Fluorescence detection device and fluorescence detection method

Abstract

The scanning microscope of the present invention includes: a pulse light generating device (or pulse light generating device) that emits excitation light of an excitation wavelength that switches with each pulse to the sample; and a detection unit that detects the fluorescence of a cell cluster (S) irradiated by the excitation light and outputs a detection signal, wherein the detection bandwidth of the detection unit is adjusted such that the detection signal attenuates during the pulse-off period of the excitation light.

Description

Technical Field

[0001] This disclosure relates to a fluorescence detection device and a fluorescence detection method. Background Technology

[0002] Patent document 1 discloses a multiphoton microscope that generates multiphoton excitation capable of simultaneously absorbing multiple excitation photons when excitation light is irradiated onto fluorescent molecules. Existing technical documents Patent documents

[0003] Patent Document 1: Japanese Patent Publication No. 2022-524180. Summary of the Invention The problem the invention aims to solve

[0004] When observing the depths of a sample using a multiphoton microscope, the laser intensity needs to be increased as the sample moves deeper. Therefore, the possibility of damaging the sample increases during deep observation. To avoid damage, the laser intensity should be reduced as much as possible, even during deep observation. However, reducing the laser intensity results in a smaller detection signal, which may prevent proper observation.

[0005] This disclosure is made in view of the above-mentioned actual situation, and its purpose is to provide a fluorescence detection device and fluorescence detection method that improves the accuracy of fluorescence observation by increasing the amount of detection signal. Technical means for solving problems

[0006] (1) A fluorescence detection device according to one aspect of the present disclosure comprises: an excitation light source that emits excitation light with an excitation wavelength that is switched on each pulse toward a sample; and a detection unit that detects fluorescence from the sample irradiated by the excitation light and outputs a detection signal, wherein the detection bandwidth of the detection unit is adjusted such that the detection signal decays during the pulse-off period of the excitation light.

[0007] In a fluorescence detection apparatus according to one aspect of the present disclosure, a detection unit detects fluorescence from a sample irradiated with excitation light whose excitation wavelength is switched with each pulse, and outputs a detection signal from the detection unit. Furthermore, in the fluorescence detection apparatus according to one aspect of the present disclosure, the detection bandwidth of the detection unit is adjusted such that the detection signal attenuates during the pulse-off period of the excitation light. By adjusting the detection bandwidth to attenuate the detection signal during the pulse-off period (before the arrival of the next pulse), different pulses are distinguished from each other in time, preventing the mixing of detection signals associated with different pulses. Therefore, structures such as wavelength-based signal filtering for distinguishing different pulses are unnecessary, thus suppressing the reduction in the amount of detection signal that would be problematic if such a structure were provided. As described above, the fluorescence detection apparatus 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 bandwidth of the detection unit can be adjusted such that the time required for fluorescence decay and the time required for detection signal decay, when added together, is shorter than the repetition interval of the excitation light pulse. Based on this structure, the time required for fluorescence and detection signal decay can be shorter than the pulse repetition interval, thereby achieving more reliable temporal separation of the different pulses.

[0009] (3) In the fluorescence detection device described in (1) or (2) above, the detection unit does not detect fluorescence by passing through a filter used for wavelength separation of fluorescence. As a result, the reduction in the amount of detection signal caused by passing through the filter can be avoided, thereby appropriately increasing the amount of detection signal.

[0010] (4) In any one of (1) to (3) above, the detection unit may be composed of a photosensor that detects any one of the fluorescence corresponding to the excitation wavelength of each pulse. With such a structure, the amount of detection signal can be increased without setting a structure to filter the signal according to the wavelength, and the structure can be simplified.

[0011] (5) In any one of (1) to (4) above, the excitation light source may also 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 light stimulation of the first excitation wavelength with each pulse. The detection unit may also not perform light detection at the fluorescence detection time from the sample corresponding to the pulse of the first excitation wavelength, but perform light detection at the fluorescence detection time from the sample corresponding to the pulse of the second excitation wavelength. With such a structure, it is possible to avoid detecting stimulating fluorescence (removing stimulating fluorescence) and only detect the desired fluorescence, thereby helping to improve the detection limit.

[0012] (6) In any one of (1) to (5) above, the fluorescence detection apparatus 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 pulse. With such a structure, it is possible to easily obtain the required information from the analog signal corresponding to each pulse without the need for a high-speed A / D converter, thereby enabling the processing of high-speed signals at low cost.

[0013] (7) In the fluorescence detection device described in (6) above, the A / D converter can be set to acquire the maximum value of the detection signal. By synchronizing the frequency as described above to always acquire the same position of the signal waveform, and further by acquiring the maximum value of the detection signal, the required 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 can be set to acquire the integral value of the detection signal. When acquiring the maximum value as described above, it may be impossible to acquire the maximum value well, for example, due to the jitter of the fluorescence signal. In this regard, by acquiring the integral value, the required information can be more appropriately obtained 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 to a sample with an excitation wavelength switched on each pulse; and detecting fluorescence from the sample irradiated by the excitation light and outputting a detection signal such that the detection bandwidth is adjusted to attenuate during the pulse-off period of the excitation light. Invention Effects

[0016] According to one aspect of this disclosure, a fluorescence detection device and a fluorescence detection method can be provided to improve the accuracy of fluorescence observation by increasing the amount of detection signal. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of a scanning microscope illustrating an embodiment. Figure 2 This diagram illustrates the problem of deep imaging. Figure 3 This is a diagram illustrating the excitation light and fluorescence of GFP and RFP. Figure 4 (a) is a diagram illustrating an example of excitation in two different frequency bands. Figure 4 (b) is an explanation and Figure 4 The graph of the fluorescence signal corresponding to the excitation light in (a). Figure 4 (c) is a graph illustrating the sensor bandwidth. Figure 5 This is a diagram schematically illustrating an example of the structure of the detector for the comparative example. Figure 6 This is a chart illustrating why the full fluorescence spectrum cannot be obtained due to the bandpass filter. Figure 7 (a) is a diagram illustrating an example of excitation in two different frequency bands. Figure 7 (b) is an explanation and Figure 7 The graph of the fluorescence signal corresponding to the excitation light in (a). Figure 7 (c) is a graph illustrating the sensor bandwidth. Figure 8 This is a diagram that schematically illustrates an example of the structure of the detector in this embodiment. Figure 9 (a) and (b) are graphs illustrating the sensor bandwidth conditions. Figure 10(a) is a graph showing the case where the sensor bandwidth is narrower than the A / D converter bandwidth. Figure 10 (b) is a graph showing the case where the sensor bandwidth is relaxed. Figure 10 (c) is a graph illustrating the method of obtaining only the maximum value of the detection signal. Figure 11 Figures (a) and (b) illustrate the effect of synchronizing the sampling frequency of the A / D converter with the repetition frequency of the pulse. Figure 12 (a) and (b) are graphs illustrating the acquisition of the integral value of the detection signal. Figure 13 This diagram illustrates the detection method used in light stimulation control. Figure 14 This is a block diagram showing a pulsed light generating device. Figure 15 (a) shows from Figure 14 A graph showing the time waveform of the ultrashort pulse light output by the oscillator. Figure 15 (b) shows from Figure 14 A graph showing the spectrum of ultrashort pulse light output by the oscillator. Figure 15 (c) is showing from Figure 14 A graph showing the time waveform of the ultrashort pulse light output by the fiber amplifier. Figure 15 (d) is showing from Figure 14 A graph showing the spectrum of ultrashort pulse light output from an optical fiber amplifier. Figure 16 It shows from Figure 14 A graph showing a specific example of the spectrum of ultrashort pulse light output from an optical fiber amplifier. Figure 17 (a) shows from Figure 14 A graph showing the time waveform of the ultrashort pulse light output by the acousto-optic modulator. Figure 17 (b) shows from Figure 14 A graph showing the spectrum of the ultrashort pulse light output by the acousto-optic modulator. Figure 18 (a) shows from Figure 14 A graph showing the time waveform of the ultrashort pulse light output from the soliton frequency-shifting fiber. Figure 18 (b) shows from Figure 14 A graph showing the spectrum of ultrashort pulse light output from a soliton frequency-shifting fiber. Figure 18 (c) is showing from Figure 14 A graph showing the time waveform of the ultrashort pulse light output by the filter. Figure 18 (d) is showing from Figure 14 A graph showing the spectrum of ultrashort pulse light output from the filter. Figure 19This is a diagram illustrating the amplification of chirped pulses. Figure 20 (a) is a diagram illustrating the method of creating an optical path difference by causing a material to pass through a material with a difference in refractive index. Figure 20 (b) is a diagram illustrating the method of generating optical path difference through diffraction. Figure 21 This is a schematic diagram of a structure related to chirped pulse amplification. Figure 22 This is a schematic diagram of a structure related to chirped pulse amplification with time-width extension in an optical fiber. Figure 23 (a) is shown Figure 22 The diagram shows the stretcher and compressor characteristics in the structure. Figure 23 (b) is shown Figure 22 A diagram of residual GVD in the structure. Figure 24 This is a diagram illustrating the generation of a double wave. Figure 25 It is a graph showing the wavelength conversion efficiency of each crystal length in each band. Figure 26 This is a diagram illustrating angular phase matching. Figure 27 This is a diagram illustrating an example of the wavelength conversion section structure. Figure 28 This is a diagram illustrating an example of the wavelength conversion section structure of a modified example. Figure 29 This is a block diagram illustrating another example of a pulsed light generating device. Figure 30 This is a flowchart illustrating the wavelength-variable method. Figure 31 (a) is a diagram used to illustrate an example of amplifying short-wavelength ultrashort pulses of light. Figure 31 (b) is used to explain Figure 31 The subsequent chart of (a). Figure 31 (c) is used to explain Figure 31 The subsequent chart of (b). Figure 31 (d) is used to explain Figure 31 The subsequent chart of (c). Figure 32 (a) is a diagram used to illustrate an example of amplifying long-wavelength ultrashort pulses of light. Figure 32 (b) is used to explain Figure 32 The subsequent chart of (a). Figure 32 (c) is used to explain Figure 32 The subsequent chart of (b). Figure 32 (d) is used to explain Figure 32The subsequent chart of (c). Figure 33 It is a graph showing the relationship between the absorption cross section, stimulated emission cross section, and transmittance of the filter and wavelength of the Tm optical fiber. Figure 34 This is a schematic diagram of a modified scanning microscope (fluorescence detection device). Figure 35 It is a graph showing the time waveforms of each signal. Figure 36 This is a diagram illustrating a delay generator. Figure 37 This is a schematic diagram illustrating another variation of a pulsed light generating device (excitation source). Figure 38 It is a graph showing the time waveforms of each signal. Detailed Implementation

[0018] Hereinafter, the embodiments will be described in detail with reference to the accompanying drawings. In each drawing, the same or equivalent parts are labeled with the same symbols, and repeated descriptions are omitted.

[0019] Figure 1 This diagram schematically illustrates a scanning microscope (fluorescence detection device) 1 according to an embodiment. The scanning microscope 1 is, for example, a laser scanning microscope (LSM). Alternatively, the scanning microscope 1 is a multiphoton microscope, or for example, a two-photon microscope. The scanning microscope 1 irradiates the cell cluster S (sample) with excitation light. The cell cluster S is then subjected to staining treatment, for example, using a fluorescent probe. Therefore, when the cell cluster S is irradiated with excitation light, the cell cluster S that has absorbed the fluorescent probe will emit fluorescence. In the above staining treatment, for example, green fluorescent protein (GFP) and red fluorescent protein (RFP) can be used as fluorescent molecules (fluorescent dyes) to label the antibody of the cell cluster S. The scanning microscope 1 detects the fluorescence emitted by the cell cluster S.

[0020] The scanning microscope 1 is equipped with a pulse light generating device 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 generating device 10 is, for example, a laser device, and in this embodiment, a femtosecond laser device. The pulsed light generating device 10 generates ultrashort pulsed light as excitation light L1. The pulsed light generating device 10 performs wavelength switching for each pulse. Furthermore, the pulsed light generating device 10 only needs to have an output form that performs wavelength switching for each pulse; in addition to this output form, it can also have different output forms, such as continuously outputting pulsed light with a single wavelength, outputting multiple pulsed light for each of the multiple wavelengths after wavelength switching, or outputting pulsed light with completely random wavelengths and frequencies, etc. That is, the pulsed light generating device 10 emits excitation light L1, which switches the excitation wavelength for each pulse, towards the cell cluster S. Details of the pulsed light generating device 10 will be described later.

[0022] Dichroic mirror 20 is a reflective mirror that reflects light of a specific wavelength while transmitting light of other wavelengths. Dichroic mirror 20 reflects light of the wavelength band that can be used as excitation light L1 in the direction of objective lens 30. In addition, dichroic mirror 20 allows fluorescence L2 from cell cluster S, described later, to pass through.

[0023] Objective lens 30 is positioned opposite cell cluster S and is a lens that focuses the excitation light L1 incident via dichroic mirror 20 to a focal point FP of cell cluster S. Fluorescence L2 generated at focal point FP passes through objective lens 30 and dichroic mirror 20. Fluorescence L2 transmitted through dichroic mirror 20 is then incident on detector 50 via condenser lens 40. Furthermore, an excitation light cutoff filter (not shown) may be provided between condenser lens 40 and detector 50 to suppress the excitation light L1 from incident on detector 50.

[0024] Detector 50 is a light sensor that detects the fluorescence L2 from the cell cluster S irradiated by the excitation light L1 and outputs a detection signal. Detector 50 is, for example, an electron tube such as a photomultiplier tube (PMT) or a hybrid photodetector (HPD) that houses a photocathode and an electron multiplier within a vacuum housing. The photomultiplier tube is connected to a control circuit (not shown), which includes a high-voltage power supply and a gain adjustment circuit for adjusting the power supplied to the electron multiplier. In this embodiment, although detector 50 is described as a photomultiplier tube, it is not limited to this; it could also be a semiconductor light-receiving element such as a SiPM (Silicon Photomultiplier), an avalanche photodiode (APD), or a photodiode (PD), as well as electron tubes, image sensors, and cameras using them. Details of 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. Details of the A / D converter 60 will be described later.

[0026] The computer 70 performs prescribed control and signal processing on the digital signal (fluorescent signal) output from the A / D converter 60. The computer 70 can also transmit the signal processing results to an external server, for example, via serial communication.

[0027] Next, refer to Figures 2 to 6 This explains the problems encountered when using two-photon microscopy or other multiphoton microscopy to observe the deep parts of cell cluster S (deep imaging).

[0028] Figure 2 This diagram illustrates the problems with deep imaging. Typically, when performing deep imaging on cell cluster S, the intensity of the excitation light L1 is increased as the focal point FP is set deep within cell cluster S. However, this can potentially damage the sample during deep observation, necessitating minimizing the laser intensity of the excitation light L1.

[0029] However, in fluorescence observation devices, because wavelength-selective filters such as bandpass filters are needed to separate the fluorescence bands, the detection signal quantity tends to decrease. From the viewpoint of ensuring the detection signal quantity, it is sometimes impossible to reduce the laser intensity sufficiently. The reasons for the decrease in detection signal quantity are explained below.

[0030] Figure 3 This diagram illustrates the excitation light and fluorescence of green fluorescent protein (GFP) and red fluorescent protein (RFP). Now, consider the case where GFP and RFP are used as fluorescent molecules (fluorescent dyes) to label the antibody of cell cluster S. Figure 3 The horizontal axis represents the wavelength. Figure 3 The image shows excitation light L11 for GFP excitation and fluorescence L21 corresponding to excitation light L11, and excitation light L12 for RFP excitation and fluorescence L22 corresponding to excitation light L12. Parts of the wavelengths of fluorescence L21 and fluorescence L22 overlap.

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

[0032] Figure 4Figure (c) illustrates the detection bandwidth of the photodetector, i.e., the sensor bandwidth 301 of the detector (photosensor). In this embodiment, the detection bandwidth of the photodetector refers to the bandwidth of the output signal extracted from the photodetector as a detection signal. In other words, a wider detection bandwidth means a wider output signal bandwidth, i.e., a shorter time from the start of the rise to the end of the fall in an output signal; a narrower detection bandwidth means a narrower output signal bandwidth, i.e., a longer time from the start of the rise to the end of the fall in an output signal. Furthermore, the width and narrowness of the detection bandwidth are more specifically determined in the circuit section of the photodetector by the width and narrowness of the frequency bandwidth of the amplifier section that multiplies the current output from the photosensor and converts it into a voltage output. That is, when the frequency bandwidth of the amplifier section is wide, the detection bandwidth is wide; when the frequency bandwidth of the amplifier section is narrow, the detection bandwidth is also narrow. Therefore, in this embodiment, it can also be replaced by the frequency bandwidth of the amplifier section that multiplies the current output from the photosensor and converts it into a voltage output in the circuit section of the photodetector. Typically, the sensor bandwidth 301 of the detector (optical sensor) is set to be much narrower than the repetition interval of the excitation light pulses (see reference). Figure 4 (a) ~ Figure 4 (c) Therefore, in the detection signal output from the optical sensor (specifically, the digital signal converted in the subsequent A / D converter), it is impossible to distinguish the components of fluorescence L21 and fluorescence L22 in time. Thus, even when switching excitation light L11 and excitation light L12 on a pulse-by-pulse basis, the narrow sensor bandwidth 301 of the optical sensor makes it impossible to properly distinguish and acquire fluorescence L21 corresponding to excitation light L11 and fluorescence L22 corresponding to excitation light L12. Furthermore, even if fluorescence L21 and fluorescence L22 experience intensity fluctuations within a shorter time than the sensor bandwidth 301, these fluctuations will not be reflected in the detection signal, and therefore, this information cannot be acquired.

[0033] To properly distinguish and obtain fluorescence L21 and fluorescence L22, the following methods can be considered: Figure 5 The structure shown includes bandpass filters 503 and 603. Figure 5In the example shown, regarding fluorescence, light in the 500nm wavelength band is reflected by a dichroic mirror 400, while light in the 600nm wavelength band is transmitted. The light reflected in the dichroic mirror 400 is incident on the photodetector 501 via lens 502 and bandpass filter 503. The light detected in the photodetector 501 is GFP fluorescence. The light transmitted through the dichroic mirror 400 is incident on the photodetector 601 via lens 602 and bandpass filter 603. The light detected in the photodetector 601 is RFP fluorescence. Thus, in cases where the sensor bandwidth is narrow and it is impossible to distinguish the fluorescence of each pulse in time, a bandpass filter is needed to decompose the fluorescence signal by wavelength.

[0034] Figure 6 This chart illustrates why the full fluorescence spectrum could not be obtained due to bandpass filters 503 and 603. Figure 6 In the graph, the horizontal axis represents wavelength, and the vertical axis represents intensity. Additionally, as... Figure 6 As shown in the legend, Figure 6 The spectrum of GFP fluorescence L21, the wavelength range of bandpass filter 503 related to the detection of fluorescence L21, the spectrum of RFP fluorescence L22, and the wavelength range of bandpass filter 603 related to the detection of fluorescence L22 are shown respectively. Figure 6 As shown, the spectral ranges detected by photodetectors 501 and 601 are limited because GFP fluorescence L21 passes through bandpass filter 503 and RFP fluorescence L22 passes through bandpass filter 603. Thus, the full spectrum of the fluorescence spectrum cannot be detected due to the use of bandpass filters 503 and 603, resulting in a smaller detected fluorescence signal. Furthermore, the amount of light passing through bandpass filters 503 and 603 is reduced. In this structure, to ensure sufficient fluorescence signal, the laser intensity of the excitation light needs to be increased to a certain extent, resulting in insufficient suppression of fluorescence generation from areas outside the focal point. Additionally, even if the distinction between fluorescence L21 and fluorescence L22 is feasible, when the sensor bandwidth 301 of the photodetector is narrow, intensity fluctuations in fluorescence L21 and fluorescence L22 within a shorter time than the sensor bandwidth 301 will not be reflected in the detection signal, and therefore this information cannot be obtained.

[0035] Therefore, in the scanning microscope 1 of this embodiment, a method is employed to distinguish the fluorescence of each pulse without using a bandpass filter in the structure that switches the excitation wavelength for each pulse. Specifically, in the scanning microscope 1, the detection bandwidth (sensor bandwidth) of the detector 50 is adjusted such that the detection signal (the signal output from the detector 50) attenuates during the pulse-off period of the excitation light L1. The pulse-off period refers to the period of closure in the pulse light that is repeatedly turned on and off. The detection signal attenuation may, for example, refer to a state where the detection signal attenuates to a signal amount that is less than 10% of its maximum value.

[0036] like Figure 7 As shown in (a), waveform 101 corresponding to excitation light L11 and waveform 102 corresponding to excitation light L12 appear sequentially for each pulse, as follows: Figure 7 As shown in (b), waveform 201 corresponding to fluorescence L21 corresponding to excitation light L11 and waveform 202 corresponding to fluorescence L22 corresponding to excitation light L12 also appear sequentially. Now, as Figure 7 As shown in (c), the sensor bandwidths 351 and 352 of the detector 50 (optical sensor) are set to be sufficiently wide (equally wide) relative to the repetition interval of the excitation light pulses. Figure 7 In (c), sensor bandwidth 351 is the sensor bandwidth corresponding to fluorescence L21, and sensor bandwidth 352 is the sensor bandwidth corresponding to fluorescence L22. Figure 7 In (c), the sensor bandwidths 351 and 352 are set to a 1:1 ratio relative to waveform 201 corresponding to fluorescence L21 and waveform 202 corresponding to fluorescence L22 corresponding to excitation light L12. Furthermore, as... Figure 7 As shown in (c), the sensor bandwidths 351 and 352 of detector 50 are adjusted so that the detection signal (the waveforms of sensor bandwidths 351 and 352) attenuates during the pulse-off period of the excitation light. In this way, the detection signal of detector 50 has already attenuated before the next pulse is detected.

[0037] Figure 8 This is a schematic diagram illustrating an example of the structure of the detector 50 in this embodiment. (See diagram for reference.) Figure 8 As shown, the detector 50 consists of a photosensor 51 that detects any one of the fluorescence (e.g., fluorescence L21 and L22 mentioned above) at the excitation wavelength of each pulse. The photosensor 51 of the detector 50 detects fluorescence without passing it through a bandpass filter for wavelength separation. This bandpass-free configuration makes it possible to set the sensor bandwidth of the detector 50 such that the detection signal of the detector 50 attenuates during the pulse-off period of the excitation light, thereby temporally distinguishing the fluorescence of different pulses.

[0038] More specifically, the sensor bandwidth of detector 50 is adjusted such that the time required for the fluorescence to decay plus the time required for the detection signal to decay is shorter than the repetition interval of the excitation light pulse. Figure 9 Figures (a) and (b) are diagrams illustrating the sensor bandwidth conditions. Figure 9 In (a) and (b), the horizontal axis represents time and the vertical axis represents intensity. Now, with the repetition interval of the excitation light pulse (the gating frequency based on AOM) set to frep, the fluorescence lifetime (the longest fluorescence lifetime in multiple staining) set to T, the full width at half maximum (FWHM) of the detection signal set to FWHM, and the sensor bandwidth (signal bandwidth of the sensor) of detector 50 set to fsensor, the following equation (1) is derived based on the conditions of detector 50 described above. Furthermore, the fluorescence lifetime here refers to the time until the initial intensity of the afterglow decays to 1 / e. Since 1 / e is approximately 37%, this time is taken as four times the amount of time required for the fluorescence signal to decay. 1 / frep>4T+2FWHM 1 / frep > 4T + 2 × 1 / (2 × fsensor) 1 / fsensor<1 / frep-4T……(1) In this way, the sensor bandwidth of detector 50 is set to converge within a range determined by the pulse repetition interval and fluorescence lifetime.

[0039] Here, when the sensor bandwidth of detector 50 is narrower than the bandwidth of the A / D converter, the detection signal from detector 50 can be sampled without any problems. That is, as Figure 10 As shown in (a), when the sensor bandwidth 301 is narrow enough than the A / D converter bandwidth to allow for setting many sampling points SP for the detected signal, the detected signal can be sampled without any problems and the analog signal can be appropriately reproduced from the A / D converter. Regarding this point, as described above, in the structure of this embodiment, due to... Figure 10 As shown in (b), the sensor bandwidth 351 of detector 50 is widened. If the A / D converter bandwidth remains the same, the detection signal from detector 50 may not be adequately sampled, potentially resulting in the inability to properly reproduce the analog signal from the A / D converter. While increasing the speed of the A / D converter could be considered to address this problem, it is technically challenging and increases costs. Furthermore, in the scanning microscope 1, since scanning at high frequencies and constructing an overall image after acquiring a large amount of signal are required, the increased sampling rate associated with increasing the speed of the A / D converter means a massive increase in data volume, which is practically fatal.

[0040] Regarding this point, in the structure of this embodiment, by synchronizing the sampling frequency of the A / D converter 60 with the repetition frequency of the excitation light pulses, it is possible to sample the same position in the detection signal. In this case, it is preferable to also provide an appropriate time delay to the sampling frequency of the A / D converter 60. Alternatively, the A / D converter 60 can be configured to acquire the maximum value of the detection signal. That is, as... Figure 10 As shown in (c), the sampling point SPM of the A / D converter 60 can also be set such that it is always near the maximum value of the detected signal. By synchronizing the frequency in this way, the A / D converter 60 can be made slower (instead of slower than before) without increasing its speed, thereby contributing to data compression by sampling the detected signal with the fewest possible times. Furthermore, the maximum value of the detected signal in this embodiment is not limited to the value at the peak of the pulsed detected signal, but also includes the range of signal values ​​that show more than 70% of the signal value relative to the peak.

[0041] Furthermore, by synchronizing the sampling frequency of the A / D converter 60 with the repetition frequency of the excitation light pulses, random noise (dark noise / stray light) can be removed. For example... Figure 11 As shown in (a), without the aforementioned synchronization, the detection signal sampled at sampling point SP will contain a component of random noise D. Regarding this point, as... Figure 11 As shown in (b), if the above synchronization is performed, and only the maximum value of the detection signal is detected at the sampling point SPM, the random noise D is likely to be outside the detection range, and the random noise D can be appropriately removed. By removing the random noise, the detection lower limit can be expanded.

[0042] The A / D converter 60 can also be set to acquire values ​​other than the maximum value of the detected signal, for example, it can be set to acquire the integral value. Figure 12 Graphs (a) and (b) illustrate the integral values ​​of the acquired detection signal. For example... Figure 12 As shown in (a), when the sensor bandwidth 351 is relatively wide, it is affected by the jitter (fluctuation) of the fluorescence signal represented by waveform 201, which may lead to increased dispersion of the value in the above-described method of obtaining the maximum value of the detection signal. Therefore, for example, it is possible to consider inputting the detection signal to a charge amplifier circuit (not shown), charging it for the time required for the fluorescence signal to decay, and sampling only one point of the integral value. Figure 12 In the example shown in (b), a point of the charging signal CS, which is related to the integral value obtained by inputting the detection signal to the charge amplifier circuit (not shown), is set as the sampling point SPM. With this structure, the effects of jitter (fluctuations) in the fluorescence signal can be suppressed. By sampling as quickly as possible after the fluorescence signal decays, the effects of stray light, etc., can be minimized.

[0043] In addition, the scanning microscope 1 can also be used for various fluorescence observations besides those described above; for example, it can also be used for light-stimulation-controlled fluorescence observation. In this case, the pulsed light generating device 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 effect of the light stimulation of the first excitation wavelength with each pulse. Then, as... Figure 13 As shown, the detector 50 can detect light without performing sampling by the A / D converter at the fluorescence detection timing corresponding to the pulse of the first excitation wavelength from the cell cluster S (the detection timing corresponding to the excitation for stimulation), and only detect light at the fluorescence detection timing corresponding to the pulse of the second excitation wavelength from the cell cluster S (the detection timing corresponding to the excitation for observation). According to this structure, since the fluorescence corresponding to the excitation for stimulation can be removed, the situation where the fluorescence corresponding to the excitation for observation cannot be properly detected due to being buried in the fluorescence corresponding to the excitation for stimulation can be avoided. This helps to improve the detection limit.

[0044] Next, refer to Figures 14-33 The details of the pulse light generating device 10, which switches the excitation wavelength according to each pulse, will be explained.

[0045] like Figure 14 As shown, the pulsed light generating device 10 of this embodiment utilizes soliton self-frequency shifting (Raman soliton shifting) to generate long-wavelength ultrashort pulsed light (pulsed light). For example, the pulsed light generating device 10 is a femtosecond laser device for a two-photon microscope. The pulsed light generating device 10 includes an oscillator 702, an fiber amplifier 703, an acousto-optic modulator 704, a compressor 705, a soliton frequency shifting fiber 706, a stretcher fiber 707, an fiber amplifier 708, a compressor 709, and a wavelength conversion unit 710.

[0046] Oscillator 702 constitutes the oscillation section that oscillates to produce ultrashort pulses of light. Oscillator 702, as... Figure 15 As shown in (a), an ultrashort pulse sequence with a specified period F1 is generated. The wavelength of the ultrashort pulse light oscillating from oscillator 702 can, for example, be a wavelength range including 1550 nm. Here, oscillator 702, as... Figure 15 As shown in (b), the oscillation is an ultrashort pulse of light with a first spectral width H1 and a first intensity K1. There is no particular limitation on the oscillator 702; various oscillators can be used.

[0047] The fiber amplifier 703 constitutes an amplification section that broadbands the spectrum of the ultrashort pulse light oscillated by the oscillator 702. The fiber amplifier 703 amplifies the ultrashort pulse light spectrum and increases its power through self-similar soliton amplification. The fiber amplifier 703 is positioned in the optical path of the ultrashort pulse light between the oscillator 702 and the soliton frequency-shifting fiber 706.

[0048] The fiber amplifier 703 includes a fiber amplification unit. This fiber amplification unit of the fiber amplifier 703 is a normal-dispersion fiber, specifically a double-clad fiber co-doped with erbium and ytterbium. That is, the fiber amplifier 703 amplifies light using the normal-dispersion double-clad fiber without broadening, inducing nonlinear effects while simultaneously obtaining ultrashort pulse light as broadband amplified light. Normal-dispersion fiber is an fiber with a negative dispersion parameter D (ps / nm / km). The additives (dopants) used in the fiber amplifier 703 are not particularly limited, and various additives can be used.

[0049] like Figure 15 (c) and Figure 15 As shown in (d), the fiber amplifier 703 broadbands the spectral width of the ultrashort pulse light to a second spectral width H2, which is wider than the first spectral width H1. The fiber amplifier 703 also increases the power of the ultrashort pulse light to a second intensity K2, which is higher than the first intensity K1. Specifically, as... Figure 16 As shown, the fiber amplifier 703 sets the spectral width of the ultrashort pulse light to be greater than 100 nm. Figure 16 In the diagram, the horizontal axis represents the wavelength of the ultrashort pulse light, and the vertical axis represents the relative value of the intensity of the ultrashort pulse light relative to a specified intensity.

[0050] The acousto-optic modulator 704 constitutes a light intensity control unit that controls the intensity of ultrashort pulse light per pulse. The acousto-optic modulator 704 is a device that uses acoustic (sound wave) power to modulate ultrashort pulse light 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 frequency-shifting fiber 706 in the optical path of the ultrashort pulse light. Furthermore, the acousto-optic modulator 704 can be disposed at any position as long as it is between the oscillator 702 and the soliton frequency-shifting fiber 706. The acousto-optic modulator 704, as... Figure 17 (a) and Figure 17 As shown in (b), the intensity of the ultrashort pulse light is controlled to change with each pulse. For example, as... Figure 17 As shown in (a), when intensity modulation named M1 and M2 is applied, as Figure 17As shown in (b), ultrashort pulses LM1 and LM2 are generated, corresponding to the intensities given by M1 and M2. The intensity modulation range and accuracy of the ultrashort pulses L(LM1, LM2) depend on the performance of the acousto-optic modulator 704. The intensity of each pulse in the pulse sequence of the ultrashort pulses can be arbitrarily modulated by the acousto-optic modulator 704.

[0051] Compressor 705 constitutes a pulse compression section that compresses the pulse duration of ultrashort pulse light. In this embodiment, compressor 705 is disposed between acousto-optic modulator 704 and soliton frequency-shifting fiber 706 in the optical path of ultrashort pulse light. Furthermore, compressor 705 can be disposed at any position as long as it is between fiber amplifier 703 and soliton frequency-shifting fiber 706. Even if, for example, the ultrashort pulse light is broadened (e.g., by several picoseconds) due to fiber amplifier 703, compressor 705 can still compress the duration of the ultrashort pulse light, outputting an ultrashort pulse light with a duration broadened to a certain value (less than 1 picosecond). There are no particular limitations on compressor 705; various compressors can be used.

[0052] The soliton frequency-shifting fiber 706 constitutes a modulation unit that utilizes soliton self-frequency shifting to modulate the wavelength of ultrashort pulse light, which is simultaneously broadbanded and has increased output power by the fiber amplifier 703. The soliton frequency-shifting fiber 706 is positioned downstream of the fiber amplifier 703 in the optical path of the ultrashort pulse light. The soliton frequency-shifting fiber 706, as... Figure 18 (a) and Figure 18 As shown in (b), the wavelength of the ultrashort pulse light is lengthened to generate a soliton S1. The wavelength band of the ultrashort pulse light L that is lengthened here can, for example, be a band containing 1800 nm to 2200 nm. The soliton frequency-shifting fiber 706 can be, for example, a single-mode anomalous dispersion fiber that exhibits anomalous dispersion in the wavelength band of the ultrashort pulse light generated by the fiber amplifier 703. Furthermore, by controlling the acousto-optic modulator 704, a soliton with a wavelength different from that of soliton S1 can also be generated. The wavelength of soliton S1, for example, when... Figure 18 When the intensity of M1 and M2 is modulated as shown in (c), as Figure 18As shown in (d), soliton S shifts to the wavelength corresponding to the intensity given by M1 and M2 (solitons S1 and S2). Furthermore, the frequency-shifting wavelength range and accuracy of soliton S depend on the performance of the acousto-optic modulator 704. The frequency-shifting wavelength of each soliton S in the soliton sequence generated from the pulse sequence of ultrashort pulse light can be arbitrarily changed by intensity modulation of the pulse sequence using the acousto-optic modulator 704. Furthermore, in the illustrated example, the ultrashort pulse light modulated by soliton self-frequency shifting contains a non-soliton component S0 (components that fail to become solitons S1 or S2). Additionally, a filter (not shown) that cuts off the non-soliton component S0 of the ultrashort pulse light L can be provided at the rear end of the soliton frequency-shifting fiber 706. Such a filter can also have an OD value of 3 or higher.

[0053] The fiber stretcher 707, fiber amplifier 708, and compressor 709 are structures related to chirped pulse amplification. For information on structures related to chirped pulse amplification, please refer to [reference needed]. Figures 19-23 Please provide an explanation.

[0054] First, the chirped pulse amplification will be explained. Figure 19 This diagram illustrates chirped pulse amplification. When strongly amplifying the output of ultrashort pulse light, a process called chirped pulse amplification is sometimes performed to avoid excessively high peak power and adverse effects caused by nonlinearity. In chirped pulse amplification, the time width of the ultrashort pulse light is first broadened (broadened), then the broadened ultrashort pulse light is amplified, and finally, the time width of the amplified ultrashort pulse light is restored to its original state (restored to the state before broadening). For example... Figure 19 As shown, in the chirped pulse amplification, firstly, the pulse light I, which is an ultrashort pulse light, input to the stretcher fiber 707, has its time width stretched using the stretcher fiber 707. Then, the pulse light II, whose time width has been stretched by the stretcher fiber 707, is amplified by the fiber amplifier 708. Finally, the pulse light III, which is an ultrashort pulse light, amplified by the fiber amplifier 708, is compressed using the compressor 9 to restore its time width to its pre-stretched state.

[0055] Next, two examples will be given regarding methods for widening (or compressing) the time width in chirped pulse amplification. Both examples widen (or compress) the time width by creating a difference in optical path length. Figure 20 (a) is a diagram illustrating the method of creating a difference in optical path length by causing the difference in refractive index of light as it passes through a material. Figure 20 (b) is a diagram illustrating the method of generating differences in optical path length through diffraction. For example... Figure 20As shown in (a), an ultrashort pulse of light, formed by multiple wavelengths of light passing through each other with their phases aligned and overlapping, experiences variations in the optical path length of each wavelength due to differences in refractive index caused by the passage through a certain material. This results in variations in the group delay time of each wavelength. This can be used to widen or conversely compress the time width. The material in this case is, for example, optical fiber. Additionally, as... Figure 20 As shown in (b), the diffraction grating creates differences in the optical path lengths of 91 and 92 (details to be described later) at each wavelength, thereby changing the group delay time at each wavelength. This allows for either widening or conversely compressing the time width.

[0056] In such chirped pulse amplification, for ultrashort pulse light, when restoring the broadened time width to its original state, compression is preferably performed in the same manner as when broadening the time width. From this perspective, it is possible to consider performing broadening and compression using the same method. That is, for example, using a method that generates differences in optical path length at each wavelength based on the difference in refractive index allowing the fiber to pass through (see [reference]). Figure 20 In case (a), both the stretcher and the compressor are made of optical fiber, and a method is used to generate differences in the optical path length at each wavelength based on the diffraction grating (see [reference]). Figure 20 In case (b), a diffraction grating pair is used as both the stretcher and the compressor. In this way, by using the same type of optical elements to perform stretching and compression separately, the characteristics of stretching and compression (details to be described later) are consistent, thereby enabling compression of ultrashort pulses to the same extent as the stretched time width.

[0057] On the other hand, from the perspective of stability or throughput, it may be more advantageous to configure the stretcher and compressor with different structures when optimizing them. For example, when using optical fiber for stretching and compression, stretching can be expected to have higher throughput (typically with almost zero loss) compared to a diffraction grating pair, but regarding compression, nonlinear effects can be anticipated, leading to light that does not possess the desired characteristics. Specifically, complex dispersion added to ultrashort pulses can sometimes prevent complete compression. As a result, the energy that should be concentrated on the main pulse is dispersed to other parts, thus becoming an undesirable state in terms of causing nonlinear effects. On the other hand, when using a diffraction grating pair for stretching and compression, regarding compression, since the beam diameter increases as the ultrashort pulse propagates in a space system, the energy density is reduced, and therefore, nonlinear effects can be expected to be less likely to occur. Regarding broadening, since the diffraction efficiency of a diffraction grating pair is typically around 90%, in the case of a diffraction grating pair with multiple diffraction gratings, energy is lost with each diffraction, and the final throughput will be reduced to about 60% of the original energy.

[0058] Based on these considerations, a structure can be considered that uses optical fiber as a stretcher and diffraction grating pairs as compressors, thereby suppressing nonlinear effects while ensuring throughput. Structures that perform stretching and compression using different methods (with different types of optical elements) are less likely to maintain consistent stretching and compression characteristics (details will be described later) compared to structures that perform stretching and compression using the same method (with the same type of optical elements), making it difficult to restore the ultrashort pulse shape to its pre-stretched form after compression. This becomes an even more serious problem in broadband light sources or broadband wavelength-variable light sources. In the chirped pulse amplification structure of the pulsed light generation device 10 associated with this embodiment, to solve this problem, the stretcher optical fiber 707 is provided with... Figure 21 The two optical fibers shown are (first optical fiber 71 and second optical fiber 72).

[0059] Figure 21 This is a schematic diagram of the structure related to the chirped pulse amplification of the pulsed light generating device 10 in this embodiment. Figure 21 As shown, the pulse light generating device 10, as a structure related to chirped pulse amplification, includes a stretcher fiber 707 (stretcher), a fiber amplifier 708 (amplifier), and a compressor 709.

[0060] The stretcher fiber 707 is a stretcher that stretches the time width of ultrashort pulse light. The wavelength range of the ultrashort pulse light for which the stretcher fiber 707 stretches the time width is, for example, 1800 nm to 2200 nm. The stretcher fiber 707 is constructed by combining a first fiber 71 that stretches the time width of the ultrashort pulse light with a first characteristic, and a second fiber 72 that stretches the time width of the ultrashort pulse light with a second characteristic different from that of the first fiber 71. The first fiber 71 and the second fiber 72 utilize the difference in refractive index of each wavelength when the ultrashort pulse light passes through to create a difference in the optical path length of each wavelength, thereby stretching the time width of the ultrashort pulse light. The first fiber 71 stretches the time width of ultrashort pulse light, for example, the wavelength range of 1800 nm to 2200 nm, output from the soliton frequency-shifting fiber 706 with the first characteristic and outputs it to the second fiber 72. The first fiber 71 can also be, for example, a normal dispersion fiber. The second optical fiber 72 is connected to the first optical fiber 71. For the ultrashort pulse light input from the first optical fiber 71, it broadens the time width using a second characteristic and outputs it to the optical fiber amplifier 708. The second optical fiber 72 can also be, for example, a normal-dispersion or anomalous-dispersion optical fiber. Details regarding the first and second characteristics will be described later.

[0061] Fiber optic amplifier 708 amplifies (high-powers) pulsed light whose time width has been broadened using stretcher fiber 707. Fiber optic amplifier 708 includes a fiber amplification unit. This fiber amplification unit of fiber optic amplifier 708 is an anomalously dispersive fiber, such as thulium-doped fiber. The laser medium added to the fiber of fiber optic amplifier 708 is not particularly limited and can be rare earth elements such as ytterbium, erbium, and neodymium, or Bi, etc. The wavelength range of the ultrashort pulsed light L amplified by fiber optic amplifier 708 is, for example, 1800 nm to 2200 nm. Fiber optic amplifier 708, capable of reliably amplifying this pulsed light in a broadband wavelength range, comprises, for example, a first fiber optic amplifier (not shown) with high gain G1 (not shown) on a first wavelength side serving as the short wavelength side of the ultrashort pulsed light L, and a second fiber optic amplifier (not shown) with high gain G2 (not shown) on a second wavelength side serving as the long wavelength side, and is configured with filters between these amplifiers to reduce noise amplified light caused by ASE (spontaneous emission amplification) and soliton self-frequency shift. As an example, short wavelengths (first wavelengths) are wavelengths above 1800 nm and below 2000 nm, while long wavelengths (second wavelengths) are wavelengths above 2000 nm and below 2200 nm. For instance, light on the second wavelength side of an ultrashort pulse light L containing the 1800 nm to 2200 nm band will generate noise in the amplification of the first fiber amplifier due to ASE or soliton self-frequency shift. Therefore, it will not only fail to achieve sufficient amplification in the second fiber amplifier, but may even become a cause of parasitic oscillations. However, since these effects can be suppressed by using filters between the fiber amplifiers, the light can be reliably amplified and output to the compressor 709.

[0062] Compressor 709 compresses the time width of the ultrashort pulse light amplified by fiber amplifier 708. Compressor 709 has diffraction grating pairs 91 and 92. The diffraction grating pairs 91 and 92 are structures that compress the time width by creating differences in the optical path length of each wavelength, thereby changing the group delay time of each wavelength. The wavelength range of the ultrashort pulse light whose time width is compressed by diffraction grating pairs 91 and 92 is, for example, 1800 nm to 2200 nm.

[0063] Next, the method for determining the first characteristic of the first optical fiber 71, the second characteristic of the second optical fiber 72, and the characteristics (compression characteristics) of the diffraction grating pairs 91 and 92 will be explained.

[0064] The first and second characteristics mentioned above determine how the ultrashort pulse light L broadens. The compression characteristic determines how the ultrashort pulse light is compressed. The first, second, and compression characteristics can be represented, for example, using information related to group velocity dispersion (GVD). Group velocity refers to the velocity of the beam associated with the ultrashort pulse light. Group velocity dispersion refers to the wavelength dispersion of the group velocity. Information related to group velocity dispersion can be the group velocity dispersion itself, or it can be the group delay dispersion (GDD) obtained by multiplying the group velocity dispersion by the length of the medium (fiber length or diffraction grating pair distance), or it can be the TOD / GVD ratio, which is the ratio of the third-order group delay dispersion (TOD) obtained by differentiating the group velocity dispersion with respect to frequency.

[0065] The above characteristics can be determined, for example, through the following steps. First, in such a case... Figure 22 In the structure shown where the second fiber 72 is absent, the residual group delay dispersion is derived when the time width of the ultrashort pulse light is broadened by the first fiber 71, the ultrashort pulse light is amplified by the fiber amplifier 708, and the time width of the ultrashort pulse light L is compressed by the diffraction gratings 91 and 92. At this time, the first characteristic of the first fiber 71 is the group delay dispersion G1 of the first fiber 71, which is determined by arbitrarily determining the fiber length (refer to...). Figure 23 (a)). Furthermore, the characteristics of the diffraction grating pairs 91 and 92 are due to the distance l between the diffraction grating pairs 91 and 92 (refer to...). Figure 20 (b) The group delay dispersion G2 of the diffraction grating pairs 91 and 92 is arbitrarily determined (refer to) Figure 23 (a)). The group delay dispersion G2 of the diffraction grating pairs 91 and 92 can be derived specifically 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 pairs 91 and 92, and θ is the incident angle of light. Group delay dispersion G2(λ)=-λ3l / πc2d2[1-(λ / d-sinθ)2]……(2)

[0066] Then, by adding the group delay dispersion G1 of the first fiber 71 to the group delay dispersion G2 of the second fiber 72, the aforementioned residual group delay dispersion G3 is derived (refer to...). Figure 23 (b)). In Figure 23 In (a) and (b), the horizontal axis represents wavelength, and the vertical axis represents the group delay dispersion value. For example... Figure 23As shown in (b), in ultrashort pulse light, the partial residual group delay dispersion G3 at wavelength X1 is 0, achieving state recovery (returning to the state before broadening) and appropriate compression of the ultrashort pulse light. On the other hand, as Figure 23 As shown in (b), in ultrashort pulse light, the value of the residual group delay dispersion G3 at wavelength X2 becomes a value deviating from 0, and the ultrashort pulse light fails to recover its state (failed to recover to the state before broadening) and fails to compress properly.

[0067] Furthermore, given that the group delay dispersion G1 (a first characteristic), the group delay dispersion G2 (a compression characteristic), and the residual group delay dispersion G3 are determined, in order to make the value of the residual group delay dispersion G3 approach a desired value (e.g., 0), the group delay dispersion G4 of the second optical fiber 72, which is a second characteristic of the second optical fiber 72, is set. Specifically, the group delay dispersion G4 can be set to the 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 optical fiber 72.

[0068] As described above, the second characteristic of the second optical fiber 72 is set such that, in a structure where the second optical fiber 72 is absent, after the time width is broadened by the first optical fiber 71 and then the time width is compressed by the diffraction grating for 91 and 92, 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 is close to a predetermined value, preferably set to close to 0.

[0069] Back Figure 14 The wavelength conversion unit 710 is a structure that performs wavelength conversion for each pulse of the ultrashort pulse light output from the compressor 709. For example, the wavelength conversion unit 710 can also perform wavelength conversion for each pulse in the 900nm~1100nm band.

[0070] The wavelength conversion unit 710 converts light, for example, from 1800 nm to 2200 nm, into wavelengths of 900 to 1100 nm suitable for use in two-photon microscopy. The wavelength conversion unit 710 can also perform this wavelength conversion using second harmonic generation, a phenomenon encompassed by nonlinear effects. Second harmonic generation refers to the phenomenon of converting the original wavelength (fundamental wave) into light of half its wavelength (double harmonic).

[0071] The conversion from the fundamental frequency to the second harmonic in the generation of second harmonics is explained. Figure 24 This is a diagram illustrating the generation of a double harmonic wave. For example... Figure 24As shown, when a high-intensity ultrashort pulse of light, serving as the fundamental wave, is incident on a crystal (a nonlinear optical medium) acting as a wavelength conversion element, a second harmonic wave, which is a harmonic component of the electron vibration, is generated due to polarization vibrations. Specifically, the harmonics (second harmonic waves) are generated at various points within the crystal through which the ultrashort pulse of light passes. The sum of these second harmonic waves generated at each point constitutes the final second harmonic wave.

[0072] Here, in Figure 24 The example shown illustrates an instance where the phases of the second harmonics at each point are aligned. However, the phases of the second harmonics at each point are not always aligned. Because the crystal responds to the fundamental wave, producing second harmonics as a result, and this process repeats sequentially, differences in the crystal's response are reflected in the phases of the second harmonics, causing them to become misaligned at each point. If the second harmonics at each point are added in a misaligned state, they will either cancel each other out or superimpose, resulting in a combined second harmonic that is not large enough. Therefore, it is important to determine the crystal conditions so that the phases of the second harmonics produced at each point are aligned as much as possible (phase matching). Phase matching is achieved when the refractive index of the fundamental wave is equal to the refractive index experienced by the harmonics.

[0073] Figure 25 This is a graph showing the wavelength conversion efficiency of nonlinear optical crystals such as single-crystal BBO (Beta-BaB2O4) at various wavelengths for each crystal length. Figure 25 In the diagram, the horizontal axis represents the wavelength band, and the vertical axis represents the normalized value of the wavelength conversion efficiency in second-harmonic generation (SHG). Figure 25 The solid lines in the diagram show the wavelength conversion efficiency of a relatively thick crystal (1 mm in length) across various wavelength bands, while the dashed lines show the wavelength conversion efficiency of a relatively thin crystal (0.2 mm in length) across various wavelength bands. Furthermore, although the thicker crystal exhibits a greater maximum wavelength conversion efficiency, in... Figure 25 Because wavelength conversion efficiency is normalized, the upper limit for wavelength conversion efficiency of all crystals is 1. For example... Figure 25 As shown, the longer (thicker) the crystal, the more stringent the condition for double harmonic overlap at each point becomes, and the narrower the band in which wavelength conversion can be performed with high efficiency. Therefore, from the perspective of relaxing the band that satisfies phase matching, it is advisable to consider using a relatively thin crystal.

[0074] However, since the intensity of the second harmonic is proportional to the square of the crystal length, it is difficult to improve wavelength conversion efficiency in relatively thin crystals. While the intensity of the second harmonic also depends on the intensity of the fundamental wave, increasing the fundamental wave intensity to ensure the second harmonic intensity is not a cost-effective solution to compensate for the square effect of the crystal length. Concentrating the light into a very small area within the crystal could spatially increase the fundamental wave intensity, but such concentration introduces third-order and higher nonlinear effects. These nonlinear effects hinder the generation of second harmonics, resulting in a decrease in wavelength conversion efficiency. Furthermore, high intensity can sometimes damage the crystal itself. In conclusion, using relatively thin crystals makes it difficult to significantly improve wavelength conversion efficiency.

[0075] Therefore, it is possible to consider using a relatively thick crystal while setting up a driving system structure to change the incident angle of light in the crystal in order to achieve phase matching in the broadband domain, which is a problem of thick crystals. Figure 26 This is a diagram illustrating angular phase matching. In Figure 26 In the structure shown, a crystal 500 with birefringence is prepared. The incident angle of light in the crystal 500 is adjusted by a driving system (not shown) to adjust the refractive index, thereby achieving phase matching in the broadband domain.

[0076] However, in settings such as Figure 26 In the structure of the driving system shown, it is impossible to drive the driving system in every pulse (e.g., every tens of MHz) of ultrashort pulse light, so it is difficult to use as a structure for wavelength conversion of ultrashort pulse light.

[0077] As mentioned above, it has been difficult to efficiently convert the wavelength of ultrashort pulses of light whose wavelength changes with each pulse.

[0078] To address this problem, the wavelength conversion unit 710 of this embodiment uses a relatively thick crystal for high-efficiency wavelength conversion, and instead of using a driving system, it disperses the light for each wavelength band to change the optical path for each band, thereby performing wavelength conversion on ultrashort pulses over a wide wavelength range. Hereinafter, refer to... Figure 27 A specific structural example of the wavelength conversion unit 710 will be described.

[0079] Figure 27 This is a diagram illustrating an example of the structure of the wavelength conversion unit 710. (See diagram for example.) Figure 27As shown, the wavelength conversion unit 710 includes a diffraction grating (dispersion section) 1101, a lens (focusing section) 1102, a wavelength conversion element (wavelength conversion unit) 1103, a lens 1104, and a diffraction grating (diffraction grating) 1105. The diffraction grating 1101 and the lens 1102 are structures that change the optical path for each wavelength band so that the light of the wavelength band to be converted at each incident position is incident at each incident position (details will be described later) of the wavelength conversion element 1103, and are optical path setting units for each wavelength.

[0080] The diffraction grating 1101 disperses the ultrashort pulse light output from the compressor 709 for each wavelength band (splitting the ultrashort pulse light), thereby changing the optical path for each wavelength band. Specifically, the diffraction grating 1101 disperses the ultrashort pulse light for each wavelength band by incidenting the light of the wavelength band to be converted at each incident position to each incident position of the wavelength conversion element 1103.

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

[0082] The wavelength conversion element 1103 is a crystal that converts to different wavelengths depending on the incident position of the light. To achieve quasi-phase matching of different wavelengths at various incident positions, the wavelength conversion element 1103 is formed in a fan-shaped structure. More specifically, the wavelength conversion element 1103 can also be a fan-shaped PPLN (Periodically Poled Lithium Niobate). That is, the wavelength conversion element 1103 can also be a so-called Fan-out PPLN. The crystal length (thickness) of the wavelength conversion element 1103 can be, for example, between 1 mm and 20 mm.

[0083] Quasi-phase matching refers to a technique that precisely achieves phase matching in additive double harmonics by either preventing the generation of the desired double harmonic or by reversing the phase of the desired double harmonic. Here, as an example of quasi-phase matching, an example of reversing the phase of the desired double harmonic (reversing the polarity of the crystal) will be explained.

[0084] Because the wavelength conversion element 1103 has a fan-shaped structure, quasi-phase matching of any wavelength band can be achieved at each incident position of the crystal. That is, although the period of the portion where the second harmonics cancel each other out varies from wavelength band to wavelength band, by using a crystal whose period is reversed according to the fan-shaped structure, the wavelength bands at each incident position of the crystal can be different from each other. In such a wavelength conversion element 1103, since it is possible to predetermine which wavelength band to achieve quasi-phase matching at each incident position, by setting the diffraction grating 1101 and lens 1102 such that light of the wavelength to be converted is incident at each incident position, wavelength conversion can be appropriately performed for light of various wavelengths.

[0085] Lens 1104 is a lens that focuses light whose wavelength has been converted by wavelength conversion element 1103 onto diffraction grating 1101. Furthermore, the distance from wavelength conversion element 1103 to lens 1104 and the distance from lens 1104 to diffraction grating 1105 are both set in conjunction with the focal length f of lens 1102 and lens 1104, for example, set to the focal length f of lens 1102 and lens 1104.

[0086] The diffraction grating 1105 outputs wavelength-converted ultrashort pulses of light that have been dispersed by the diffraction grating 1101 for each wavelength band and then reach the light via the lens 1102, wavelength conversion element 1103, and lens 1104. Furthermore, to restore the dispersion of light by the diffraction grating 1101, the number of grooves in the diffraction grating 1105 can be 2N, which is twice the number of grooves N that disperses the light in the diffraction grating 1101.

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

[0088] BBO 1202 and 1205 are nonlinear optical crystals used for wavelength conversion. BBO 1202 is configured with an angle (phase-matching angle) suitable for wavelength conversion in the first band. BBO 1205 crystals are connected in series with BBO 1202 and configured with an angle (phase-matching angle) suitable for wavelength conversion in a second band different from the first band.

[0089] According to this structure, by simply connecting BBO 1202 and 1205 in a straight line, BBO 1202 and 1205 can each perform wavelength conversion in their corresponding bands. In this structure, without using a drive system to move BBO 1202 and 1205, wavelength conversion can be appropriately performed for multiple bands. Since wavelength conversion can be performed without using a drive system, even when performing wavelength conversion of ultrashort pulse light in a very short time (where movement of the wavelength conversion element based on the drive system would result in insufficient time for wavelength conversion), wavelength conversion can be performed appropriately. Furthermore, in this structure, since the thickness and shape of BBO 1202 and 1205 can be appropriately set for efficient wavelength conversion, wavelength conversion can be performed efficiently. As described above, in the wavelength conversion section related to this modification, wavelength conversion can be performed efficiently in a structure that performs wavelength conversion in a short time over a wide band.

[0090] Furthermore, while it's possible to connect BBOs 1202 and 1205 side-by-side instead of in a straight line, this would render the light in the band between the first and second wavelengths unusable. Therefore, from the viewpoint of using a wide-bandwidth spectrum, it is preferable for BBOs 1202 and 1205 to be connected in a straight line. Although two types of BBOs are used in the above example, the number of BBOs can be increased depending on the number of bands being converted.

[0091] The structure of the pulsed light generating device is not limited to the above-described manner; for example, it can also employ... Figure 29 The pulsed light generating device 10A shown is illustrated below. Referring hereafter, [further details are provided]. Figures 29-33 The method of pulse light generating device 10A will be described.

[0092] like Figure 29As shown, the pulsed light generating device 10A includes an oscillator 702, a first fiber amplifier 753, an acousto-optic modulator 704, a compressor 705, a soliton frequency-shifting fiber 706, a second fiber amplifier 757, a filter 758, a third fiber amplifier 759, and a wavelength conversion unit 710. Hereinafter, the first fiber amplifier 753, the second fiber amplifier 757, the filter 758, and the third fiber amplifier 759, which have structures different from those of the pulsed light generating device 10 described above, will be explained.

[0093] The first fiber amplifier 753 constitutes an amplification section that broadbands the spectrum of the ultrashort pulse light oscillating in the oscillator 702. The first fiber amplifier 753 amplifies the ultrashort pulse light by using a self-similar soliton, thereby broadbanding the spectrum and increasing the power of the ultrashort pulse light. The first fiber amplifier 753 is positioned between the oscillator 702 and the soliton frequency-shifting fiber 706 in the optical path of the ultrashort pulse light.

[0094] The first fiber amplifier 753 comprises a normally dispersive fiber and an excitation source. The normally dispersive fiber is a double-clad fiber co-doped with erbium and ytterbium. That is, the first fiber amplifier 753 amplifies ultrashort pulse light as broadband amplified light by inducing nonlinear effects while passing the normally dispersive double-clad fiber in a manner that does not cause broadening. The normally dispersive fiber is an fiber with a negative dispersion parameter D (ps / nm / km). There are no particular limitations on the additives used in the first fiber amplifier 753, and various additives can be used.

[0095] The second fiber amplifier 757 constitutes a first optical amplification unit that amplifies the ultrashort pulse light whose wavelength has been altered by the soliton frequency-shifting fiber 706. The second fiber amplifier 757 comprises a rare-earth-doped fiber and an excitation source. The rare-earth-doped fiber is, for example, a Tm fiber. The excitation source is, for example, a laser diode that outputs excitation light with a wavelength of 1550 nm. The second fiber amplifier 757 is an amplifier optimized for the short wavelength side. The second fiber amplifier 757 amplifies the ultrashort pulse light, which serves as the signal light (seed light), by reducing its absorption gain.

[0096] The second fiber amplifier 757 has a higher gain G1 on the first wavelength side, which is the short wavelength side of the ultrashort pulse light, than on the second wavelength side, which is the long wavelength side (see reference). Figure 31 (b)). The characteristics of such a second fiber amplifier 757 can be achieved, for example, by adjusting the length of the rare-earth-doped fiber and the concentration of rare-earth doping. As an example, the short wavelength (first wavelength) is a wavelength of 1800 nm or more and less than 2000 nm, and the long wavelength (second wavelength) is a wavelength of 2000 nm or more and less than 2200 nm.

[0097] Filter 758 constitutes a filter section for filtering the ultrashort pulse light amplified by the second fiber amplifier 757. Filter 758 attenuates ASE (Amplified Spontaneous Emission). In this embodiment, filter 758 attenuates not only the ASE generated by the amplification based on the second fiber amplifier 757, but also the light with amplified noise components and light in the high-gain region wavelengths generated by the amplification based on the second fiber amplifier 757. Furthermore, in this embodiment, light in the high-gain region wavelengths refers to light in a specific band of short wavelengths, and light in a band with a particularly high amplification ratio compared to other bands. If this light is not attenuated, it will result in preferential amplification of only this light, thus hindering optical amplification across a wide wavelength range. The ASE attenuated by filter 758 here is the ASE in a defined band covering the wavelength range between short and long wavelengths. In addition to hindering amplification, light with amplified ASE or noise components may also cause parasitic oscillations, thereby damaging the amplifier system. ASE (Acousto-Optical Precipitation) is easily generated, for example, when the wavelength of the seed light is mismatched with that of the gain, or when the power of the seed light is weak. Furthermore, in filter 758, pulses in the short-wavelength region are absorbed, becoming the gain for subsequent pulses in the long-wavelength region. There are also processes that shift energy from pulses in the gain region of the gain fiber to pulses in the non-gain region. In addition, filter 758 can be a component constructed using an AOM (Acousto-Optical Modulator) or EOM (Electro-Optical Modulator), or a gain fiber such as a Tm fiber, Ho fiber, or Tm / Ho fiber where no excitation light is incident. Furthermore, filter 758 can also be a gain fiber in the second fiber amplifier 757 where the excitation light is attenuated by more than 20 dB.

[0098] The third fiber amplifier 759 constitutes a second optical amplification section for amplifying the ultrashort pulse light filtered by the filter 758. The third fiber amplifier 759 comprises a rare-earth-doped fiber and an excitation source. The rare-earth-doped fiber is, for example, a Tm fiber. The excitation source is, for example, a laser diode that outputs excitation light with a wavelength of 790 nm, or a CW (continuous wave) laser that outputs excitation 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 pulse light L, which serves as the signal light, by increasing its absorption gain. The third fiber amplifier 759 has a higher gain G2 (see reference) on the second wavelength side, which is the long wavelength side of the ultrashort pulse light L, than on the first wavelength side, which is the short wavelength side. Figure 31 (d). The characteristics of such a third fiber amplifier 759 can be achieved, for example, by adjusting the length of the rare-earth-doped fiber and the concentration of rare-earth doping.

[0099] Next, the wavelength-variable method implemented using the pulsed light generating device 10A will be discussed, while referring to... Figure 30 process Figure 1 The explanation will be provided later.

[0100] First, the ultrashort pulse light L is oscillated in oscillator 702 to generate an ultrashort pulse sequence with a specified period (oscillation step: step S1). The ultrashort pulse light L is then high-powered via a first fiber amplifier 753, simultaneously broadbanding its spectrum (amplification step: step S2). The intensity of the ultrashort pulse light L is controlled per pulse via acousto-optic modulator 704, for example, according to required specifications or conditions (step S3). The time width of the ultrashort pulse light L is compressed via compressor 705 (step S4). The wavelength of the broadbanded ultrashort pulse light L is modulated using soliton self-frequency shifting via soliton frequency-shifting fiber 706, thereby changing the wavelength (step S5).

[0101] Subsequently, the ultrashort pulse light L, whose wavelength was changed in step S5, is amplified by the second fiber amplifier 757 (step S6). In step S6, the ultrashort pulse light L is amplified with a higher gain G1 on the short wavelength side than on the long wavelength side. The ultrashort pulse light L amplified in step S6 is then filtered by the filter 758 (step S7). This reduces the amplified ASE in the specified wavelength band, the amplified noise components, and the high-gain wavelength light generated by the amplification based on the second fiber amplifier 757.

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

[0103] In the pulsed light generating device 10A described above, for example, Figure 31 As shown in (a), changing the wavelength of the ultrashort pulse light L to a short wavelength λ1 has the following effect. That is, as... Figure 31 As shown in (b), the short-wavelength λ1 ultrashort pulse light L is amplified (pre-amplified) by a second fiber amplifier 757 with gain G1 without the generation of ASE. The pre-amplified short-wavelength λ1 ultrashort pulse light L, as shown in (b), is amplified by (c) by (d) by (c) by (d) by (d) by (b). Figure 31 As shown in (c), the light passes directly through filter 758 without being attenuated. Then, the ultrashort pulse light L passing through filter 758, as... Figure 31As shown in (d), the light is further amplified by the third fiber amplifier 759 with gain G2. At this time, although the gain G2 is high on the long wavelength side, the ultrashort pulse light L with short wavelength λ1 is in a state of easy energy absorption because it has already been amplified in the second fiber amplifier 757. Therefore, it is amplified in the third fiber amplifier 759 while suppressing the generation of ASE (without worrying about ASE).

[0104] On the other hand, for example, Figure 32 As shown in (a), changing the wavelength of the ultrashort pulse light L to a longer wavelength λ2 will have the following effect. That is, as Figure 32 As shown in (b), the long-wavelength λ2 ultrashort pulse light L is not amplified much in the preamplifier of the second fiber amplifier 757 with gain G1 compared to the short-wavelength λ1 ultrashort pulse light L, resulting in ASE 11. Subsequently, as... Figure 32 As shown in (c), ASE 11 is attenuated in filter 758. Then, the long-wavelength λ2 ultrashort pulse light L is amplified by a third fiber amplifier 759 with gain G2 without the generation of ASE. Therefore, according to this embodiment, in the wavelength-variable method where the wavelength of the output ultrashort pulse light L is variable, it is possible to reliably amplify the ultrashort pulse light L while suppressing ASE 11. This is particularly effective in cases of optical amplification across a wide wavelength range, and in cases of optical amplification in bands where it is difficult to use optical amplifiers with suitable conditions.

[0105] Figure 33 This is a graph showing the absorption cross-section, stimulated emission cross-section of Tm optical fiber, and the relationship between the transmittance of the filter and wavelength. Figure 33 In the diagram, the horizontal axis represents the wavelength of the ultrashort pulse light L, and the vertical axis represents the absorption cross-section, stimulated emission cross-section, and transmittance of the Tm fiber and filter 758. Tm fiber, when amplifying light with wavelengths of 1800–2200 nm, exhibits… Figure 33 The absorption cross section 922 and stimulated emission cross section 921 are shown. In a fiber amplifier system containing Tm fiber, if the fiber length is short, insufficient gain on the long wavelength side will easily lead to ASE (associated optic scattering) during amplification of light on the long wavelength side. On the other hand, if the fiber length is long, the absorption will also increase, resulting in excessive absorption of light on the short wavelength side. Therefore, ASE is easily generated during amplification of light on the short wavelength side. In this embodiment, the fiber amplifier containing Tm fiber is divided into two segments, with a second fiber amplifier 757 and a third fiber amplifier 759. Furthermore, by inserting a filter 758 with a transmittance 923 (dashed line in the figure) between the second fiber amplifier 757 and the third fiber amplifier 759, ASE is removed, and amplification of wavelength-variable laser light over a wide band is achieved.

[0106] In this embodiment, amplification of a wide-band ultrashort pulse light L is achieved without the need for a separate mechanism. Amplification of the wide-band ultrashort pulse light L is achieved without utilizing nonlinear optical effects such as Raman amplification. This embodiment enables the construction of a wide-band and high-output (e.g., 30 dBm or more) amplifier system. According to this embodiment, parasitic oscillations caused by increased ASE (associated ester) can also be prevented. In this embodiment, as described above, a wavelength-variable light source is used in a two-photon microscope. In this case, since high-output amplification can be achieved in a wide wavelength range, high-output and multi-wavelength imaging can be realized in a two-photon microscope by cooperating with a downstream wavelength conversion system.

[0107] Finally, the effects of the structure in this embodiment will be explained.

[0108] The scanning microscope 1 includes: a pulse light generating device 10 (or a pulse light generating device 10A) that emits excitation light with an excitation wavelength that switches according to each pulse to the sample; and a detector 50 that detects the fluorescence of the cell cluster S irradiated by the excitation light and outputs a detection signal, wherein the detection bandwidth of the detector 50 is adjusted to attenuate the detection signal during the pulse-off period of the excitation light.

[0109] In the scanning microscope 1 of this embodiment, the detector 50 detects the fluorescence of cell clusters S irradiated by excitation light whose excitation wavelength is switched with each pulse, and outputs a detection signal from the detector 50. Furthermore, in the scanning microscope 1 of this embodiment, the detection bandwidth of the detector 50 is adjusted such that the detection signal attenuates during the pulse-off period of the excitation light. By adjusting the detection bandwidth so that the detection signal attenuates during the pulse-off period (before the arrival of the next pulse), different pulses are distinguished from each other in time, avoiding the mixing of detection signals associated with different pulses. Therefore, since there is no need for structures such as those that filter signals according to wavelength to distinguish different pulses, the reduction in the amount of detection signal, which would be problematic when such structures are provided, can be suppressed. By ensuring the amount of detection signal, since there is no need to excessively increase the laser intensity of the excitation light, the generation of fluorescence from outside the focal point can be sufficiently suppressed. As described above, the scanning microscope 1 according to this embodiment can improve the accuracy of fluorescence observation by increasing the amount of detection signal.

[0110] The intensity distribution of the focused beam with respect to depth does not change even when the laser power is reduced. Therefore, even with reduced laser power, the ratio of fluorescence generated at the focal point to the total amount of fluorescence generated outside the focal point (near the surface) remains unchanged. This ratio can only be reduced by decreasing losses due to absorption and scattering. The reason for reducing laser power is to avoid damaging the sample. In deep imaging, laser power exceeding 100 mW is applied to the sample, but in the mouse brain, even hundreds of mW can cause damage such as inflammation. Furthermore, to separate multicolor fluorescence using multiple filters and to prevent spectral overlap using multiple detectors, 40% to 50% of the generated fluorescence is discarded. Therefore, the detection efficiency is low. Furthermore, the increased distance between the objective lens and detector due to the multiple filters and detectors reduces the detection efficiency of fluorescence scattered within the sample. The structure of this embodiment can be used to address these problems.

[0111] In confocal microscopy, fluorescence intensity is directly proportional to laser intensity, while in two-photon microscopy, fluorescence intensity is directly proportional to the square of the laser intensity. For example, by reducing the repetition frequency from 80 MHz to 8 MHz, the pulse energy of each pulse becomes tenfold while maintaining the same average laser power, resulting in a 100-fold increase in fluorescence intensity. Therefore, compared to confocal microscopy, using pulse synchronization techniques in two-photon (multiphoton) microscopy can significantly increase the amount of fluorescence signal.

[0112] The detection bandwidth (sensor bandwidth) of detector 50 can also be adjusted so that the sum of the time required for fluorescence decay and the time required for detection signal decay is shorter than the repetition interval of the excitation light pulse. With this structure, the time required for fluorescence and detection signal decay can be shorter than the pulse repetition interval, thereby achieving more reliable temporal separation of the different pulses.

[0113] Detector 50 can also detect fluorescence without passing through filters (such as bandpass filters) used for wavelength separation of fluorescence. This avoids the reduction in detection signal intensity caused by passing through filters, thereby appropriately increasing the detection signal intensity.

[0114] The detector 50 can also be composed of a single photosensor 51 that detects any one of the fluorescence at the excitation wavelength corresponding to each pulse. With this structure, the amount of detected signal can be increased without the need for a structure that filters the signal based on wavelength, while simultaneously simplifying the overall structure. Furthermore, with only one photosensor, the detection efficiency of scattered fluorescence can be improved because it can be positioned closest to the objective lens.

[0115] The pulsed light generating device 10 (or pulsed light generating device 10A) can also emit excitation light by switching between a first excitation wavelength for stimulating cell clusters S and a second excitation wavelength for observing the effect of light stimulation from the first excitation wavelength on a pulse-by-pulse basis. The detector 50 can also perform light detection only when fluorescence from cell clusters S is detected corresponding to the pulse of the first excitation wavelength, and only when fluorescence from cell clusters S is detected corresponding to the pulse of the second excitation wavelength. With this structure, only the desired fluorescence can be detected while avoiding detection of stimulating fluorescence (removing stimulating fluorescence), thereby helping to improve the detection limit. Furthermore, the pulsed light generating device 10 uses a structure that switches the excitation wavelength on a pulse-by-pulse based on pulsed light obtained from the same oscillator (generator), which improves convenience compared to a structure that uses multiple wavelengths of excitation laser to switch the excitation wavelength on a pulse-by-pulse basis. In the method of using two different lasers, it is difficult to match the frequencies using different resonators, and the interval between each pulse gradually deviates over time, which may not achieve the desired effect. However, by using pulsed light obtained from the same oscillator (generator), this problem can be solved.

[0116] The scanning microscope 1 also includes an A / D converter 60 that converts the detection signal output from the detector 50 into a digital signal. The sampling frequency of the A / D converter 60 can also be synchronized with the repetition frequency of the excitation light pulses. With this structure, the required information can be easily obtained from the analog signal corresponding to each pulse without the need for a high-speed A / D converter, thus enabling low-cost processing of high-speed signals. Furthermore, a suitable fixed delay can be provided, and the microscope can be configured to sample at ideal timing.

[0117] The A / D converter 60 can also be set 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 by acquiring the maximum value of the detection signal, the required information can be more appropriately obtained from the analog signal corresponding to each pulse.

[0118] The A / D converter 60 can also be set to acquire the integral value of the detection signal. When acquiring the maximum value as described above, it may be difficult to obtain the maximum value accurately due to factors such as fluorescence signal jitter. In this case, acquiring the integral value allows for more appropriate acquisition of the required information from the analog signal corresponding to each pulse. Alternatively, the A / D converter can also be set to acquire the time waveform of the detection signal. In this case, information such as fluorescence lifetime can also be extracted from the time waveform.

[0119] This disclosure is not limited to the embodiments described above. For the structure of the modified examples, please refer to... Figures 34-38 Please provide an explanation.

[0120] Figure 34 This is a schematic diagram of a modified scanning microscope 2001 (fluorescence detection device). Figure 34 For ease of explanation, some structural diagrams have been omitted. Figure 34 The scanning microscope 2001 shown includes a pulsed light generating device 2010, a microscope body 2080, a fluorescence detector 2050, and an A / D converter 2060 (see reference). Figure 35 ).

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

[0122] Oscillator 2011 oscillates to generate pulsed light. First fiber amplifier / compressor 2012 amplifies the pulsed light output from oscillator 2011 (increasing power) while compressing the pulse duration. The fiber amplifier can be, for example, a normal dispersive fiber containing erbium. AOM 2013 is located after the first fiber amplifier / compressor 2012, using the power of sound (sound waves) to modulate the ultrashort pulsed light. Soliton frequency-shifting fiber 2014 is located after the AOM 2013, using soliton self-frequency shifting to modulate the wavelength of the pulsed light. Second fiber amplifier / compressor 2015 is located after the soliton frequency-shifting fiber 2014, amplifying the pulsed light while compressing the pulse duration. Wavelength conversion unit 2016 performs wavelength conversion, for example, through second harmonic generation (SHG).

[0123] The pulsed light output from the wavelength conversion unit 2016 is irradiated onto the cell cluster S via a dichroic mirror 2020 included in the microscope body 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). A / D converter 2060 (see reference) Figure 35 It converts the detection signal (analog signal) output from detector 3050 into a digital signal and outputs the digital signal.

[0124] Here, in addition to the detection signal, the A / D converter 2060 also receives a synchronization signal generated by delaying the pulse by a specified time. Figure 35 This is a graph showing the time waveforms of each signal. In Figure 35 In the graph, the horizontal axis represents time, and the vertical axis represents intensity. Figure 35In the image, from top to bottom, are shown the following: “1”, the pulse train oscillating from the oscillator 2011 (e.g., light containing the 1550nm band), “2”, the light passing through the AOM 2013, “3”, the light input to the microscope body 2080, “4”, the light input to the fluorescence detector 2050, “5”, the signal output from the fluorescence detector 2050, and the synchronization signal. Figure 34 The positions of “1” to “4” in the text correspond to Figure 35 The time waveforms “1” to “4” shown are shown.

[0125] like Figure 36 As shown, the synchronization signal can also be obtained, for example, by... Figure 34 A portion of the pulse train of "1", "2", and "3" is branched (sampled) and generated based on the signal detected and output by a detector 2055, such as a photodetector. For example, a synchronization signal can also be generated by delaying the signal output from the detector 2055 by the delay generator 2090 for an arbitrary (specified) time. The synchronization signal output from the delay generator 2090 is input to the A / D converter 2060.

[0126] like Figure 35 As shown, by inputting a synchronization signal generated by delaying the pulse for a specified time in addition to the detection signal to the A / D converter 2060, the A / D converter 2060 outputs digital signals for both the detection signal and the synchronization signal. Based on the digital signals output from the A / D converter 2060 (the digital signals for both the detection signal and the synchronization signal), the computer (not shown) samples the signal acquisition of the fluorescence detector 2050, thereby suppressing noise in the fluorescence detector 2050 and appropriately capturing the peak value of the fluorescence signal of the cell cluster S generated by pulse irradiation.

[0127] Figure 37 This diagram schematically illustrates another modified example of a pulsed light generating device 3010. The pulsed light generating device 3010 includes an oscillator 3011, a first fiber amplifier / compressor 3012, an AOM 3013, a soliton frequency-shifting fiber 3014, a WDM (wavelength division multiplexer) 3017, second fiber amplifiers / compressors 3015, wavelength conversion units 3016, and a WDM 3018. Figure 37In the structure shown, the intensity of each pulse is rapidly modulated by AOM 3013, and other pulse trains are generated in soliton frequency-shifting fiber 3014 by varying the wavelength according to each pulse. Then, in WDM 3017, two wavelengths of light are separated, and each light reaches WDM 3018 via a second fiber amplifier / compressor 3015 and a wavelength conversion unit 3016, respectively. WDM 3018 integrates the two wavelengths of light and outputs them. Furthermore, although this embodiment alternates between two wavelengths, the wavelengths may not alternate, and there may be more than two wavelengths.

[0128] Figure 38 This is a graph showing the time waveforms of each signal. In Figure 38 In the graph, the horizontal axis represents time, and the vertical axis represents intensity. Figure 38 In the diagram, from top to bottom, are shown: “1”, the pulsed light oscillating from oscillator 3011 (e.g., light containing the 1550nm band); “1”, the modulation signal input to AOM 3013; “2”, the light passing through AOM 3013; “4”, the light output from WDM 3018; the signal output from detector (not shown); the signal that delays one wavelength of light (light A); the signal that delays another wavelength of light (light B); and the synchronization signal generated by delaying by a different amount according to each excitation wavelength.

[0129] like Figure 38 As shown, a synchronization signal is generated by delaying pulses by a different amount of delay for each excitation wavelength. That is, a synchronization signal is generated by delaying light A by an arbitrary amount of delay and delaying light B by a different amount of delay than light A. For example, in the case of generating a synchronization signal based on light transmitted through an AOM, deviations in pulse period due to the fiber length, refractive index, and optical path length of the fiber amplifier (AMP) will occur in the pulse train after integrating different wavelengths. Therefore, by using a synchronization signal with unequal periods (a synchronization signal delayed by a different amount of delay for each wavelength), the peak value of the fluorescence signal can be appropriately captured.

[0130] Here, when only the detection signal and synchronization signal from the detector are observed, it can be considered impossible to distinguish which excitation wavelength each sample represents. In this case, by having a computer (not shown) identify the excitation wavelength associated with the detection signal based on an identification signal that identifies the excitation wavelength, it is possible to specify which excitation wavelength each sample represents. Alternatively, the identification signal can also be generated based on a synchronization signal of any excitation wavelength. By using a synchronization signal of any excitation wavelength 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 pulse light output from oscillator 3011 before it is input to the first fiber amplifier / compressor 3012, can also be input to the A / D converter 3060. Regarding the acquisition location of the pulse for generating the synchronization signal, for example, the front end of the first fiber amplifier / compressor 3012 and the rear end of the WDM 3018 can be considered. The optical pulse is converted into an analog signal, for example, by a detector such as a photodetector. A certain threshold is set for the peak value of the analog signal and binarized, and then a delay is added to generate the synchronization signal. Here, if the pulse light is acquired at the rear end of the WDM 3018, if the difference in light intensity between light A and light B becomes large, it becomes difficult to set the threshold, potentially causing the following problems: the delay may deviate; and when the output is changed by the amplifier, the threshold used to generate the synchronization signal must also be changed. In this respect, if the pulse light is acquired at the front end of the first fiber amplifier / compressor 3012, although it is necessary to make it have different delay values ​​for each final output wavelength, a synchronization signal can be generated based on pulse light with stable light intensity that is not affected by the amplifier. Symbol Explanation

[0132] 1, 2001… Scanning microscope (fluorescence detection device), 10, 10A, 2010, 3010… Pulse light generation device (excitation source), 50… Detector, 51… Optical sensor, 60, 2060, 3060… A / D converter, 2050… Detector for fluorescence.

Claims

1. A fluorescence detection device, wherein, have: An excitation light source emits excitation light onto the sample, with the excitation wavelength switching according to each pulse; and The detection unit detects the fluorescence of the sample irradiated by the excitation light and outputs a detection signal. The detection unit adjusts the detection bandwidth in such a way that the detection signal attenuates during the pulse-off period of the excitation light.

2. The fluorescence detection device according to claim 1, wherein, The detection unit adjusts the detection bandwidth such that the sum of the time required for fluorescence decay and the time required for detection signal decay is shorter than the repetition interval of the excitation light pulse.

3. The fluorescence detection device according to claim 1 or 2, wherein, The detection unit does not detect the fluorescence using a filter for wavelength separation.

4. The fluorescence detection device according to claim 1 or 2, wherein, The detection unit consists of a photosensor that detects any one of the fluorescence corresponding to the excitation wavelength of each pulse.

5. The fluorescence detection device according to claim 1 or 2, wherein, 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 the effect of the first excitation wavelength's light stimulation on each pulse. The detection unit does not detect light during the fluorescence detection timing of the sample corresponding to the pulse of the first excitation wavelength, but performs light detection during the fluorescence detection timing of the sample corresponding to the pulse of the second excitation wavelength.

6. The fluorescence detection device according to claim 1 or 2, wherein, It also includes an A / D converter, which converts the detection signal output from the detection unit into a digital signal. The sampling frequency of the A / D converter is synchronized with the repetition frequency of the excitation light pulse.

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

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

9. The fluorescence detection device according to claim 1, wherein, It also includes an A / D converter, which converts the detection signal output from the detection unit into a digital signal. In addition to the detection signal, the A / D converter also inputs a synchronization signal generated by delaying the pulse by a specified time.

10. The fluorescence detection device according to claim 9, wherein, In addition to the detection signal, the A / D converter also inputs a synchronization signal generated by delaying the pulse by a different amount of delay according to each excitation wavelength.

11. The fluorescence detection device according to claim 10, wherein, It also includes a signal processing unit that performs prescribed signal processing on the signal output from the A / D converter. The signal processing unit identifies the excitation wavelength of the detection signal based on the identification signal that identifies the excitation wavelength.

12. The fluorescence detection device according to any one of claims 9 to 11, wherein, The excitation light source has an oscillator that oscillates to produce pulses and an amplifier that amplifies the pulses output from the oscillator. The A / D converter input is a synchronization signal generated by delaying the pulse output from the oscillator before it is input to the amplifier.

13. A fluorescence detection method, in, include: Excitation light with a wavelength switched for each pulse is emitted towards the sample; and The fluorescence from the sample irradiated by the excitation light is detected, and a detection signal is output. The detection bandwidth is adjusted in such a way that the detection signal decays during the pulse-off period of the excitation light.

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