to perform wavelength conversion of ultrashort pulsed light.

[0077] As described above, in the past, it was difficult to perform wavelength conversion with high efficiency in a configuration that performs wavelength conversion over a wide band in a short time, such as when performing wavelength conversion of ultrashort pulsed light in which the wavelength is changed for each pulse.

[0078] To address these issues, the wavelength conversion unit 710 according to this embodiment performs highly efficient wavelength conversion using a relatively thick crystal, and also performs broadband wavelength conversion of ultrashort pulsed light by dispersing light for each wavelength band and changing the optical path for each wavelength band without using a driving system. Below, a detailed configuration example of the wavelength conversion unit 710 will be described with reference to FIG.

[0079] Fig. 27 is a diagram illustrating an example of the configuration of the wavelength conversion unit 710. As shown in Fig. 27, the wavelength conversion unit 710 includes a diffraction grating (dispersion unit) 1101, a lens (light-collecting unit) 1102, a wavelength conversion element (wavelength conversion unit) 1103, a lens 1104, and a diffraction grating 1105. The diffraction grating 1101 and the lens 1102 constitute a wavelength-specific optical path setting unit that changes the optical path for each wavelength band so that light of a wavelength band to be converted at each incident position (described in detail later) is incident on the wavelength conversion element 1103.

[0080] The diffraction grating 1101 disperses the ultrashort pulsed light output from the compressor 709 into each wavelength band (spectroscopes the ultrashort pulsed light), thereby changing the optical path for each wavelength band. Specifically, the diffraction grating 1101 disperses the ultrashort pulsed light into each wavelength band so that light of a wavelength band to be converted at each incident position is incident on each incident position of the wavelength conversion element 1103.

[0081] The lens 1102 is a lens that focuses light from the diffraction grating 1101 onto the incident position of the wavelength conversion element 1103. The distance from the diffraction grating 1101 to the lens 1102 and the distance from the lens 1102 to the wavelength conversion element 1103 are both set to match the focal length f of the lens 1102, for example, the focal length f of the lens 1102.

[0082] The wavelength conversion element 1103 is a crystal that converts different wavelength bands depending on the incident position of light. The wavelength conversion element 1103 has a fan-shaped structure so that quasi-phase matching of different wavelength bands can be achieved at each incident position. More specifically, the wavelength conversion element 1103 may be a PPLN (Periodically Poled Lithium Niobate) with a fan-shaped structure. That is, the wavelength conversion element 1103 may be a so-called fan-out PPLN. The crystal length (thickness) of the wavelength conversion element 1103 may be, for example, about 1 mm to 20 mm.

[0083] Quasi-phase matching is a technique for achieving pseudo-phase matching by preventing the generation of the doubled wave to be canceled out of the doubled waves that are added together, or by inverting the phase of the doubled wave to be canceled. Here, we will explain an example of quasi-phase matching in which the phase of the doubled wave to be canceled out is inverted (the polarity of the crystal is inverted).

[0084] The wavelength conversion element 1103 has a fan-shaped structure, which makes it possible to achieve quasi-phase matching of any wavelength band at each incident position of the crystal. That is, the period of the part where the double waves cancel each other varies depending on the wavelength band, and by using a crystal whose period is inverted according to the fan-shaped structure, the wavelength bands at which quasi-phase matching is achieved at each incident position of the crystal can be made different from each other. In such a wavelength conversion element 1103, it is possible to specify in advance which wavelength band quasi-phase matching is achieved at each incident position, and by setting the above-mentioned diffraction grating 1101 and lens 1102 so that light of the wavelength band to be converted is incident at each incident position, it is possible to appropriately perform wavelength conversion for each of the light of various wavelength bands.

[0085] The lens 1104 is a lens that focuses the light wavelength-converted by the wavelength conversion element 1103 onto the diffraction grating 1101. The distance from the wavelength conversion element 1103 to the lens 1104 and the distance from the lens 1104 to the diffraction grating 1105 are both set to match the focal length f of the lenses 1102 and 1104, for example, the focal length f of the lenses 1102 and 1104.

[0086] Diffraction grating 1105 outputs, as wavelength-converted ultrashort pulse light, light that has been dispersed for each wavelength band by diffraction grating 1101 and then reaches it via lens 1102, wavelength conversion element 1103, and lens 1104. Note that the number of grooves in diffraction grating 1105 may be 2N, which is twice the number N of grooves in diffraction grating 1101, in order to restore the original dispersion of the light after wavelength conversion.

[0087] The configuration of the wavelength conversion unit 710 is not limited to the above embodiment and may be, for example, the configuration shown in Fig. 28 . As shown in Fig. 28 , the wavelength conversion unit according to the modified example includes a lens 1201, a BBO (Beta-BaB2O4) 1202 (first conversion element unit), a lens 1203, a lens 1204, a BBO 1205 (second conversion element unit), and a lens 1206. The lens 1201 is a lens that focuses light onto the BBO 1202. The lenses 1203 and 1204 are lenses that focus light from the BBO 1202 onto the BBO 1205. The lens 1206 is a lens that focuses light from the BBO 1205 and emits it.

[0088] The BBO crystals 1202 and 1205 are nonlinear optical crystals that perform wavelength conversion. The BBO crystal 1202 is arranged at an angle (phase matching angle) suitable for wavelength conversion of a first wavelength band. The BBO crystal 1205 is connected in series to the BBO crystal 1202 and arranged at an angle (phase matching angle) suitable for wavelength conversion of a second wavelength band different from the first wavelength band.

[0089] This configuration allows wavelength conversion of the wavelength bands corresponding to the BBOs 1202 and 1205, respectively, with a simple configuration in which the BBOs 1202 and 1205 are connected in series. This configuration allows appropriate wavelength conversion of multiple wavelength bands without using a drive system to move the BBOs 1202 and 1205. Because wavelength conversion can be performed without using a drive system, appropriate wavelength conversion can be performed even in cases where wavelength conversion is required in an extremely short time, such as with ultrashort pulsed light (when the wavelength conversion cannot be completed in time by moving the wavelength conversion element using a drive system). Furthermore, this configuration allows the thickness and shape of the BBOs 1202 and 1205 to be appropriately set for highly efficient wavelength conversion, thereby enabling highly efficient wavelength conversion. As described above, the wavelength conversion unit of this modified example allows highly efficient wavelength conversion in a configuration that performs wavelength conversion over a wide band in a short time.

[0090] It is possible to connect the BBOs 1202 and 1205 in parallel rather than in series, but in this case, some wavelength bands will become unusable when the wavelength bands are divided. That is, the light in the wavelength band between the first wavelength band and the second wavelength band will become unusable. For this reason, from the perspective of using a wide wavelength band, it is preferable that the BBOs 1202 and 1205 are connected in series. In the above example, two types of BBOs were used, but the number of BBOs may be increased depending on the number of wavelength bands to be converted.

[0091] The configuration of the pulsed light generator is not limited to the above-described embodiment, and for example, a pulsed light generator 10A shown in Fig. 29 may be employed. Embodiments of the pulsed light generator 10A will be described below with reference to Figs. 29 to 33.

[0092] 29 , the pulsed light generator 10A includes an oscillator 702, a first fiber amplifier 753, an acousto-optic modulator 704, a compressor 705, a soliton shift fiber 706, a second fiber amplifier 757, a filter 758, a third fiber amplifier 759, and a wavelength converter 710. The following describes the first fiber amplifier 753, the second fiber amplifier 757, the filter 758, and the third fiber amplifier 759, which are different from the pulsed light generator 10 described above.

[0093] The first fiber amplifier 753 constitutes an amplifying section that broadens the spectrum of the ultrashort optical pulse generated by the oscillator 702. The first fiber amplifier 753 broadens the spectrum of the ultrashort optical pulse and increases the output power of the ultrashort optical pulse by similariton amplification. The first fiber amplifier 753 is disposed between the oscillator 702 and the soliton-shifted fiber 706 in the optical path of the ultrashort optical pulse.

[0094] The first fiber amplifier 753 includes a normal dispersion fiber and a pumping light source. The normal dispersion fiber is a double-clad fiber co-doped with erbium and ytterbium. That is, the first fiber amplifier 753 performs amplification while generating a nonlinear effect using the normal dispersion double-clad fiber to prevent stretching, and obtains ultrashort pulse light as broadband amplified light. The normal dispersion fiber is a fiber with a negative dispersion parameter D (ps / nm / km). There are no particular limitations on the dopant used in the first fiber amplifier 753, and various dopant types may be used.

[0095] The second fiber amplifier 757 constitutes a first optical amplification section that amplifies the ultrashort pulse light whose wavelength has been tuned by the soliton shift fiber 706. The second fiber amplifier 757 includes a rare-earth doped fiber and a pumping light source. The rare-earth doped fiber is, for example, a Tm fiber. The pumping light source is, for example, a laser diode that outputs pumping light having a wavelength of 1550 nm. The second fiber amplifier 757 is an amplifier optimized for short wavelengths. The second fiber amplifier 757 amplifies the ultrashort pulse light as signal light (seed light) by absorbing the gain.

[0096] The second fiber amplifier 757 has a higher gain G1 (see FIG. 31( b)) on the first wavelength side, which is the short wavelength side of the ultrashort pulsed light, than on the second wavelength side, which is the long wavelength side. Such characteristics of the second fiber amplifier 757 can be achieved, for example, by adjusting the length of the rare-earth doped fiber and the concentration of the doped rare earth. As an example, the short wavelength (first wavelength) is a wavelength not less than 1800 nm and less than 2000 nm, and the long wavelength (second wavelength) is a wavelength not less than 2000 nm and not more than 2200 nm.

[0097] The filter 758 constitutes a filter unit that filters the ultrashort pulse light amplified by the second fiber amplifier 757. The filter 758 attenuates ASE (Amplified Spontaneous Emission). In this embodiment, the filter 758 not only attenuates ASE generated by the amplification by the second fiber amplifier 757, but also attenuates light resulting from amplified noise components generated by the amplification by the second fiber amplifier 757 and light with wavelengths in the high gain region. In this embodiment, light with wavelengths in the high gain region refers to light in a predetermined wavelength band at short wavelengths that has a particularly high amplification rate compared to light in other wavelength bands. If this light is not attenuated, only this light will be preferentially amplified, which will hinder optical amplification over a wide wavelength range. Here, the ASE attenuated by the filter 758 is ASE in a predetermined wavelength band that includes a wavelength range between short and long wavelengths. Light resulting from amplified ASE and noise components not only hinders amplification, but may also cause parasitic oscillation and damage the amplifier system. ASE tends to occur, for example, when the wavelength bands of the seed light and the gain do not match, or when the power of the seed light is weak. Furthermore, in the filter 758, pulses in the short wavelength region are absorbed, resulting in the gain of pulses in the following long wavelength region, and a process of energy transfer from pulses in the gain region of the gain fiber to pulses in the gainless region exists. The filter 758 may be, for example, an acousto-optic modulator (AOM) or an electro-optic modulator (EOM), or may be a gain fiber such as a Tm fiber, a Ho fiber, or a Tm / Ho fiber that does not allow the pump light to enter. The filter 758 may also be a gain fiber in the region of the gain fiber of the second fiber amplifier 757 where the pump light is attenuated by 20 dB or more.

[0098] The third fiber amplifier 759 constitutes a second optical amplifier section that amplifies the ultrashort pulsed light filtered by the filter 758. The third fiber amplifier 759 includes a rare-earth-doped fiber and a pumping light source. The rare-earth-doped fiber is, for example, a Tm fiber. The pumping light source is, for example, a laser diode that outputs pumping light with a wavelength of 790 nm or a CW laser that outputs pumping light with a wavelength of 1550 nm. The third fiber amplifier 759 is an amplifier optimized for the long wavelength side. The third fiber amplifier 759 amplifies the ultrashort pulsed light L as signal light by absorbing the gain. The third fiber amplifier 759 has a higher gain G2 (see FIG. 31( d) ) on the second wavelength side, which is the long wavelength side of the ultrashort pulsed light L, than on the first wavelength side, which is the short wavelength side. Such characteristics of the third fiber amplifier 759 can be achieved, for example, by adjusting the length of the rare-earth-doped fiber and the concentration of the rare earth added.

[0099] Next, a wavelength tuning method performed using the pulsed light generating device 10A will be described with reference to the flowchart of FIG.

[0100] First, an oscillator 702 oscillates an ultrashort pulse light L to generate an ultrashort pulse train with a predetermined period (oscillation step: step S1). A first fiber amplifier 753 increases the output power of the ultrashort pulse light L and broadens the spectrum of the ultrashort pulse light L (amplification step: step S2). An acousto-optic modulator 704 controls the intensity of the ultrashort pulse light L for each pulse, for example, according to required specifications or conditions (step S3). A compressor 705 compresses the time width of the ultrashort pulse light L (step S4). A soliton shift fiber 706 modulates and variates the wavelength of the spectrally broadened ultrashort pulse light L using soliton self-frequency shifting (step S5).

[0101] Next, the ultrashort pulsed light L whose wavelength has been tuned in step S5 is amplified by the second fiber amplifier 757 (step S6). In step S6, the ultrashort pulsed light L is amplified at a gain G1 that is higher on the short wavelength side than on the long wavelength side. The ultrashort pulsed light L amplified in step S6 is filtered by the filter 758 (step S7). This attenuates ASE in a predetermined wavelength band generated by the amplification by the second fiber amplifier 757, light in which noise components have been amplified, and light with wavelengths in the high gain region.

[0102] Next, the ultrashort pulsed light L filtered in step S7 is amplified by the third fiber amplifier 759 (step S8). In step S8, the ultrashort pulsed light L is amplified at a gain G2 that is higher on the long wavelength side than on the short wavelength side. Then, the wavelength of the amplified ultrashort pulsed light L is converted to a target wavelength by the wavelength conversion unit 710 (step S9).

[0103] The pulsed light generator 10A described above exhibits the following advantageous effects when the wavelength of the ultrashort pulsed light L is tuned to a short wavelength λ1, as shown in Fig. 31(a). Specifically, as shown in Fig. 31(b), the ultrashort pulsed light L of the short wavelength λ1 is amplified (preamplifier) ​​by the second fiber amplifier 757 having a gain G1 without generating ASE. As shown in Fig. 31(c), the preamplified ultrashort pulsed light L of the short wavelength λ1 passes through the filter 758 without being attenuated. The ultrashort pulsed light L that has passed through the filter 758 is then further amplified by the third fiber amplifier 759 having a gain G2, as shown in Fig. 31(d). At this time, although the gain G2 is high on the long wavelength side, the ultrashort pulse light L of the short wavelength λ1 has already been amplified by the second fiber amplifier 757 and is therefore more likely to absorb energy, and therefore is amplified in the third fiber amplifier 759 while suppressing the occurrence of ASE (without worrying about ASE).

[0104] On the other hand, for example, when the wavelength of the ultrashort pulse light L is tuned to the long wavelength λ2 as shown in FIG. 32( a), the following effect is achieved. That is, as shown in FIG. 32( b), the ultrashort pulse light L with the long wavelength λ2 is not amplified as much as the ultrashort pulse light L with the short wavelength λ1 by the preamplifier of the second fiber amplifier 757 having a gain G1, resulting in the generation of ASE11. Thereafter, as shown in FIG. 32( c), the ASE11 is attenuated by the filter 758. Then, the ultrashort pulse light L with the long wavelength λ2 is amplified by the third fiber amplifier 759 having a gain G2 without generating ASE. Therefore, according to this embodiment, in a wavelength tuning method in which the wavelength of the output ultrashort pulse light L is tunable, it is possible to reliably amplify the ultrashort pulse light L while suppressing ASE11. This is particularly effective when optical amplification is performed over a wide wavelength band or in a wavelength band in which it is difficult to use an optical amplifier suited to the conditions.

[0105] FIG. 33 is a graph showing the relationship between wavelength and the absorption cross section, stimulated emission cross section, and filter transmittance of a Tm fiber. In FIG. 33, the horizontal axis represents the wavelength of the ultrashort pulsed light L, and the vertical axis represents the absorption cross section, stimulated emission cross section, and transmittance of the filter 758 of the Tm fiber. When amplifying light with a wavelength of 1800 to 2200 nm, the Tm fiber has the absorption cross section 922 and stimulated emission cross section 921 shown in FIG. 33. In a fiber amplifier system including a Tm fiber, if the fiber length is short, the gain on the long wavelength side is insufficient, and ASE is likely to occur when amplifying light on the long wavelength side. On the other hand, if the fiber length is long, absorption increases, and light on the short wavelength side is excessively absorbed. Therefore, ASE is likely to occur when amplifying light on the short wavelength side. In this regard, in this embodiment, a fiber amplifier including a Tm fiber is arranged in two stages, with a second fiber amplifier 757 and a third fiber amplifier 759. Then, a filter 758 with a transmittance of 923 (dotted line in the figure) is inserted between the second fiber amplifier 757 and the third fiber amplifier 759 to remove ASE, thereby enabling amplification of a wideband wavelength-tunable laser light.

[0106] In this embodiment, broadband ultrashort pulsed light L can be amplified without using a separate mechanism. Broadband ultrashort pulsed light L can be amplified without using nonlinear optical effects such as Raman amplification. This embodiment can configure a broadband and high-output (e.g., 30 dBm or more) amplifier system. This embodiment can also prevent parasitic oscillation due to increased ASE. In this embodiment, as described above, a wavelength-tunable light source is used in a two-photon microscope. In this case, high-output amplification is possible in a broad wavelength range, and high-output and multi-wavelength imaging is possible in the two-photon microscope in combination with a downstream wavelength conversion system.

[0107] Finally, the effects of the configuration according to this embodiment will be described.

[0108] The scanning microscope 1 comprises a pulsed light generator 10 (or pulsed light generator 10A) that emits excitation light, the excitation wavelength of which changes with each pulse, toward a sample, and a detector 50 that detects fluorescence from a cell mass S irradiated with the excitation light and outputs a detection signal, and the sensor bandwidth of the detector 50 is adjusted so that the detection signal attenuates within the pulse-off period of the excitation light.

[0109] In the scanning microscope 1 according to this embodiment, fluorescence from the cell cluster S irradiated with excitation light whose excitation wavelength changes with each pulse is detected by the detector 50, and a detection signal is output from the detector 50. In the scanning microscope 1 according to this embodiment, the sensor bandwidth of the detector 50 is adjusted so that the detection signal decays within the pulse-off period of the excitation light. By adjusting the detection bandwidth so that the detection signal decays within the pulse-off period (before the next pulse arrives), different pulses are temporally distinguished from one another, preventing the mixing of detection signals associated with different pulses. This eliminates the need for a configuration that filters signals according to wavelength to distinguish between different pulses, thereby suppressing the reduction in the amount of detection signal, which can be a problem when such a configuration is used. Ensuring the amount of detection signal eliminates the need to excessively increase the laser intensity of the excitation light, thereby appropriately suppressing fluorescence generation from sources other than the focal point. As described above, the scanning microscope 1 according to this embodiment improves the amount of detection signal, thereby improving the accuracy of fluorescence observation.

[0110] The intensity distribution of the focused beam with respect to depth does not change even when the laser power is reduced. Therefore, the ratio of the fluorescence generated at the focal point to the total fluorescence generated outside the focal point (near the surface) remains unchanged even when the laser power is reduced. The only way to reduce this ratio is to reduce losses due to absorption and scattering. The reason for reducing the laser power is to avoid sample damage. In deep imaging, a laser power exceeding 100 mW is irradiated onto the sample, but in the case of a mouse brain, a power of several hundred mW can cause damage such as inflammation. Furthermore, to separate the multicolor fluorescence using multiple filters and prevent spectral overlap between multiple detectors, 40–50% of the generated fluorescence is discarded. This results in low detection efficiency. Furthermore, the placement of multiple filters and detectors increases the distance from the objective lens to the detector, reducing the detection efficiency of fluorescence scattered within the sample. The configuration of this embodiment can be adopted to solve these problems.

[0111] In confocal microscopes, fluorescence intensity is proportional to the laser intensity, whereas in two-photon microscopes, fluorescence intensity is proportional to the square of the laser intensity. For example, by lowering the repetition rate from 80 MHz to 8 MHz, the pulse energy per pulse increases tenfold for the same average laser power, resulting in 100 times the fluorescence intensity. Therefore, using pulse synchronization techniques in two-photon (multiphoton) microscopes rather than confocal microscopes can increase the amount of fluorescence signal.

[0112] The sensor bandwidth of the detector 50 may be adjusted so that the combined time taken for the fluorescence to decay and the detection signal to decay is shorter than the repetition interval of the excitation light pulses. With this configuration, the time taken for the fluorescence and detection signal to decay is made shorter than the repetition interval of the pulses, thereby more reliably achieving the temporal separation of the different pulses described above.

[0113] The detector 50 may detect the fluorescence without passing through a filter (such as a bandpass filter) for wavelength separation of the fluorescence, thereby avoiding a reduction in the amount of detected signal that would otherwise be caused by passing the fluorescence through a filter, and thereby appropriately improving the amount of detected signal.

[0114] The detector 50 may be configured with a single optical sensor 51 that detects all of the fluorescence corresponding to the excitation wavelength of each pulse. This configuration allows for a simpler configuration while improving the amount of detected signal without requiring a configuration for filtering signals according to wavelength. Furthermore, with only a single optical sensor, the sensor can be positioned closest to the objective lens, improving the detection efficiency of scattered fluorescence.

[0115] The pulsed light generator 10 (or the pulsed light generator 10A) emits excitation light by switching between a first excitation wavelength for stimulating the cell cluster S and a second excitation wavelength for observing the effect of the optical stimulation of the first excitation wavelength for each pulse. The detector 50 may not detect light at the timing for detecting fluorescence from the cell cluster S corresponding to the pulse of the first excitation wavelength, but may detect light at the timing for detecting fluorescence from the cell cluster S corresponding to the pulse of the second excitation wavelength. This configuration can detect only necessary fluorescence while avoiding detection of stimulated fluorescence (removing stimulated fluorescence), thereby contributing to an improvement in the lower detection limit. Note that the pulsed light generator 10 is configured to switch the excitation wavelength for each pulse using pulsed light obtained from the same oscillator, which is more convenient than a configuration in which the excitation wavelength is switched for each pulse using excitation lasers of multiple wavelengths. In a method using two different lasers, it is difficult to align the frequencies in the different resonators, and the intervals between pulses may gradually shift over time, making it difficult to fully achieve the expected effect. However, this problem can be solved by using pulsed light obtained from the same oscillator.

[0116] The scanning microscope 1 further includes an A / D converter 60 that converts the detection signal output from the detector 50 into a digital signal, and the sampling frequency of the A / D converter 60 may be synchronized with the repetition frequency of the excitation light pulse. This configuration makes it easier to obtain the necessary information from the analog signal corresponding to each pulse without providing a high-speed A / D converter, enabling high-speed signal processing at low cost. Furthermore, an appropriate fixed delay may be provided to enable sampling at ideal timing.

[0117] The A / D converter 60 may be configured to acquire the maximum value of the detection signal. As described above, by synchronizing the frequency to always acquire the same position of the signal waveform and further acquiring the maximum value of the detection signal, it is possible to more appropriately acquire the necessary information from the analog signal corresponding to each pulse.

[0118] The A / D converter 60 may be configured to acquire an integral value of the detection signal. When acquiring the maximum value as described above, there is a risk that the maximum value may not be acquired successfully due to, for example, fluctuations in the fluorescent signal. In this regard, acquiring an integral value makes it possible to more appropriately acquire necessary information from the analog signal corresponding to each pulse. The A / D converter may also be configured to acquire a time waveform of the detection signal. In this case, information such as the fluorescence lifetime can also be extracted from the time waveform.

[0119] The present disclosure is not limited to the above-described embodiment. Modified examples will be described with reference to FIGS.

[0120] Fig. 34 is a diagram schematically illustrating a scanning microscope 2001 (fluorescence detection device) according to a modified example. For ease of explanation, some components are not shown in Fig. 34. The scanning microscope 2001 shown in Fig. 34 includes a pulsed light generator 2010, a microscope housing 2080, a fluorescence detector 2050, and an A / D converter 2060 (see Fig. 35).

[0121] The pulsed light generator 2010 includes an oscillator 2011 , a first fiber amplifier / compressor 2012 , an AOM (acousto-optic modulator) 2013 , a soliton shift fiber 2014 , a second fiber amplifier / compressor 2015 , and a wavelength conversion unit 2016 .

[0122] The oscillator 2011 generates pulsed light. The first fiber amplifier / compressor 2012 amplifies (increases the output power of) the pulsed light output from the oscillator 2011 and compresses the pulse duration of the pulsed light. The fiber amplifier may be, for example, a normal dispersion fiber containing erbium. The AOM 2013 is provided downstream of the first fiber amplifier / compressor 2012 and modulates the ultrashort pulsed light using acoustic (sound wave) power. The soliton shift fiber 2014 is provided downstream of the AOM 2013 and modulates the wavelength of the pulsed light using soliton self-frequency shift. The second fiber amplifier / compressor 2015 is provided downstream of the soliton shift fiber 2014 and amplifies the pulsed light and compresses the pulse duration of the pulsed light. The wavelength conversion unit 2016 performs wavelength conversion using, for example, second harmonic generation (SHG).

[0123] The pulsed light output through the wavelength conversion unit 2016 is irradiated onto the cell cluster S via a dichroic mirror 2020 included in the microscope housing 2080. Furthermore, the fluorescence from the cell cluster S is detected by a fluorescence detector 2050 via the dichroic mirror 2020. The fluorescence detector 2050 may be, for example, a photomultiplier tube or a photodiode (PD). The A / D converter 2060 (see FIG. 35 ) converts the detection signal (analog signal) output from the detector 3050 into a digital signal and outputs the digital signal.

[0124] Here, in addition to the detection signal, a synchronization signal generated by delaying the pulsed light by a predetermined time is input to the A / D converter 2060. FIG. 35 is a diagram showing the time waveforms of each signal. In FIG. 35, the horizontal axis represents time and the vertical axis represents intensity. From top to bottom, FIG. 35 shows pulsed light (e.g., light including a wavelength band of 1550 nm) emitted from the oscillator 2011, “1,” light that has passed through the AOM 2013, “3,” light input to the microscope housing 2080, “4,” light input to the fluorescence detector 2050, “4,” a signal output from the fluorescence detector 2050, and a synchronization signal. The positions of “1” to “4” in FIG. 34 correspond to the time waveforms “1” to “4” shown in FIG. 35.

[0125] 36, the synchronization signal may be generated based on a signal that is detected and output by a detector 2055 such as a photodetector after branching (sampling) a portion of the pulse train of "1," "2," and "3" in FIG. 34. For example, the synchronization signal may be generated by delaying the signal output from the detector 2055 by an arbitrary (predetermined) time using a delay generator 2090. The synchronization signal output from the delay generator 2090 is input to an A / D converter 2060.

[0126] 35 , a synchronization signal generated by delaying a pulse by a predetermined time in addition to the detection signal is input to the A / D converter 2060, causing the A / D converter 2060 to output digital signals of the detection signal and the synchronization signal. A computer (not shown) samples the signal acquisition by the fluorescence detector 2050 based on the digital signals output from the A / D converter 2060 (digital signals of the detection signal and the synchronization signal), thereby suppressing noise from the fluorescence detector 2050 and appropriately capturing the peak of the fluorescence signal of the cell cluster S generated by pulse irradiation.

[0127] Fig. 37 is a schematic diagram showing a pulsed light generator 3010 according to another modification. The pulsed light generator 3010 includes an oscillator 3011, a first fiber amplifier / compressor 3012, an AOM 3013, a soliton-shifted fiber 3014, a WDM (Wavelength Division Multiplexing) 3017, second fiber amplifier / compressors 3015, wavelength converters 3016, and a WDM 3018. In the configuration shown in Fig. 37, the AOM 3013 performs high-speed intensity modulation for each pulse, and the soliton-shifted fiber 3014 modulates the wavelength for each pulse to generate a pulse train. The WDM 3017 then splits the light into two wavelengths, and each light passes through the second fiber amplifier / compressor 3015 and the wavelength converter 3016 before reaching the WDM 3018. The WDM 3018 combines and outputs the light of two wavelengths. Although this embodiment alternates between two wavelengths, the wavelengths do not have to alternate, and more than two wavelengths may be used.

[0128] Fig. 38 is a diagram showing the time waveforms of each signal. In Fig. 38, the horizontal axis represents time and the vertical axis represents intensity. From the top, Fig. 38 shows pulsed light (e.g., light including a wavelength band of 1550 nm) emitted from oscillator 3011, modulated signal (I) input to AOM 3013, light (2) passed through AOM 3013, light (4) output from WDM 3018, a signal output from a detector (not shown), a signal obtained by delaying light of one wavelength (light A), a signal obtained by delaying light of the other wavelength (light B), and a synchronization signal generated by delaying each pump wavelength by a different delay amount.

[0129] As shown in Figure 38, the synchronization signal is generated by delaying the pulsed light by a different delay amount for each excitation wavelength. That is, the synchronization signal is generated by delaying light A by an arbitrary delay amount and delaying light B by a delay amount different from that of light A. For example, when generating a synchronization signal based on light that has passed through an AOM, the pulse train obtained after combining different wavelengths will have a shift in pulse period due to the AMP fiber length, refractive index, and optical path length. Therefore, by using a synchronization signal with an unequal period (a synchronization signal delayed by a different delay amount for each wavelength), the peak of the fluorescence signal can be properly captured.

[0130] Here, if only the detection signal and synchronization signal from the detector are considered, it may be impossible to distinguish which excitation wavelength each sample represents. In this regard, a computer (not shown) can identify the excitation wavelength associated with each sample based on an identification signal that identifies the excitation wavelength, thereby identifying which excitation wavelength each sample represents. In this case, the identification signal may be generated based on a synchronization signal for one of the excitation wavelengths. By using the synchronization signal for one of the excitation wavelengths to control the start timing of signal acquisition, the excitation wavelength of each sampled signal can be appropriately identified.

[0131] A synchronization signal generated by delaying the pulsed light output from the oscillator 3011 and before input to the first fiber amplifier / compressor 3012 may be input to the A / D converter 3060. The position where the pulsed light from which the synchronization signal is generated can be obtained may be, for example, before the first fiber amplifier / compressor 3012 or after the WDM 3018. The optical pulse is converted into an analog signal using a detector such as a photodetector, and the analog signal is binarized by setting a certain threshold for the peak value of the analog signal, and a delay amount is added to generate the synchronization signal. Here, when the pulsed light is obtained after the WDM 3018, problems arise, such as the difficulty in setting the threshold and the risk of the delay amount being off when the difference in light intensity between the above-mentioned light A and light B increases, and the need to change the threshold for generating the synchronization signal when the output is changed by the amplifier. In this regard, when the pulsed light is obtained before the first fiber amplifier / compressor 3012, although a different delay amount is required for each final output wavelength, the synchronization signal can be generated based on the pulsed light with a stable light intensity without being affected by the amplifier.

[0132] 1, 2001...Scanning microscope (fluorescence detection device), 10, 10A, 2010, 3010...Pulse light generator (excitation light source), 50...Detector, 51...Photosensor, 60, 2060, 3060...A / D converter, 2050...Fluorescence detector.

Claims

1. an excitation light source that emits excitation light, the excitation wavelength of which is switched for each pulse, toward a sample; a detection unit that detects fluorescence from the sample irradiated with the excitation light and outputs a detection signal; the detection unit has a detection band adjusted so that the detection signal attenuates during a pulse-off period of the excitation light; an A / D converter that converts the detection signal output from the detection unit into a digital signal; In addition to the detection signal, a synchronization signal generated by delaying a pulse by a predetermined time is input to the A / D converter, the excitation light source includes an oscillator that generates a pulse, and an amplifier that amplifies the pulse output from the oscillator; A fluorescence detection device, wherein a synchronization signal generated by delaying a pulse output from the oscillator and before being input to the amplifier is input to the A / D converter.

2. 2. The fluorescence detection device according to claim 1, wherein the detection section adjusts a detection band so that the sum of the time taken for the fluorescence to decay and the time taken for the detection signal to decay is shorter than the repetition interval of the pulses of the excitation light.

3. 3. The fluorescence detecting device according to claim 1, wherein the detecting section detects the fluorescence without passing through a filter for wavelength separation of the fluorescence.

4. 3. The fluorescence detecting device according to claim 1, wherein said detecting section is composed of a single optical sensor that detects all of said fluorescence corresponding to the excitation wavelength of each pulse.

5. the excitation light source emits excitation light by switching between a first excitation wavelength for stimulating the sample and a second excitation wavelength for observing an effect of optical stimulation of the first excitation wavelength for each pulse; 3. The fluorescence detection device according to claim 1, wherein the detection unit does not detect light at a timing for detecting fluorescence from the sample in response to the pulse of the first excitation wavelength, but detects light at a timing for detecting fluorescence from the sample in response to the pulse of the second excitation wavelength.

6. an A / D converter that converts the detection signal output from the detection unit into a digital signal; 3. The fluorescence detection device according to claim 1, wherein the sampling frequency of said A / D converter is synchronized with the repetition frequency of the pulses of said excitation light.

7. 7. The fluorescence detection device according to claim 6, wherein the A / D converter is set to obtain a maximum value of the detection signal.

8. 7. The fluorescence detection device according to claim 6, wherein the A / D converter is configured to obtain an integral value of the detection signal.

9. 2. The fluorescence detection device according to claim 1, wherein a synchronization signal generated by delaying a pulse by a delay amount that differs for each excitation wavelength is input to said A / D converter in addition to said detection signal.

10. a processing unit that performs predetermined signal processing on the signal output from the A / D converter; 10. The fluorescence detection device according to claim 9, wherein the processing unit identifies the excitation wavelength associated with the detection signal based on an identification signal that identifies the excitation wavelength.

11. Emitting excitation light, the excitation wavelength of which is switched for each pulse, toward the sample; detecting fluorescence from the sample irradiated with the excitation light and outputting a detection signal; a detection band is adjusted so that the detection signal attenuates during a pulse-off period of the excitation light; The method further includes converting the output detection signal into a digital signal by an A / D converter; In addition to the detection signal, a synchronization signal generated by delaying a pulse by a predetermined time is input to the A / D converter, A fluorescence detection method, wherein a synchronization signal generated by delaying a pulse oscillated by an oscillator and before being amplified by an amplifier is input to the A / D converter.