JP2011257589A - Laser microscope, laser microscope system and laser beam transmitting means - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform the simultaneous beam transmission of a plurality of ultrashort pulse laser beams having different single wavelengths while suppressing an extension of time width, thereby making it possible to perform a high-speed observation on a microscope.SOLUTION: Prechirp (3A, 3B) outputs from a ultrashort pulse laser beam source (2A) operating in the vicinity of the zero dispersion wavelength and from a ultrashort pulse laser beam source (2B) of a different wavelength are multiplexed by means of a dichroic mirror (5), and the transmission wavelength and average strength are controlled at a light modulator (6). Then, the resultant light is let incident to HC-PCF (8) and is transmitted therein. Then, the light output is input into a microscope body (1C).

Description

  The present invention relates to a laser microscope, a laser microscope system, and laser light transmission means for observing a specimen with a plurality of laser beams.

  Conventionally, a sample is irradiated with an ultrashort pulse laser beam having an average output of several mW to several W and a pulse width of several tens of fs to several hundred fs through an objective lens, thereby generating a multiphoton excitation effect on the focal plane of the objective lens. A multi-photon excitation type laser microscope is known. In particular, a laser scanning microscope that obtains a scanning image of a specimen by generating fluorescence by the multiphoton excitation effect is used for biological research of biological samples and the like. When a laser system that emits high-power and high-peak ultrashort pulse laser light is combined with the microscope main body to form a microscope system, the laser light from the light source can be emitted using an optical fiber that is easy to handle and highly convenient. It is desired to introduce it after transmitting it appropriately.

  However, when an ultrashort pulse laser beam having a pulse width on the order of femtoseconds is introduced into an optical fiber, a nonlinear effect (self-phase modulation: spectral broadening) is induced by increasing the photon density inside the fiber, and group velocity dispersion. In combination with this, the pulse width of the ultrashort pulse laser beam emitted from the tip of the objective lens is enlarged, and the multiphoton excitation efficiency is impaired and the multiphoton fluorescence luminance is lowered.

  As a technique to solve this problem, an anomalous dispersion optical system is provided between the optical fiber and the laser light source that emits the ultrashort pulse laser light in order to introduce the laser light using an optical fiber whose group velocity has normal dispersion. An arranged laser scanning microscope is known (for example, see Patent Document 1). According to such a laser microscope, since the pulse width of the ultrashort pulse laser beam is previously made to enter the optical fiber by the anomalous dispersion optical system, it is possible to avoid the occurrence of nonlinear effects in the optical fiber.

A titanium sapphire laser is widely used as an ultrashort pulse laser light source capable of changing the center wavelength. In the vicinity of the end of the optical fiber, the recompression of the pulse width of the ultrashort pulse laser light proceeds. Therefore, in order to avoid the occurrence of the nonlinear effect associated therewith, a normal dispersion element disposed on the downstream side of the optical fiber Thus, normal dispersion is applied, and the pulse width after emission from the normal dispersion element is made substantially equal to that before incidence on the anomalous dispersion optical system.
Japanese Patent Laid-Open No. 10-68889

In recent years, many ultrashort pulse laser light sources operating at a single wavelength have been announced. These light sources cannot change the center wavelength like titanium sapphire lasers, but they do not require a water-cooled cooling device, are very cheap compared to titanium sapphire lasers, It also has advantages such as being small. The titanium sapphire laser as the light source can change the center wavelength in a wide band. However, since the mechanical part in the optical resonator is moved in the wavelength change, the wavelength cannot be switched at high speed. Also, since the oscillation wavelength of the optical resonator is changed in principle, the directivity of the ultrashort pulse laser light changes slightly, so that the coupling of the optical fiber with the core that guides the light is finely adjusted each time. There is a need. In addition, the light input to the microscope main body by the conventional optical fiber needs to mechanically adjust the dispersion amount of the anomalous dispersion optical system for each wavelength, and the wavelength cannot be switched at high speed.

  Further, when a titanium sapphire laser is used in principle, it is impossible to oscillate a plurality of ultrashort pulse laser beams having different wavelengths. It was impossible to transmit on fiber.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a laser microscope capable of switching a plurality of ultrashort pulse laser beams having different wavelengths at high speed.

  The present invention has been made in view of the above circumstances, and transmits a plurality of single-wavelength ultrashort pulse laser beams having different wavelengths while minimizing the spread of time width, thereby providing a multi-wavelength laser. An object of the present invention is to provide a laser microscope that can quickly perform observation with light. In one embodiment of the present invention, a plurality of single-wavelength ultrashort pulse laser beams having different wavelengths are transmitted by the same (common) transmission means while minimizing the spread of time width, An object of the present invention is to provide a laser microscope and a system that perform quick and high-quality observation with a multi-wavelength laser beam.

The invention of the laser microscope according to claim 1, which achieves the above object, is a laser microscope for detecting an optical signal generated from a sample by irradiating the sample with a laser beam, and a plurality of ultrashort pulse laser beams having different wavelengths. A dispersion means for adding chromatic dispersion to the light, a multiplexing means for combining the plurality of ultrashort pulse laser lights including the laser light provided with chromatic dispersion, and the combined ultrashort pulse laser light is propagated. A laser transmission means comprising a hollow core photonic crystal fiber, and for irradiating the specimen with an ultrashort pulse laser beam propagated by the laser transmission means, a light emitting end of the hollow core photonic crystal fiber is provided. It is optically connected to the entrance of the irradiation optical system of the microscope.
According to a second aspect of the present invention, the laser microscope according to the first aspect further comprises a light modulation means capable of switching a transmitted wavelength and modulating an average intensity. The invention according to claim 3 is the laser microscope according to claim 1, wherein at least one of the plurality of laser beams is near a zero dispersion wavelength of the hollow core photonic crystal fiber. It is what. According to a fourth aspect of the present invention, there is provided the laser microscope according to the first or third aspect, wherein the dispersing means is configured to use a part of the plurality of laser beams in the hollow core photonic crystal fiber. Dispersion compensation that cancels the group velocity dispersion amount is provided. The invention according to claim 5 is the laser microscope according to claim 3, wherein the core of the hollow core photonic crystal fiber for propagating the ultrashort pulse laser beam is elliptical. Is. Furthermore, the invention according to claim 6 is the laser microscope according to any one of claims 1 to 5, wherein the core of the hollow core photonic crystal fiber is a hollow body filled with gas. It is.
A seventh aspect of the invention comprises the laser microscope according to any one of the first to sixth aspects, wherein a plurality of ultrashort pulse laser light sources having different oscillation wavelengths and the wavelength dispersion means are provided for each of the light sources. This is a laser microscope system characterized by the above. The invention according to claim 8 is a transmission means for the laser microscope according to claim 2, wherein an optical connection portion on the incident side for optically connecting a plurality of ultrashort pulse laser beams in a combined state, and an incident Light modulation means arranged on a combined optical path of a plurality of later ultrashort pulse laser beams, and light incidence to propagate the plurality of ultrashort pulse laser lights modulated by the light modulation means through a common hollow core Laser light transmission comprising: a hollow-core photonic crystal fiber having an end disposed; and an output-side optical connection for optical connection to a microscope main body disposed at the light exit end of the fiber Means.

  According to the first aspect of the present invention, the hollow core photonic crystal fiber having no nonlinear optical effect is used, and the group velocity dispersion of the hollow core photonic crystal fiber and the group velocity dispersion after the hollow core photonic crystal fiber are compensated. As described above, dispersion compensation is performed for each of a plurality of ultrashort pulse laser light sources having different wavelengths, and after being combined, the ultrashort pulse laser light is input to the hollow core photonic crystal fiber and propagated therethrough. The laser light source and the microscope main body can be connected by an optical fiber, and ultrashort pulse laser beams having a plurality of wavelengths can be propagated simultaneously. Furthermore, efficient multiphoton excitation can be performed on the specimen while minimizing the time width of the ultrashort pulse laser light.

    According to the second aspect of the present invention, in addition to the function and effect of the first aspect of the invention, the light modulation means is provided so that the wavelength and the average intensity irradiated on the sample can be adjusted. The selected marker can be selected to excite multiphotons, and the brightness of the multiphoton fluorescence image can be adjusted. Furthermore, by making it possible to adjust the wavelength and the average intensity at high speed, it is possible to observe temporal changes for different markers of the sample.

  According to the invention described in claim 3, in addition to the function and effect of the invention described in claim 1, at least one wavelength of a plurality of ultrashort pulse laser light sources having different wavelengths is set in the hollow core photonic crystal fiber. Near zero-dispersion wavelength and input to the hollow core photonic crystal fiber. At this wavelength, there is no group velocity dispersion of the hollow core photonic crystal fiber, so compensation for group velocity dispersion after the hollow core photonic crystal fiber In applications where it is not necessary to perform this, the dispersion compensation means can be eliminated at this wavelength, and the number of components used can be reduced.

  According to the invention described in claim 4, in addition to the operational effects of the invention described in claim 1 or 3, the spread of the time width of the ultrashort pulse laser beam due to group velocity dispersion can be effectively suppressed. In addition, efficient multiphoton excitation is performed while suppressing a decrease in peak light intensity due to the spread of the time width of the ultrashort pulse laser light.

  According to the invention described in claim 5, in addition to the function and effect of the invention described in claim 3, the hollow core photonic crystal fiber has an elliptical core so that the polarization direction of the ultrashort pulse laser beam can be changed. It can be stored and propagated, so that optical components with polarization characteristics can be used in the microscope body, and the marker of the polarization-dependent specimen can be excited more efficiently by multiphoton, and bright multiphoton A fluorescent image can be obtained.

  According to the invention described in claim 6, in addition to the operational effects of the invention described in any one of claims 1 to 5, laser beams having different wavelengths can be transmitted in a uniform space without a medium.

  According to the seventh aspect of the present invention, not only a laser microscope system in which a plurality of ultrashort pulse light sources are combined with the laser microscope according to any one of the first to sixth aspects, but also good for each light source. Can be propagated through a common hollow core photonic crystal fiber in a well dispersed state.

According to the invention described in claim 8, the laser beam capable of transmitting a plurality of ultrashort pulse lights having different wavelengths while suppressing the nonlinear effect to the laser microscope according to claim 2 while performing optical modulation. A transmission means can be provided.

It is a block diagram which shows the whole structure of the laser microscope which concerns on embodiment of this invention. It is sectional drawing of the hollow core photonic crystal fiber of the laser microscope of FIG. It is a figure which shows the example of the pre-chirper of the laser microscope of FIG. It is a figure which shows the other example of the pre-chirper of the laser microscope of FIG. It is a figure which shows the relationship between the wavelength of a hollow core photonic crystal fiber of the laser microscope of FIG. 1, and group velocity dispersion | distribution. It is a figure which shows the example of the relationship between time and image acquisition in operation | movement of the laser microscope of FIG. It is a block diagram of the whole structure which shows the modification of the several ultra-short pulse laser light source of a different wavelength of the laser microscope of FIG.

  A laser scanning microscope system 1 according to an embodiment of the present invention will be described below with reference to FIGS. As shown in FIG. 1, a laser scanning microscope system 1 according to the present embodiment is a multiphoton excitation laser scanning microscope that generates a multiphoton excitation effect by irradiating a specimen A with an ultrashort pulse laser beam. The ultrashort pulse laser light source 2 in the light source device 1A and the microscope apparatus main body 1C are connected via a hollow core photonic crystal fiber 8 which is an optical transmission means of the optical transmission device 1B. The ultrashort pulse laser light source 2 includes laser light sources 2A and 2B that emit a plurality of ultrashort pulse laser beams having different single wavelengths.

  In the light source device 1A, on the light emitting side of the laser light source 2A, first, a pre-chirper 3A for imparting wavelength dispersion to the laser light source 2A is disposed, and a dichroic mirror 4A is disposed at the subsequent stage. On the other hand, a pre-chirper 3B for providing wavelength dispersion to the laser light source 2B is disposed on the light emitting side of the laser light source 2B, and a folding mirror 4B is disposed at the subsequent stage. The folding mirror 4B is arranged so that the optical path 5B faces the dichroic mirror 4A, and the dichroic mirror 4A combines the optical paths 5A and 5B to form a single combined optical path 5C.

  The optical transmission device 1B is a transmission means for a laser microscope, and includes an incident-side optical connection portion that optically connects a plurality of ultrashort pulse laser beams in a combined state, and a plurality of ultrashort pulse laser beams after incidence. And a hollow core photonic having a light incident end disposed so as to propagate a plurality of ultra-short pulse laser beams light-modulated by the light modulation means through a common hollow core. It is necessary to have a crystal fiber and an optical connection part on the emission side which is arranged at the light emission end of the fiber and is optically connected to the microscope body. Here, as shown in the figure, the optical transmission device 1B includes an optical modulator 6 for applying optical modulation as will be described later to two types of laser light passing through the combined optical path 5C, and a coupling optical system. 7 and the hollow core photonic crystal fiber 8 are arranged in order along the optical path. Here, the two types of ultrashort pulse laser beams are chirped by the dispersion means of the light source device 1A, and then are combined and enter from the incident side of the optical transmission device 1B, and are further optically modulated. The coupling optical system 7 is a single hollow core that functions as the only light reflection transmission path of the optical path from the light source device 1A of the microscope system 1 to the microscope main body 1C. The arrangement and configuration are such that laser light can be efficiently introduced from the light incident end of the photonic crystal fiber 8 and introduced into one hollow core. Here, the optical modulator 6 which is a light modulation means increases the propagation performance when it is incident on the hollow core photonic crystal fiber 8 while switching the wavelength of two or more ultrashort pulse laser beams. Although it is preferable to arrange in the front stage of the nick crystal fiber 8, it does not necessarily have to be in the front stage, and may be arranged in the rear stage or both.

  On the other hand, at the rear stage of the emission end 8B of the hollow core photonic crystal fiber 8, along the optical path 5D through which the two laser beams emitted from the hollow core pass, a collimating optical system 9, a beam shaping optical system 10, and Is arranged so that each laser beam is incident on the irradiation optical path of the microscope main body 1C through this connection means. Here, the collimating optical system 9 and the beam shaping optical system 10 are constituent elements attached to the microscope main body 1C as shown in FIG. 1, and are the only optical transmission means of the optical transmission apparatus 1B via an appropriate optical connection port. It is optically connected to the light emitting end 8B of the hollow core photonic crystal fiber 8 that is. Depending on the case, an adapter that connects the light incident connection with the microscope main body 1 </ b> C and the light emitting end of the hollow core photonic crystal fiber 8 may be used.

    Each of the laser light sources 2A and 2B is a light source that oscillates a single-wavelength laser beam, and is effective for observing a sample such as a living tissue or a living cell, and a wavelength band (for example, 700 nm to 1300 nm); Ultra-short pulse laser light having an average output (for example, several mW to several W) and a pulse width (for example, several tens of fs to several hundred fs), which is different from an ultra short pulse laser beam (or simply called a short pulse laser). Each of the pulsed laser beams is emitted independently. Here, examples of the light source that oscillates a single wavelength laser beam include a mode-locked solid-state laser, a mode-locked fiber laser, a mode-locked semiconductor laser, and a gain-switched semiconductor laser. May be a higher harmonic. In addition, for a wavelength tunable laser light source such as a titanium sapphire laser, when two or more wavelength tunable laser light sources are emitted in a combination in which the wavelengths of the light sources do not always overlap each other. May be included as one embodiment of the present invention. In the present embodiment, the ultrashort pulse laser light source 2 includes two light sources, laser light sources 2A and 2B that oscillate a single wavelength laser beam. The laser light source 2A is a gain-switched semiconductor laser that oscillates a single-wavelength laser beam having a wavelength band of 980 nm, is optically amplified by a fiber optical amplifier, and is a time width of an ultrashort pulse laser beam by nonlinear pulse compression. Is set to about 200 femtoseconds. Similarly, the laser light source 2B is a mode-locked solid-state laser that oscillates a single-wavelength laser beam having a wavelength band of 1064 nm, and the time width of the ultrashort pulse laser beam is set to about 200 femtoseconds. It is.

  The pre-chirpers 3A and 3B are dispersion means for adding wavelength dispersion to the ultrashort pulse laser beam. For example, in the example shown in FIG. 3, the pair of opposed transmission gratings 3a and 3b and the height in the direction perpendicular to the paper surface The mirror pair 3c is turned back by changing the direction, and the transmission gratings 3a and 3b and the mirror pair 3c. The transmission gratings 3b and 3a are mirrors 3d for taking out the ultrashort pulse laser beam whose height direction is perpendicular to the paper surface. ing. Depending on the wavelength of the ultrashort pulse laser light emitted from the laser light sources 2A and 2B, the type of the hollow core photonic crystal fiber 8, and the type of the objective lens 12 provided in the microscope body 1C, the light is emitted from the tip of the objective lens 12. The amount of anomalous dispersion imparted to the ultrashort pulse laser beam is adjusted by adjusting the distance between the transmission type gratings 3a and 3b so that the ultrashort pulse laser beam has a predetermined pulse width.

  Further, in the example shown in FIG. 4 which is a modified example of the pre-chirpers 3A and 3B, a pair of transmissive gratings 3a and 3b facing the letter C, and a mirror pair 3c which is folded back by changing the height direction in the direction perpendicular to the paper surface, A first lens 3d, a second lens 3e, a transmissive grating 3a, a first lens 3d, a second lens 3e, a transmissive grating 3b, and a mirror between a pair of transmissive gratings 3a, 3b facing the letter C. A pair 3c, a transmissive grating 3b, and a second lens 3e, a first lens 3d, and a mirror 3d that takes out an ultrashort pulse laser beam whose height direction is changed perpendicular to the paper surface by the transmissive grating 3a. Depending on the wavelength of the ultrashort pulse laser light emitted from the laser light sources 2A and 2B, the type of the hollow core photonic crystal fiber 8, and the type of the objective lens 12 provided in the microscope body 1C, the light is emitted from the tip of the objective lens 12. The transmission grating 3a and the first lens 3d or the distance between the transmission grating 3b and the second lens 3e is set so that the ultrashort pulse laser beam having a predetermined pulse width is the same as the distance between the first lens 3d and the second lens 3e. Adjustment is made without changing the interval, and the amount of positive or negative dispersion imparted to the ultrashort pulse laser beam is adjusted.

  The folding mirror 4B emits an ultrashort pulse laser beam emitted from the laser light source 2B and added with dispersion by the pre-chirper 3B, and an ultrashort pulse laser beam having a different wavelength emitted from the laser light source 2A and added with dispersion by the pre-chirper 3A. The folding direction can be adjusted to be coaxial with the mirror 4A. The dichroic mirror 4A is configured to transmit and transmit the wavelength band of the ultrashort pulse laser light emitted from the laser light source 2A and reflect the wavelength band of the ultrashort pulse laser light emitted from the laser light source 2B. The ultrashort pulse laser beam and the reflected ultrashort pulse laser beam can be adjusted to propagate coaxially.

  In the present embodiment, the optical modulator 6 is configured by an acoustooptic element (acoustooptic variable filter: AOTF). In this acoustooptic device, the average output of the ultrashort pulse laser beam emitted from the laser light sources 2A and 2B and transmitted through the optical modulator 6 is input to the acoustooptic device so that the target fluorescence intensity can be obtained. It is possible to change the sound wave frequency to be input to the acousto-optic device by adjusting by changing the wavelength of the laser light sources 2A and 2B transmitted through the optical modulator 6.

  The coupling optical system 7 has at least one lens, and is arranged so as to be movable in the optical axis direction and in the optical axis cross section, so that the ultrashort pulse laser beam emitted from the optical modulator 6 is hollow core photonic crystal. By adjusting the predetermined converging position at the incident end of the fiber 8 so that no deviation occurs in the direction of the optical axis and in the cross section of the optical axis, the ultra-short pulse laser light is efficiently incident into the hollow core. It is.

  The hollow core photonic crystal fiber 8 is an embodiment of an optical transmission means for transmitting ultrashort pulse laser light oscillated from a short pulse laser light source toward the light irradiation optical system of the microscope body, and is a gas (preferably Is a light guide fiber having an optical hollow passage as a core portion that has a constant cross-sectional shape and dimensions over the entire length. In particular, the hollow core photonic crystal fiber 8 has a cross-sectional structure in which holes of a periodic arrangement pattern are arranged in a cladding portion so as to surround the hollow core so as to form a photonic band gap. When the core portion of the fiber whose cladding portion is a photonic band gap is hollow, one or more laser beams incident on the hollow core serving as an optical transmission path are almost completely exuded to the outside (cladding portion) of the hollow core. The laser beam is transmitted without leakage over the entire length of the fiber (from the incident end 8A to the exit end 8B). In addition, since the hollow core photonic crystal fiber has almost no nonlinear effect, the inventors have noted that it is convenient only to transmit a plurality of types of laser light included in the transmission band in a single mode. Is. In the present invention, a plurality of ultrashort pulse laser beams having different single wavelengths can be combined into a common hollow core photonic crystal fiber, and can be transmitted to the microscope body after propagating through the core. The present invention has been reached after intensive investigations into various configurations.

  The hollow core photonic crystal fiber 8 in the present embodiment is a fiber having a transmission band in the near-infrared wavelength band, for example. An example of a cross-sectional structure of the hollow core photonic crystal fiber 8 is shown in FIG. In FIG. 2, a cladding 22 in which hollow bodies (holes) 21 for determining a transmission band are regularly arranged around a hollow core 20 forming a circular optical path for guiding light. There is an outer peripheral portion 23 made of glass around the cladding 22. As shown in FIG. 1, the hollow core photonic crystal fiber of the present invention has an appropriate length (for example, 1 m or more, preferably 3 m) so that the ultrashort pulse laser light source 2 and the microscope body 1C can be installed in a flexible arrangement. And a diameter (for example, 1 mm or less, preferably 0.4 mm or less), and a material having high light transmittance (for example, quartz) is preferable. In this embodiment, since the hollow core photonic crystal fiber 8 has a sufficiently long overall length and a sufficiently thin diameter, the fiber can be bent relatively freely, so that the layout as a microscope system is flexible.

  The hollow core photonic crystal fiber 8 illustrated as an example has a core 20 composed of a very long cylindrical (or elliptical cylinder) through-hole having a uniform shape and dimensions over the entire length of the fiber, and this There is no particular limitation other than the fiber structure designed so that the core 20 has a property of confining light energy in the through-hole by a so-called photonic band gap. However, as will be described later, in an actual design example, it is shown that there are some shape characteristics. For example, a large number of hollow bodies 21 are provided in the cladding portion 22 which means an outer peripheral region from the core 20. The average diameter of the core 20 is larger than the average diameter of the hollow body 21 and the number of the cores 20 is preferably one.

  The hollow core photonic crystal fiber has a manufacturing method, and finally forms a photonic band gap that determines the wavelength band that transmits light, and bundles the glass tube that becomes the cladding part, and the part that becomes the core at the center of this bundle The preform is made by removing the glass tube that determines the thickness of the preform, and the preform is drawn in the same manner as in the ordinary optical fiber manufacturing method. In other words, the size and shape of the hollow core are determined by how many glass tubes (generally expressed as the number of cells) are removed.

  The dimension of the hollow core 20 has, for example, 2 to 20 cells (diameter of 2 to 30 μm). In the example examined by the inventors, there are cases where the nonlinear effect cannot be ignored when there are two cells. In addition, the single mode tends to be difficult to maintain as the number of cells is increased to 7 cells (diameter of 10 μm) or more. For this reason, the number of cells in the hollow core is preferably 3 or more (3 μm or more), and preferably 7 cells or less. The numerous hollow bodies 21 forming the clad are not particularly limited as long as they are arrangement patterns that form a photonic band gap.

  Another important requirement of the present invention is that the transmission means for transmitting the ultrashort pulse laser beam consists essentially of a hollow core photonic crystal fiber. That is, unlike a fiber having a normal solid core (including a case where the cladding has a hollow structure), the hollow core photonic crystal fiber guides the core 20 in which most of the irradiated ultrashort pulse laser light is hollow. Waves and hollows (especially air) have a very low nonlinear refractive index compared to the glass body forming the solid core, so nonlinear optical effects (self-phase modulation: light) induced by increasing the photon density of the core (Spreading of the spectrum) has negligible characteristics (substantially no nonlinear effect occurs). Thereby, it is not necessary to deal with the nonlinear optical effect as in a normal solid core fiber. On the other hand, when it is convenient to give the hollow core photonic crystal fiber 8 the property of maintaining polarization, it is preferable to use a hollow core photonic crystal fiber in which the core 20 has an elliptical shape.

  On the other hand, other fibers called simply hollow fibers (or hollow fibers or holey fibers) do not have a photonic band gap of the cladding, and a reflection film is formed on the inner wall of the hollow portion where light is guided. In this hollow fiber, the wavelength band through which light is transmitted by the reflection film is determined, and a large transmission band cannot be obtained as compared with the hollow core photonic crystal fiber. Further, since the aperture is large, it is not easy to propagate only the single mode. There are other photonic crystal fibers, but it is difficult in principle to use a low nonlinear material for the core of photonic crystal fibers, and many of them actively use nonlinear effects, contrary to the present invention. Used for purposes. It is also possible to increase the core and lower the light intensity density of the core to avoid the nonlinear effect, but in this case, it becomes difficult to guide light only in the single mode.

  FIG. 5 shows the relationship between the wavelength and the group velocity dispersion (β2) of the hollow core photonic crystal fiber 8. As shown in FIG. 5, the group velocity dispersion of the hollow core photonic crystal fiber 8 has a negative (abnormal) dispersion on the longer wavelength side than the wavelength at which the group velocity dispersion is zero, and is positive (normal) on the opposite short wavelength side. ) Have variance. Light with a band in the optical spectrum, such as ultrashort pulse laser light, propagates through a medium with group velocity dispersion, and in the normal dispersion region, the long wavelength component propagates through the medium faster than the short wavelength component. The time width of light spreads, and in the anomalous dispersion region, the short wavelength component propagates through the medium earlier than the long wavelength component, thereby widening the time width of the ultrashort pulse laser light.

  In the present embodiment, the hollow core photonic crystal is such that the wavelength of the laser light source 2 </ b> A that oscillates one of the two ultrashort pulse laser beams is close to the zero dispersion wavelength of the hollow core photonic crystal fiber 8. Fiber 8 is selected. Since the wavelength of the laser light source 2B having a wavelength different from that of the laser light source 2A is other than the zero dispersion wavelength of the hollow core photonic crystal fiber 8, the prechirper 3B is reversed so as to cancel the group velocity dispersion amount of the hollow core photonic crystal fiber 8. The group velocity dispersion is generated. In the present embodiment, as shown in FIG. 5, the wavelength of the laser light source 2 </ b> B becomes an anomalous dispersion region of the hollow core photonic crystal fiber 8, so the pre-chirper 3 generates normal dispersion that cancels this.

  The transmission band of the hollow core photonic crystal fiber 8 is designed to have a propagation loss amount of 1 dB / m or less, and the zero dispersion wavelength shown in FIG. Further, when moisture or foreign matter enters the hollow body of the core 20 or the clad 21, the amount of propagation loss increases. The end face of the hollow core photonic crystal fiber 8 is blocked so as not to be exposed to the outside air, and the ultrashort pulse laser light is provided with a non-reflective coating, for example, provided with a certain distance from the end face. A transparent glass window comes in and out. Alternatively, a very thin glass body is fused to the end face in the direction of the optical axis, and the very thin glass body is subjected to oblique polishing or antireflection coating so that ultra-short pulse laser light enters and exits this very thin glass body. It has become.

  The hollow core photonic crystal fiber 8 (abbreviated as HC-PCF) actually studied in this embodiment is a photonic bandgap fiber (for example, model number HC-1060-02) obtained from NKT Photonics, for example. And having a hollow core 20 having a diameter of about 10 μm at the center of a cladding portion having a diameter of about 100 μm, and a plurality of hollow bodies 21 having a diameter of about 7.5 μm within a range of about 50 μm from the center, It has a cross-sectional shape (see FIG. 2) arranged at high density. In addition, the photonic bandgap fiber of this embodiment has optical characteristics such that the wavelength band having anomalous dispersion is a wavelength of 1064 nm band, and the transmission loss at this anomalous dispersion wavelength is about 0.1 dB / m or less. ing. Further, the photonic bandgap fiber of this embodiment has an optical characteristic that the zero dispersion wavelength is about 980 nm band and the transmission loss at the zero dispersion wavelength is about 0.3 dB / m or less. Preferably, the outer periphery of the fiber is covered with a protective coating and has an outer diameter of about 2.5 mm or less.

  The collimating optical system 9 converts at least one lens in the optical axis direction to convert the ultrashort pulse laser beam emitted from the emission end of the hollow core photonic crystal fiber 8 into substantially parallel light. Yes. The beam shaping optical system 10 is composed of at least two lenses, and is shaped so that the beam diameter of the ultrashort pulse laser beam fills the pupil of the objective lens 12 by moving at least one lens in the optical axis direction. It is supposed to be.

  The microscope main body 1 </ b> C exits the hollow core photonic crystal fiber 8, and is a scanner 13 that two-dimensionally scans the ultrashort pulse laser light that is collimated by the collimating optical system 9 and shaped by the beam shaping optical system 10. An objective lens 12 for condensing the multi-photon fluorescence generated in the specimen A while condensing the ultrashort pulse laser light further shaped by the pupil projection lens 14 and the imaging lens 15 and irradiating the specimen A; A dichroic mirror 16 that branches the multiphoton fluorescence collected by the objective lens 12 from the optical path of the ultrashort pulse laser beam; a condensing lens 17 that collects the multiphoton fluorescence branched by the dichroic mirror 16; A photodetector 18 for detecting the emitted multiphoton fluorescence, and a table for constructing and displaying a multiphoton fluorescence image based on the multiphoton fluorescence detected by the photodetector 18. And a part 19.

  The operation of the laser scanning microscope 1 according to the present embodiment configured as described above will be described below.

  The ultrashort pulse laser beam emitted from the laser light source 2B is added with anomalous dispersion so as to cancel out the total dispersion amount of the hollow core photonic crystal fiber 8 and the normal dispersion amount of the microscope body 1C by the pre-chirper 3B2. Then, the light is reflected by the folding mirror 4B and enters the dichroic mirror 4A. The dichroic mirror 4A is reflected and enters the optical modulator 6. On the other hand, the ultrashort pulse laser beam emitted from the laser light source 2A is added with anomalous dispersion so as to cancel the normal dispersion amount of the microscope main body 1C by the pre-chirper 3A, passes through the dichroic mirror 4A, and enters the optical modulator 6.

  One of the ultrashort pulse laser beams of the laser light sources 2A and 2B incident on the optical modulator 6 is transmitted, and the average intensity thereof is adjusted. The ultrashort pulse laser beam transmitted through the optical modulator 6 is condensed by the coupling optical system 7 and coupled to the core of the hollow core photonic crystal fiber 8. The ultrashort pulse laser beam propagating through the hollow core photonic crystal fiber 8 receives and emits the group velocity dispersion of the hollow core photonic crystal fiber 8, is collimated by the collimating optical system 9, and then becomes beam shaping optics. The beam diameter of the ultrashort pulse laser beam is adjusted by the system 10 so as to fill the pupil of the objective lens 12 of the microscope main body 1C.

  The ultrashort pulse laser beam incident on the microscope main body 1C is two-dimensionally optically scanned by the scanner 13 of the illumination optical system 11 for irradiating at least a part of the specimen with the laser beam, and the pupil projection lens 14 and the image are formed. The light is made substantially parallel by the lens 15, condensed by the objective lens 12, and irradiated on a specific portion of the specimen A to perform multiphoton excitation. A biological sample (such as a cell or tissue) that is a specimen A has a marker (GFP: green fluorescent protein) that can be excited by multiphotons at the wavelength of the laser light source 2A and a marker that can be excited by multiphotons at the wavelength of the laser light source 2B ( RFP (red fluorescent protein) is incorporated in a specific part of the specimen A of interest.

  The time width of the ultrashort pulse laser light on the specimen A is zero dispersion wavelength with respect to the wavelength of the laser light source 2A, so that dispersion due to this is not taken into consideration, and the hollow core photonic is not considered. Since the anomalous dispersion is imparted by the pre-chirper 3A so as to cancel the normal dispersion after the crystal fiber, the spread of the ultrashort pulse laser beam due to the group velocity dispersion is suppressed to 1 to 2 times. In addition, the normal dispersion is added to the wavelength of the laser light source 2B so that the pre-chirper 3B cancels out the anomalous dispersion of the hollow core photonic crystal fiber and the normal dispersion after the hollow core fiber (which becomes anomalous dispersion). Therefore, the spread of the time width of the ultrashort pulse laser beam due to the group velocity dispersion is suppressed to 1 to 2 times, and the reduction of the peak light intensity due to the spread of the time width of the ultrashort pulse laser beam is suppressed, which is efficient. Multiphoton excitation is performed. Thus, in the present embodiment, a part of the plurality of pre-chirpers (3A, 3B) serving as the dispersion means respectively corresponding to the laser beams having a plurality of wavelengths, that is, the pre-chirpers (3A) corresponding to the zero dispersion wavelength. May be chirped so as to compensate only the normal dispersion after the hollow core photonic crystal fiber 8. In other words, for some of the other pre-chirpers, that is, the remaining pre-chirpers (3B) other than the pre-chirper (3A) corresponding to the zero dispersion wavelength, a char that compensates for anomalous dispersion in the hollow-core photonic crystal fiber 8 is used. You can ping.

  The multiphoton fluorescence generated in the sample A is collected by the objective lens 12, branched from the optical path of the ultrashort pulse laser light by the dichroic mirror 16, condensed by the condenser lens 17, and incident on the photodetector 18 for detection. A multi-photon fluorescence image is displayed on the display unit 19 based on the multi-photon fluorescence.

  FIG. 6 shows the relationship between the operation of the acousto-optic variable filter that is the light modulator 6 and the multiphoton fluorescence image obtained on the display unit 19. In FIG. 6, the horizontal axis indicates time, and the vertical axis indicates the transmission state of the optical modulators 6 of the laser light sources 2A and 2B. First, the optical modulator 6 transmits the ultrashort pulse laser light from the laser light source 2A while adjusting the transmission average intensity so that the intensity of the obtained multiphoton fluorescence image is optimized. At this time, the detection signal from the marker 30 capable of multi-photon excitation with the ultrashort pulse laser beam having the wavelength of the laser light source 2A of the sample A is detected by the photodetector 18, and the multi-photon excitation fluorescence image is displayed on the display unit 19. Is done. At the next moment, the optical modulator 6 switches the input sound wave frequency of the acousto-optic variable filter, and transmits the ultrashort pulse laser light having the wavelength of the laser light source 2B so that the intensity of the obtained multiphoton fluorescence image becomes optimum. Adjust to make it transparent. Then, a detection signal from the marker 31 that can be excited by multi-photon light with an ultrashort pulse laser beam having the wavelength of the laser light source 2B of the sample A is detected by the photodetector 18, and a multi-photon excited fluorescence image is displayed on the display unit 19. The

  As described above, the ultrashort pulse laser beam having two kinds of wavelengths is incident on the microscope main body substantially simultaneously by repeatedly transmitting the hollow core photonic crystal fiber 8 while being switched at high speed by the optical modulator 6. Multi-color fluorescence images can be detected and displayed in real time. Here, the term “simultaneous” means that the hollow core of the hollow core photonic crystal fiber, which is a common propagation path, is switched in a mixed state or in a short time in an optical path where two or more laser beams having different wavelengths are combined. By transmitting in an alternating propagation state, it indicates a propagation state in which the specimen A arranged on the microscope main body 1C can be irradiated with a short pulse laser beam almost simultaneously. The irradiation position on the specimen A may be the same or different depending on the role for each wavelength.

  As described above, a hollow-core photonic crystal fiber with a negligible nonlinear optical effect, a single-wavelength ultrashort pulse laser light source near the zero dispersion wavelength, and a hollow-core photonic crystal fiber zero Dispersion compensation is applied to the ultrashort pulse laser light of a single-wavelength ultrashort pulse laser light source other than the dispersion wavelength so as to cancel the group velocity dispersion after the hollow core photonic crystal fiber and the hollow core photonic crystal fiber with a pre-chirper. By inputting into a hollow core photonic crystal fiber, a single optical fiber transmits multiple ultra-short pulse laser beams with a single wavelength while minimizing the spread of the ultra-short pulse laser beam on the sample. In addition, the laser light source and the microscope main body can be connected with an optical fiber, so that a free laser scanning microscope can be used. The layout is possible.

Furthermore, by providing an optical modulator, it is possible to switch the wavelength for irradiating the sample and exciting multiple photons at high speed by switching the electrical signal input to the optical modulator, and within the sample in which different markers are incorporated. It becomes possible to observe the time change of a specific part at high speed.
(Modification)
A modification of a plurality of ultrashort pulse laser light sources having different wavelengths is shown in FIG. In FIG. 7, the laser scanning microscope system 1 includes ultrashort pulse laser light sources 2A and 2C having different single wavelengths, and the laser light source 2C uses the ultrashort pulse laser light of the other ultrashort pulse laser light source 2A. A part of the laser beam is input, and the ultrashort pulse laser beam is configured to emit an ultrashort pulse laser beam having a single wavelength different from that of the laser light source 2A. Here, in order to change the ultrashort pulse laser beam of the laser light source 2A from an ultrashort pulse laser beam having a different wavelength, the two photons of the ultrashort pulse laser beam of the laser light source 2A are converted to one harmonic photon. Low harmonic generation, optical parametric oscillation that converts one photon of the ultrashort pulse laser beam of the laser light source 2A into two photons of low-frequency signal light and idler light, and the Raman scattering effect of the medium. It can be realized by Raman shift light to high frequency Stokes light or Raman shift light to high frequency anti-Stokes light. By doing so, it is possible to generate an ultrashort pulse laser beam having a wavelength without a laser medium suitable for generating an ultrashort pulse laser beam.

  Further, in FIG. 7, the wavelength of the laser light source 2A is set to the vicinity of the zero dispersion wavelength of the hollow core photonic crystal fiber 8, and the pre-chirper 3A shown in FIG. If the spread of the ultrashort pulse laser beam on the specimen A due to dispersion after the hollow core photonic crystal fiber does not cause any problems in use, reduction and adjustment of the components used due to the elimination of the pre-chirper 3A By reducing the procedure, the laser microscope becomes cheaper and simpler. In addition, there is an advantage that a laser microscope with high energy efficiency can be realized by effectively using the ultrashort pulse laser beam of the laser light source 2A.

  In the above description, the present invention is not limited to the description of the above-described embodiments and modifications, and includes various modifications and equivalents based on the gist of the present invention. Here, the present invention includes a laser microscope system from a light source to light detection (and display means), and an optical transmission device for a laser microscope apparatus that is detachable between the light source and the microscope main body. . For the optical transmission apparatus, the light dispersion means, the hollow core photonic crystal fiber described in the transmission means 1B, and the optical connection means for connecting (or relaying) the optical path to the light source device 1A and the microscope main body 1C; Is the main requirement.

  For example, the term “laser microscope” in the present invention is not limited to the microscopes exemplified in the above-described embodiments and the like, and an ultrashort pulse laser beam is used at two or more wavelengths, and at least one laser beam is used for image formation. Other spectroscopic image acquisition devices can be included, including any type of microscope that contributes and having a configuration equivalent to the illumination optics of the microscope. Further, even in a light measurement (or light measurement) microscope that does not mainly observe images, such as an optical microscope, the laser of the present invention is used when data based on a microscope image is used in at least a part of the calculation process. Included in the microscope. Further, the present invention also includes a laser microscope that observes and / or measures a biological sample by stimulating a biological sample with one wavelength among a plurality of ultrashort pulse laser beams and acquiring an image of the response using another wavelength. .

Further, as shown in FIG. 1, the specimen A does not have to be an arbitrary substance that can be accommodated in an object or a container having a size that can be placed on a stage or the like below the illumination optical system 11 of the microscope main body 1C. . That is, when observing a living organism such as a small animal, at least a part of the objective lens and the illumination optical path is arranged so that the tip of the objective lens is as close as possible to the light irradiation site at an arbitrary position inside and outside the organism. You may make it accommodate in an elongate cylinder. In this case, by arranging the optical path of the illumination optical system 11 of the microscope body 1C as an illumination optical path so as to be transmitted by an optical transmission means equivalent to the hollow core photonic crystal fiber 8 as described in the above embodiment, It is also possible to build a system that allows direct and non-invasive access to the specimen. In this case, the second hollow core photonic crystal fiber 8 can be arranged in the rear stage of the beam shaping optical system 10 in the microscope main body 1C, and the emission end can be extended to just before the scanner 13. It is preferable to design the distance from the scanner 13 to the scanner 13 as long as possible.
When transmitting the second fiber, it is preferable to set dispersion compensation for anomalous dispersion for this purpose by the dispersion means. In the case of a microscope system in which the optical transmission means 1B and the microscope main body 1C are integrated, the hollow core photonic crystal fiber 8 in the transmission means 1B is arranged to extend into the incident optical system 11 of the microscope main body 1C. This is preferable because it is not necessary to provide a separate fiber and there is no need for further light dispersion control or dispersion means.

  In the above embodiment, single wavelength light sources having two different wavelengths are used. However, different single wavelengths corresponding to an arbitrary number necessary for obtaining detection light (for example, multicolor fluorescence) to be detected in the specimen. Any light source may be used as long as the laser light can be simultaneously oscillated through separate optical paths. For example, when a light source device that individually oscillates three or four or more wavelengths is used, image observation and / or image analysis (measurement) corresponding to all color information is possible, and various light response reactions and light stimulations are possible. Can be performed simultaneously with imaging. Here, for example, when the number of single-wavelength light sources is three, the configuration of the light source device further includes a third single-wavelength super-wavelength on the optical path 5B between the folding mirror 4A and the dichroic mirror 4B shown in FIG. By arranging the short pulse laser light source and the third pre-chirper in the same manner, the laser light oscillated from the second single-wavelength ultrashort pulse laser light source 2B is combined, and then the combined laser light May be configured to reach the dichroic mirror 4B of FIG.

  In this embodiment, an acousto-optic variable filter is used as the optical modulator 6. However, when wavelength switching is not required, an acousto-optic element (AOM, AOBS), an electro-optic element (EOM), an ND filter, or λ A combination of a / 2 plate and a polarizer can also be used as a light modulation means. In the present embodiment, the transmission grating pair is used as the pre-chirper 3, but a reflection grating pair or a prism pair may be used. In addition, an isolator or λ / 4 plate is placed immediately after the laser light source to prevent damage due to the return light to the ultrashort pulse laser light source due to the reflected light from the hollow core photonic crystal fiber and other optical components whose end surfaces are processed You may do it. Furthermore, the operation of the scanner 13 for each laser beam whose wavelength is switched by light modulation is not limited to two-dimensional scanning, and may be configured to perform three-dimensional scanning.

    In the above-described embodiment, the ultrashort pulse laser light sources having different single wavelengths are exemplified as laser microscopes for image observation used for multiphoton excitation of different markers, but one of the light sources is stimulated by (multiphoton) stimulation. It is also possible to use a multi-function type laser microscope in which the other light source is used for acquiring a multiphoton fluorescence image. The marker that emits fluorescence by photoexcitation is not limited to a fluorescent protein such as GFP, and may be various fluorescent dyes or autofluorescent substances, or a marker capable of detecting light other than fluorescence. In addition, a substance that responds to stimulation by laser light does not need to fluoresce itself, but any observation site corresponding to morphological, biochemical, or physicochemical changes induced by the optical response and / or It may be a measurement substance.

DESCRIPTION OF SYMBOLS 1 Laser scanning microscope system 2 Ultra-short pulse laser light source 3 Pre-chirper 3a, 3b Transmission type grating 3c Folding mirror pair 3d Extraction mirror 3d, 3e Lens 4A Dichroic mirror 4B Folding mirror 6 Optical modulator 7 Coupling optical system 8 Hollow core Photonic crystal fiber 9 Collimating optical system 10 Beam shaping optical system 1, 70 Microscope main body 12 Objective lens 13 Scanner 14 Pupil projection lens 15 Imaging lens 16 Dichroic mirror 17 Condensing lens 18 Photo detector 19 Display unit 20 Core 21 Hollow body (Vacancy)
22 Cladding 23 Outer periphery

Claims (8)

In a laser microscope for detecting an optical signal generated from a sample by irradiating the sample with laser light,
Dispersion means for adding chromatic dispersion to a plurality of ultrashort pulse laser beams having different wavelengths;
A multiplexing means for multiplexing the plurality of ultrashort pulse laser beams including a laser beam provided with wavelength dispersion;
A laser transmission means comprising a hollow core photonic crystal fiber for propagating the combined ultrashort pulse laser light,
In order to irradiate the specimen with the ultrashort pulse laser beam propagated by the laser transmission means, the light exit end of the hollow core photonic crystal fiber is optically connected to the entrance portion of the irradiation optical system of the microscope. A laser microscope.   2. The laser microscope according to claim 1, further comprising light modulation means capable of switching a wavelength to be transmitted and modulating an average intensity.   The laser microscope according to claim 1, wherein at least one of the plurality of laser beams is near a zero dispersion wavelength of the hollow core photonic crystal fiber.   The dispersion means provides dispersion compensation for canceling out a group velocity dispersion amount in the hollow core photonic crystal fiber with respect to a part of the plurality of laser beams. 3. The laser microscope according to 3.   4. The laser microscope according to claim 3, wherein a core of the hollow core photonic crystal fiber for propagating the ultrashort pulse laser beam has an elliptical shape.   The laser microscope according to any one of claims 1 to 5, wherein a core of the hollow core photonic crystal fiber is a hollow body filled with a gas.   A laser microscope comprising the laser microscope according to any one of claims 1 to 6, wherein a plurality of ultrashort pulse laser light sources having different oscillation wavelengths and the wavelength dispersion means are provided for each of the light sources. system. The transmission means for a laser microscope according to claim 2, wherein an incident-side optical connecting portion for optically connecting a plurality of ultrashort pulse laser beams in a combined state, and a plurality of ultrashort pulse laser beams after incidence And a hollow core photonic having a light incident end disposed so as to propagate a plurality of ultra-short pulse laser beams light-modulated by the light modulation means through a common hollow core. A laser light transmitting means comprising: a crystal fiber; and an optical connection portion on an emission side which is disposed at a light emission end portion of the fiber and is optically connected to a microscope main body.

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