JP2011141311A - Multi-photon microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prevent respective images from being shifted relative to one another when images are formed by detecting fluorescence generated by each dye, in a specimen dyed with a plurality of kinds of dyes. <P>SOLUTION: Three kinds of ultra-short pulsed light beams L<SB>1</SB>, L<SB>2</SB>, and L<SB>3</SB>respectively emitted from laser light sources 11<SB>1</SB>to 11<SB>3</SB>are mixed by dichroic mirrors 13 and 14. The ultra-short pulsed light beams L<SB>1</SB>, L<SB>2</SB>, and L<SB>3</SB>are adjusted by delay circuits 12<SB>1</SB>and 12<SB>2</SB>so as to be arranged in order at regular intervals. A laser scanning section 17 condenses the ultra-short pulsed light beams L<SB>1</SB>, L<SB>2</SB>, and L<SB>3</SB>onto a specimen S dyed with three kinds of dyes to scan them. The intensities of fluorescence, having three kinds of different spectra generated from the dyes excited thereby, are separated by dichroic mirrors 21 and 24, and are simultaneously detected by optical detecting sections 23<SB>1</SB>to 23<SB>3</SB>. Thus, fluorescence images, corresponding to fluorescence generated from each dyes, can be obtained simultaneously by carrying out excitation light focal scanning by once. Thus, the application to a two-photon excitation fluorescence microscope can be achieved. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a multiphoton microscope.

  Usually, in a two-photon excitation fluorescence microscope, imaging is performed by staining a sample with a dye, exciting the dye with a near-infrared pulsed laser (pulse light) by two-photon absorption, and detecting the generated fluorescence. (For example, refer to Patent Document 1). At this time, since fluorescence is generated only from the vicinity of the focal point of the excitation light, unlike the linear confocal microscope, there is a feature that it has a three-dimensional resolution ability even if a pinhole is not arranged in front of the detector.

  In such a two-photon excitation fluorescence microscope, a sample is often stained with a plurality of types of dyes, and excited with light having a wavelength that has a high two-photon absorption efficiency of each dye, fluorescence having different wavelengths generated from the respective dyes can be obtained. Detection and imaging for each dye is performed.

  When obtaining images of each dye in a two-photon excitation fluorescence microscope, change the wavelength with a variable wavelength light source that can be changed to multiple wavelengths, or prepare multiple light sources with different wavelengths and change the excitation wavelength sequentially. In general, an image is acquired by laser scanning.

Japanese Patent No. 2848952

  However, when laser scanning is performed by the above method when acquiring an image, the scanning range is, for example, the first time and the second time even if the same location of the sample is scanned due to scanning means such as a galvanometer mirror. It is normal that they do not match completely and are slightly shifted. Therefore, the acquired images are shifted from each other, and a method that does not cause the shift of the images has been required.

  The present invention has been made in view of such a situation. When a sample stained with a plurality of types of dyes is detected and imaged with fluorescence generated from the dyes, the images are not shifted from each other. It is to make.

  The multi-photon microscope according to the present invention includes a plurality of laser light sources that emit pulsed light whose center wavelengths are fixed to be different from each other, and a scanning that two-dimensionally scans the plurality of pulsed light emitted from each of the plurality of laser light sources. And an illumination optical system having an objective lens for condensing the plurality of pulsed lights on a sample stained with a plurality of types of dyes, and on the sample scanned by the scanning unit and condensed by the objective lens The central wavelength is different from the adjustment means for adjusting the timing at which the plurality of pulse lights are directed to the sample so that the plurality of pulse lights having different center wavelengths are sequentially irradiated for each condensing position. Detecting means for detecting, via the objective lens, a plurality of fluorescences respectively generated from a plurality of types of dyes of the sample excited in response to the plurality of pulsed lights. The features.

  According to the present invention, image shift can be prevented.

It is a block diagram which shows the example of a structure of the two-photon excitation fluorescence microscope which is the 1st Embodiment of this invention. It is a block diagram which shows the example of a structure of the two-photon excitation fluorescence microscope which is the 2nd Embodiment of this invention.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a block diagram showing an example of the configuration of a two-photon excitation fluorescence microscope according to the first embodiment of the present invention.

  The two-photon excitation fluorescence microscope 1 uses the principle of two-photon absorption to simultaneously irradiate a sample S with two photons having a wavelength twice that of excitation light in the case of single-photon excitation. It is a multiphoton microscope which observes the fluorescence from.

The two-photon excitation fluorescence microscope 1 includes a laser light source 11 ₁ to 11 ₃ , delay circuits 12 ₁ and 12 ₂ , dichroic mirrors 13, 14, 16, 21, and 24, a mirror 15, a laser scanning unit 17, a scanning lens 18, 2 The objective lens 19, the objective lens 20, the imaging lenses 22 ₁ to 22 ₃ , and the light detection units 23 ₁ to 23 ₃ are included.

As the laser light sources 11 ₁ to 11 ₃ , for example, a micro laser is used. This ultra-small laser is composed of an ultra-short pulse laser that emits ultra-short pulse light having a pulse width of femtosecond units (for example, 100 femtoseconds). More specifically, ultra-small lasers include, for example, negative dispersion mirrors composed of multilayer mirrors, laser crystals, supersaturated absorption mirrors (SESAM), and yttrium (Yb) -based solid-state lasers combined with SESAM. By using the soliton mode-locking (soliton mode-locking) technology, a small ultrashort pulse laser that can stably maintain the mode-locking state even if the resonator length is shortened to several centimeters. Composed. For details of such a small ultrashort pulse laser, see, for example, “Sho Yamazoe, Masaki Kato, Naofumi Kasamatsu,“ Small Ultrashort Pulse Laser ”, Fuji Film Research Report No.54, 2009, P.43-46” (Hereinafter referred to as Non-Patent Document 1).

  Since the wavelength of the ultrashort pulse light is fixed in the small ultrashort pulse laser described in Non-Patent Document 1, it is not necessary to provide a movable part for changing the wavelength, and the emission angle of the ultrashort pulse light In addition, the above-described technical features make it possible to make the laser diode very small as compared with a conventional ultrashort pulse laser with a variable wavelength. In addition, since this small ultrashort pulse laser has a short resonator length, the repetition frequency of ultrashort pulse light can be set significantly higher than that of a conventional ultrashort pulse laser. For example, Non-Patent Document 1 describes that the resonator length can be set to less than 5 cm and the maximum repetition frequency of ultrashort pulse light can be set to 2.9 GHz.

The laser light sources 11 ₁ , 11 ₂ , 11 ₃ configured as described above use ultrashort pulse lights L ₁ , L ₂ , L ₃ fixed at different center wavelengths (wavelengths λ ₁ , λ ₂ , λ ₃ ). Inject each. In the present embodiment, the sample S is dyed with three types of dyes, and ultrashort pulse lights L ₁ , L ₂ , L are used as light having a wavelength with high two-photon absorption efficiency of these dyes. ₃ is used.

The laser light source 11 ₁ are emitted from the ultrashort pulse light _{L 1} (wavelength lambda ₁₎ is transmitted through the dichroic mirror 13 and 14, it reaches the mirror 15.

The laser light source 11 ₂ emitted from the ultrashort pulse light L _{2 (wavelength} lambda ₂₎ is delayed by a predetermined time by the delay circuit 12 _1, the dichroic mirror 13 is reflected in the direction of the dichroic mirror 14, the dichroic mirror 14, and reaches the mirror 15.

The laser light source 11 ₃ ultrashort pulse light L ₃ emitted from the _(wavelength lambda ₃₎ is delayed by the delay circuit 12 ₂ for a predetermined time, it is reflected in the direction of the mirror 15 by the dichroic mirror 13, the mirror 15 To reach.

That is, the delay circuits 12 ₁ and 12 ₂ respectively delay the ultrashort pulse lights L ₂ and L ₃ by a predetermined time with respect to the ultrashort pulse light L ₁ and mix them by the dichroic mirrors 13 and 14. As shown in the excitation pulse train A in the figure, the timing is adjusted so that the three types of ultrashort pulse lights L ₁ , L ₂ , and L ₃ are sequentially repeated at equal intervals. For example, in the excitation pulse train A, the ultrashort pulse lights L ₁ , L ₂ , L ₃ , L ₁ , L ₂ , L ₃ ,... Has been repeated. Hereinafter, the adjusted ultrashort pulse lights L ₁ , L ₂ , and L ₃ will also be referred to as ultrashort pulse light L.

  The ultrashort pulse light L is reflected in the direction of the dichroic mirror 16 by the mirror 15, then passes through the dichroic mirror 16 and enters the laser scanning unit 17 configured by, for example, a galvanometer mirror.

  The laser scanning unit 17 applies the ultrashort pulse light L from the dichroic mirror 16 to the XY plane of the sample S stained with three types of dyes (on the optical axis of the ultrashort pulse light L irradiated to the sample S). Scanning in an orthogonal plane). That is, by the two-dimensional scanning by the laser scanning unit 17, the ultrashort pulsed light L is condensed by the objective lens 20 through the scanning lens 18 and the second objective lens 19 to form a spot on the sample S.

Thus, the sample S is dyed with three dyes, ultra-short pulse to pulse light L of the spot is formed a region (condensing position), 3 kinds of ultra-short pulse light L ₁ which center wavelengths are _different, L ₂ and L _{3 are} sequentially excited, and fluorescences having different spectra (wavelengths) are emitted from the respective dyes. That is, the laser scanning unit 17 sequentially irradiates a certain point (condensing position) on the sample S with three types of ultrashort pulse lights L ₁ , L ₂ , and L ₃ having different center wavelengths. When the irradiation for is completed, the irradiation is repeated in the same manner for the points after the next point.

  Fluorescence (fluorescence having three different spectra) generated from the dye thus excited becomes observation light, passes through the objective lens 20, the second objective lens 19, and the scanning lens 18, and the laser scanning unit 17. Is incident on. The laser scanning unit 17 reflects the fluorescence from the sample S at the same angle as when the ultrashort pulse light L irradiated to the sample S is scanned, so that the fluorescence is descanned and incident on the dichroic mirror 16.

The dichroic mirror 16 reflects the fluorescence having three different spectra generated from the excited dye of the sample S incident from the laser scanning unit 17 in the direction of the dichroic mirror 21, and is reflected by the dichroic mirrors 21 and 24. The fluorescence intensity is separated. That is, the dichroic mirror 21 transmits the fluorescence generated by being excited by the ultrashort pulse light L ₁ among the fluorescence incident from the dichroic mirror 16, and reflects the other fluorescence in the direction of the dichroic mirror 24.

Fluorescence transmitted by the dichroic mirror 21, passes through the imaging lens 22 _1, incident on the light detecting unit 23 _1. Light detector 23 _1, for example PMT: constituted by (photo mltiplier tube photomultiplier tube), etc., and receives fluorescence imaged on the light receiving surface of the light detecting portion 23 ₁ by the imaging lens 22 _1, the fluorescence An electric signal corresponding to the strength is output.

The dichroic mirror 24 reflects the fluorescence generated by being excited by the ultrashort pulse light L ₂ out of the fluorescence reflected by the dichroic mirror 21 in the direction of the imaging lens 22 ₂ , and the other ultrashort pulse light L _3. Fluorescence generated when excited by is transmitted.

Fluorescence reflected by the dichroic mirror 24 passes through the imaging lens 22 _2, for example, by PMT is incident on configured light detector 23 _2, is output as an electric signal corresponding to the intensity of the fluorescence. Meanwhile, the fluorescence which is transmitted by the dichroic mirror 24 passes through the imaging lens 22 _3, for example, is incident on the composed light detector 23 ₃ due PMT, is output as an electric signal corresponding to the intensity of the fluorescence The

The electrical signals output from the light detection units 23 ₁ to 23 ₃ are input to the personal computer 2A. Since these electric signals are in accordance with the intensity of the fluorescence generated by being excited by the ultrashort pulse lights L ₁ to L ₃ , the electric signals simultaneously output from the light detection units 23 ₁ to 23 ₃ are The intensity of the fluorescence generated from the dye at the same light collection position. Thus, the personal computer 2A, by performing predetermined image processing from the light detection unit 23 ₁ to 23 ₃ to the electric signal outputted at the same time, as an observation image, to generate a fluorescence image generated from each dye it can. The personal computer 2A displays the generated fluorescent image on the monitor 2B and records the fluorescent image data. In this fluorescence image, when a dye is excited by three types of ultrashort pulsed light L at a certain point on the sample S, fluorescence generated from the three types of dye is detected, and two of the three types are detected. Fluorescence generated from two kinds of dyes is detected when the dye is excited by a kind of ultrashort pulsed light L, and one kind of dye if the dye is excited by one of the three kinds of ultrashort pulsed light L Since the fluorescence generated from is detected, an image corresponding to the detected electrical signal is obtained. In addition, no fluorescence is detected from a point on the sample S that does not appear even when the three types of ultrashort pulsed light L are applied.

  Since this fluorescence image is obtained by one scan without scanning a certain point on the sample S a plurality of times, it does not occur that the scanning range does not match by a plurality of scans. Accordingly, when the fluorescence generated from each dye is detected and imaged for the sample S stained with three kinds of dyes, the images can be prevented from shifting from each other.

As described above, in the two-photon excitation fluorescence microscope 1 of FIG. 1, the laser light source 11 ₁ to 11 ₃ different center wavelengths emitted from (wavelength lambda _1, lambda _2, lambda ₃₎ identical repeated three of having Ultrashort pulse lights L ₁ , L ₂ , L ₃ are mixed by dichroic mirrors 13 and 14, and three types of ultrashort pulse lights L ₁ , L ₂ , L ₃ are sequentially added by delay circuits 12 ₁ and 12 _2. It is adjusted to line up at intervals. Then, the ultrashort pulse lights L ₁ , L ₂ , and L ₃ are collected on the sample S stained with three kinds of dyes, scanned two-dimensionally, and three kinds generated from the dyes excited thereby. The fluorescence intensities having different spectra are separated by the dichroic mirrors 21 and 24, and each of the fluorescence having different spectra is simultaneously detected by the _three light detectors 23 ₁ to 233. Thereby, a fluorescence image corresponding to the fluorescence generated from each dye is simultaneously acquired by one excitation light focus scanning.

  As described above, in the two-photon excitation fluorescence microscope 1 according to the first embodiment, the sample S stained with a plurality of types of dyes is excited with the ultrashort pulse light L, and the fluorescence generated from each dye is detected. At the time of imaging, since the measurement ranges of the images match, it is possible to prevent image displacement.

Further, in the two-photon excitation fluorescence microscope 1, as the laser light sources 11 ₁ to 11 ₃ , a small ultrashort pulse that has a shorter resonator length and can set a repetitive frequency of the ultrashort pulse light significantly higher than the conventional _one. Since a laser is used, it is necessary to prepare a plurality of small ultrashort pulse lasers having fixed wavelengths. However, the laser can be configured at a lower cost than when an expensive wavelength variable light source is used.

The ultrashort pulse lights L ₁ , L ₂ , and L ₃ emitted from the laser light sources 11 ₁ to 11 ₃ do not need to be completely equidistant as shown in the excitation pulse train of FIG. to the extent that their pulse train do not overlap may be adjusted delay amount by the delay circuit 12 ₁ and 12 _2. In this case, per one point on the sample S, as three pulses is equal incident, so that to the galvanometer mirror and the light detecting portion 23 ₁ that constitutes the laser scanning unit 17 performs control to synchronize the 23 _3. However, since the ultrashort pulsed light L is a high repetition pulse laser, even if laser scanning is continuously performed with a galvanometer mirror without synchronization, an error of at most one pulse is caused. It will not be.

In the first embodiment, the sample S is irradiated with three types of ultrashort pulse lights L ₁ , L ₂ , and L ₃ having different center wavelengths by the _three laser light sources 11 ₁ to 11 ₃ , thereby Although the example in which the obtained fluorescence is detected by the three light detection units 23 ₁ to 23 ₃ has been described, the number of the ultrashort pulse lights L is not limited to three. That is, the three types of ultrashort pulse light L is an example, and it is sufficient that the sample S can be irradiated with a plurality of types of ultrashort pulse light L having different center wavelengths. The laser light source 11, the delay circuit 12, the light detection unit 23, and other optical members corresponding to the types are arranged. In that case, the sample S is stained with a plurality of types of dyes corresponding to the plurality of types of ultrashort pulsed light L.

  FIG. 2 is a block diagram showing an example of the configuration of a two-photon excitation fluorescence microscope according to the second embodiment of the present invention. In the figure, portions corresponding to those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted as appropriate.

Compared with the two-photon excitation fluorescence microscope 1 of FIG. 1, the two-photon excitation fluorescence microscope 51 of FIG. 2 is an ultrashort pulse emitted from the laser light sources 11 ₁ to 11 ₃ instead of the delay circuits 12 ₁ and 12 _2. The difference is that a shutter 62 ₁ to 62 ₃ for adjusting the order of the light L ₁ , L ₂ and L ₃ and a controller 61 for controlling the shutters 62 ₁ to 62 ₃ are provided. In addition, the number of the light detection units 23 is reduced from three to one, and accordingly, the imaging lens 22 is also one. Furthermore, it is only necessary to form an image of fluorescence only on the light detection unit 23. Another difference is that the dichroic mirrors 21 and 24 are removed.

The shutter 62 ₁ to 62 _3, for example, AOM is faster shutter element such as (acoustooptic device) or AOFT (acousto-optic tunable filter), under the control of the controller 61, emitted from the laser light source 11 ₁ to 11 ₃ The irradiation of the sample S with the ultrashort pulse lights L ₁ to L _{3 is} adjusted by turning on / off, respectively.

That is, the shutters 62 ₁ to 62 ₃ transmit the ultrashort pulse lights L ₁ to L ₃ emitted from the laser light sources 11 ₁ to 11 _{3 in} order, respectively, and are mixed by the dichroic mirrors 13 and 14, respectively. As shown in the excitation pulse train B in the figure, the timing is adjusted so that three types of ultrashort pulse lights L ₁ , L ₂ , and L ₃ are repeated in order by a predetermined number. For example, in the excitation pulse train B, the ultrashort pulse is represented by ultrashort pulse lights L ₁ , L ₁ , L ₂ , L ₂ , L ₃ , L ₃ ,. _Two light beams L ₁ , L ₂ , and L ₃ are repeated in order. Note that the unit of the number of repetitions of each ultrashort pulsed light L is arbitrary, and the unit does not necessarily need to match every ultrashort pulsed light. Because, for example, even if the ultrashort pulse lights L ₁ , L ₂ , and L ₃ are repeated in units of 10, 10, and 11 respectively, an error of at most one pulse is caused. This is because the observation is not particularly affected.

Three types of ultrashort pulse lights L ₁ , L ₂ , and L ₃ are incident on the laser scanning unit 17 in the order shown in the excitation pulse train B in the figure. The laser scanning unit 17 condenses the ultrashort pulse light L onto the sample S stained with three types of dyes, and performs two-dimensional scanning. That is, three types of ultrashort pulsed light L having different center wavelengths are repeatedly irradiated to a certain point (condensing position) on the sample S in a predetermined unit, and the three types of ultrashort pulsed light L at a certain point are irradiated. When the irradiation is completed, the same irradiation is repeated for the next and subsequent points. Then, as described above, the sample S emits fluorescence from the excited dye, and the fluorescence is descanned by the laser scanning unit 17 and then incident on the dichroic mirror 16.

  The dichroic mirror 16 reflects the fluorescence incident from the laser scanning unit 17 in the direction of the imaging lens 22, so that the fluorescence imaged on the imaging lens 22 is received on the light receiving surface of the light detection unit 23. . This fluorescence is fluorescence generated from an excited dye by repeatedly irradiating three types of ultrashort pulse light L in a predetermined unit, and three types of fluorescence are emitted depending on each irradiated ultrashort pulse light L. It will have a different spectrum (wavelength). The light detection unit 23 outputs an electrical signal corresponding to the intensity of the received fluorescence to the personal computer 2A.

The personal computer 2 </ b> A is supplied with a signal (hereinafter referred to as a notification signal) for notifying in what order the ultrashort pulsed light L is emitted from the controller 61 together with the electrical signal from the light detection unit 23. . That is, the controller 61, by controlling the to 62 ₃ shutter 62 _1, and performs the order adjustment of the laser light source 11 ₁ to 11 ₃ ultrashort pulse light _L 1 emitted _from, L 2, _{L 3,} by outputting the notification signal indicating the order in the personal computer 2A, the personal computer 2A is the intensity of fluorescence indicating the electrical signal output by the time sequence from the light detection unit 23, the shutter 62 ₁ to 62 ₃ control, In other words, the sample S can be sequentially acquired in synchronization with the notification signal that is information on the ultrashort pulse light L applied to the sample S.

Specifically, under the control of the shutters 62 ₁ to 62 ₃ by the controller 61, for example, as shown in the excitation pulse train B in the figure, _two ultrashort pulse lights L ₁ , L ₂ , and L ₃ are sequentially repeated. In this case, the personal computer 2A synchronizes a notification signal indicating the order of the ultrashort pulsed light L and an electrical signal corresponding to the intensity of fluorescence generated by the excitation by the ultrashort pulsed light L in sequence. Will be entered. Based on the notification signal from the controller 61, the personal computer 2A performs predetermined image processing on the electrical signal input from the light detection unit 23, thereby generating a fluorescence image corresponding to the fluorescence generated from each dye. it can.

  Since this fluorescence image is obtained by one scan without scanning a certain point on the sample S a plurality of times, it does not occur that the scanning range does not match by a plurality of scans. Accordingly, when the fluorescence generated from each dye is detected and imaged for the sample S stained with three kinds of dyes, the images can be prevented from shifting from each other.

As described above, in the two-photon excitation fluorescence microscopy 51 in FIG. 2, the laser light source 11 ₁ to 11 ₃ different center wavelengths emitted from (wavelength lambda _1, lambda _2, lambda ₃₎ identical repeated three of having ultrashort pulse light _{L 1,} L 2, _{L 3} is, to transmit the shutter 62 ₁ to 62 ₃ in sequence, is adjusted by the controller 61, it is mixed by the dichroic mirror 13 and 14. Then, the ultrashort pulse lights L ₁ , L ₂ , and L _{3 that} are repeated in order by a predetermined number are condensed on the sample S stained with three kinds of dyes, scanned two-dimensionally, and thereby excited. The intensity of the fluorescence having three different spectra generated from the applied dye is control of the shutters 62 ₁ to 62 ₃ by the controller 61, that is, a notification signal that is information on the ultrashort pulsed light L applied to the sample S The signals are detected in order in synchronization with (the transmission timing of the ultrashort pulsed light L). Thereby, the fluorescence image corresponding to the fluorescence which generate | occur | produces from three types of pigment | dyes with which the measurement range corresponded is acquired simultaneously by one excitation light focus scan.

  As described above, in the two-photon excitation fluorescence microscope 51 according to the second embodiment, the sample S stained with a plurality of types of dyes is excited with the ultrashort pulse light L, and the fluorescence generated from each dye is detected. At the time of imaging, since the measurement ranges of the images match, it is possible to prevent image displacement.

In the second embodiment, the three laser light sources 11 ₁ to 11 ₃ irradiate the sample S with three types of ultrashort pulse lights L ₁ , L ₂ , and L ₃ having different center wavelengths. Although an example in which the obtained fluorescence is detected by one light detection unit 23 has been described, the number of ultrashort pulse lights L is not limited to three. That is, the three types of ultrashort pulsed light L are examples, and it is only necessary to irradiate the sample S with a plurality of types of ultrashort pulsed light L having different center wavelengths. In the two-photon excitation fluorescence microscope 51, the controller 61 applies the sample S to the sample S. Since it is possible to acquire information on the irradiating ultrashort pulsed light L and synchronize it with the electrical signal output from the photodetecting unit 23, the photodetecting unit 23 remains the same and the type of the ultrashort pulsed light L is maintained. The corresponding laser light source 11 and shutter 62 and other optical members are arranged. In that case, the sample S is stained with a plurality of types of dyes corresponding to the plurality of types of ultrashort pulsed light L.

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

1, 51 two-photon excitation fluorescence microscope, 2A personal computer, 2B monitor, 11 ₁ , 11 ₂ , 11 ₃ laser light source, 12 ₁ , 12 ₂ delay circuit, 13, 14, 16, 21, 24 dichroic mirror, 15 mirror, 17 laser scanning unit, 18 scanning lens, 19 second objective lens, 20 objective lens, 22 and 22 _{1, 22} 2, 22 ₃ imaging lens 23 _{1, 23} 2, 23 ₃ photodetection unit, 61 controller, 62 ₁ , 62 ₂ , 62 ₃ shutter, L ₁ , L ₂ , L ₃ ultrashort pulse light

Claims (4)

A plurality of laser light sources that emit pulsed light whose center wavelengths are fixed to be different from each other;
Illumination having scanning means for two-dimensionally scanning a plurality of pulse lights respectively emitted from the plurality of laser light sources, and an objective lens for condensing the plurality of pulse lights on a sample stained with a plurality of types of dyes Optical system,
The plurality of pulse lights are irradiated so that the plurality of pulse lights having different center wavelengths are sequentially irradiated for each condensing position on the sample that is scanned by the scanning unit and collected by the objective lens. Adjusting means for adjusting the timing toward the sample;
Detecting means for detecting, via the objective lens, a plurality of fluorescences respectively generated from a plurality of types of dyes of the sample excited according to the plurality of pulse lights having different center wavelengths. Multiphoton microscope. The adjusting means delays another pulsed light with respect to a reference pulsed light among the plurality of pulsed lights respectively emitted from the plurality of laser light sources, and the plurality of pulsed lights are different for different central wavelengths. Adjust it so that it is repeated in turn,
The multi-photon microscope according to claim 1, wherein the detection unit simultaneously detects fluorescence having a plurality of different spectra generated from a plurality of types of dyes of the sample for each fluorescence having a different spectrum. The adjusting means transmits a plurality of pulse lights respectively emitted from the plurality of laser light sources at a predetermined timing, and the plurality of pulse lights are sequentially repeated in units of a predetermined number for each different center wavelength. Adjust so that
2. The detection unit according to claim 1, wherein fluorescence having a plurality of different spectra generated from a plurality of types of dyes of the sample is sequentially detected in synchronization with a transmission timing by the adjustment unit. Multiphoton microscope. The multi-photon microscope according to any one of claims 1 to 3, wherein the laser light source is an ultrashort pulse laser light source having a short resonator length and emitting ultrashort pulse light.

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