JP4845279B2 - Biological function measurement method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
  The present invention provides a fluorescence from a very small region under conditions where an observation method using a normal microscope cannot be applied, for example, when the observation target is a large animal or when it is intended to obtain information in the brain when the animal is active. We have developed biological function measurement devices and fiber probe light detection devices that can obtain signals and apply multi-photon excitation using ultrashort pulse laser light. For example, a near infrared ultrashort pulse laser light source and a fiber probe The present invention relates to a used fiber probe light detection device and a biological function measurement device using the same.
[0002]
[Prior art]
  A scanning confocal microscope for acquiring an image by introducing laser light through a fiber into a microscope or probe having a scanning function is disclosed in Japanese Patent Laid-Open No. 3-87804.
[0003]
  A multiphoton excitation laser microscope apparatus combining an ultrashort pulse laser and a microscope is disclosed in Japanese Patent No. 2848952, and further, an ultrashort pulse laser and a fiber are used to guide laser light to an optical device and irradiate a specimen. Japanese Patent Laid-Open No. 10-186424 discloses an ultrashort light pulse transmission device, a generation device, and a transmission method.
[0004]
  FIG. 5 is a diagram showing a scanning confocal microscope apparatus disclosed in Japanese Patent Laid-Open No. 3-87804 (first prior example).
[0005]
  Laser light from the laser light source 51 is introduced into the fiber 53 through the lens 52.
[0006]
  The laser beam is held by a holder 57 on a piezoelectric element 59 installed inside the housing 56 via a coupler 54 and a fiber 55.
[0007]
  The laser beam emitted from the fiber 55 is condensed at the focal position 58a of the sample 58 by the lenses 60a and 60b.
[0008]
  Reflected light or fluorescence from the focal position 58 a passes through the lenses 60 a and 60 b and the fiber 55 again and enters the fiber 61 by the coupler 54.
[0009]
  Further, the wavelength is selected by the filter 62, converted into an electric quantity by the photoelectric conversion element 63, and input to the computer 64.
[0010]
  The computer 64 controls the piezoelectric element 59 via the driver 65 and the cable 67.
[0011]
  The tip of the fiber 55 is tilted by the piezoelectric element 59, the incident angle of the laser with respect to the lens 60a can be changed, and the focal position 58a in the sample 58 can be changed in the direction perpendicular to the optical axis.
[0012]
  It is described that the fluorescence amount obtained by scanning the tip of the fiber 55 as described above can be constructed by the computer 64 and displayed on the monitor 66.
[0013]
  FIG. 6 is a diagram showing an ultrashort optical pulse transmission device disclosed in Japanese Patent Laid-Open No. 10-186424 (second prior example).
[0014]
  The laser light source 1 is an ultrashort pulse laser.
[0015]
  A pulse stretcher optical system 2 is installed between the laser light source 1 and the fiber 3.
[0016]
  The laser light that has passed through the fiber 3 passes through the pulse compressor optical system 4 and enters the optical device 5.
[0017]
  The operation of this preceding example will be described.
[0018]
  The ultrashort pulse laser beam emitted from the laser light source 1 is incident on the pulse stretcher optical system 2.
[0019]
  In the pulse stretcher optical system 2, the pulse width of the laser light emitted from the light source is extended and the peak power per pulse is reduced.
[0020]
  The pulsed laser light has a wavelength width of about several nanometers.
[0021]
  When transmitting through a medium such as a fiber, light of the wavelength component on the long wavelength side advances first, so the shorter one of the wavelengths of the incident laser light is output earlier and the longer one is output later. For example, a spectroscopic optical element such as a prism or a grating is used so that the laser beam incident on the light can be dispersed and the optical path length can be adjusted in accordance with the wavelength.
[0022]
  The laser light that has passed through the pulse stretcher optical system 2 enters the fiber 3 on the short wavelength side early and on the long wavelength side late.
[0023]
  Since the long wavelength side advances faster during transmission through the fiber 3, the pulse width becomes shorter than that at the time of incidence and is emitted from the fiber 3.
[0024]
  The laser beam whose pulse length is shortened is further introduced into the pulse compressor optical system 4 to compress the pulse width.
[0025]
  However, the pulse compressor optical system 4 emits an ultrashort pulse laser beam slightly shorter on the short wavelength side.
[0026]
  Further, since the light on the short wavelength side is further delayed while being incident on the optical device 5 and passing through an optical system such as a relay optical system and an objective lens (not shown) before the specimen, the specimen surface is consequently obtained. In this case, the sample can be irradiated with laser light with a pulse width close to that of the original laser light.
[0027]
  Here, the optical device 5 is assumed to be a scanning microscope, and a deflection optical system that scans laser light is provided in a normal microscope, and an image is acquired by scanning the sample with laser light.
[0028]
  If the sample is irradiated with a pulse width close to the pulse width of the original laser light, the multiphoton excitation phenomenon on the sample surface can be efficiently caused, and the sample image can be acquired efficiently.
[0029]
[Problems to be solved by the invention]
  In the first preceding example, since continuous laser light in the visible wavelength range is used as the laser light source, excitation at a position deeper than the specimen surface cannot be performed as compared with the IR ultrashort pulse laser, and fluorescence at that position is not generated. It cannot be detected.
[0030]
  In the case of the first prior example, the specimen is a tooth and the probe is fixed by being sandwiched between the upper and lower teeth. The fixing method is very unstable, and a signal or an image is acquired. There was a problem that was not practical.
[0031]
  In the second prior example, it is possible to obtain a sample image with a good S / N by introducing ultrashort pulse laser light into a microscope with a fiber and increasing the probability of occurrence of multiphoton excitation phenomenon on the sample surface. It is.
[0032]
  However, in the second preceding example, a microscope is used as an example of the optical device. As a sample in the microscope, a micro sample, for example, a section sample, a cultured sample, or a small animal that can be placed under the microscope, such as a mouse, is used. To observe.
[0033]
  However, when considering observation of relatively large animals, it is difficult to observe under an ordinary commercially available microscope.
[0034]
  Moreover, even when observing small animals, they were fixed on a specially prepared stage under anesthesia so that they could be placed under a microscope.
[0035]
  The measurement in the state of anesthesia has a problem that it may be different from obtaining information in the brain, for example, when the animal is originally active.
[0036]
  Furthermore, the length of the fiber may be variously changed depending on the limitation of the laser light source and the specimen installation location and the size of the specimen, but in the second prior example, these are not considered and there are practical restrictions. There was a problem.
[0037]
  Furthermore, the advent of a photodetector capable of effectively detecting fluorescence and the like from the inside of the brain and various specimens, and a biological function measuring device using the same has been strongly desired.
[0038]
  The present invention has been made in view of the above circumstances, and provides a biological function measuring apparatus capable of stable observation under conditions where an observation method using a normal microscope cannot be applied, for example, in a state where an animal is active. With the goal.
[0039]
  Therefore, in order to achieve the above object, according to the first aspect,An optical system of the probe assembly that emits an ultrashort pulse laser beam causing multi-photon excitation and transmits the ultrashort pulse laser beam to a probe assembly fixed so as to come into contact with a specimen using a single mode optical fiber. The transmitted ultrashort pulse laser beam is focused on the specimen, and the fluorescence from the specimen generated by multiphoton excitation based on the irradiation of the ultrashort pulse laser light is converted into the probe assembly and the single mode. The information in the brain of the specimen is obtained by transmitting to an optical detector through an optical fiber and measuring the fluorescence by the optical detector, and the optical system of the probe assembly is 1000 μm from the surface of the specimen. Used to observe moving animals (except humans), characterized by having a focal length that can measure fluorescence emitted from a distance Biological function measuring method for body function measuring apparatus.
[0040]
DETAILED DESCRIPTION OF THE INVENTION
  FIG. 1 is a diagram showing a fiber probe photodetector of the biological function measuring device according to the first embodiment of the present invention.
[0041]
  The present invention provides anesthesia of information in the brain under conditions where a normal microscope observation method cannot be applied, for example, when an observation target is a large animal and cannot be observed under a normal microscope, or when an animal is active. A biological function measuring device developed by applying multi-photon excitation using ultrashort pulse laser light that can obtain fluorescence signals from a very small area even when trying to obtain with a normal microscope without using it. is there.
[0042]
  Embodiments of the present invention will be described below with reference to the drawings.
[0043]
In the figure, a laser light source 11 is a laser light source that oscillates with near-infrared ultrashort pulses.
[0044]
A mirror 10a is disposed on the optical path of the laser light emitted from the laser light source 11, and a mirror 10b is disposed on the optical path of the laser light reflected by the mirror 10a.
[0045]
A pulse stretcher 12 is provided on the optical path of the laser light reflected by the mirror 10b.
[0046]
The pulse stretcher 12 extends the pulse width of the laser light emitted from the laser light source 11 and decreases the peak power per pulse.
[0047]
Further, in the pulse stretcher 12, the incident laser beam is spectrally dispersed corresponding to the wavelength so that the shorter one of the wavelengths of the incident laser beam is output earlier and the longer one is output later. Spectral optical elements such as prisms and gratings are used to adjust the optical path.
[0048]
On the optical path of the laser beam emitted from the pulse stretcher 12, the pulse width of which is extended, an optical branching section is disposed on the reflected optical path of the mirror 10c.
[0049]
This optical branching unit includes an optical system using a dichroic filter, a dichroic mirror, and the like. The dichroic mirror 13 used in the present embodiment has a long-path characteristic, and laser light from the pulse stretcher 12 ( Excitation light) is transmitted, and reflected light / fluorescence from the specimen is reflected.
[0050]
  On the optical path of the laser beam whose pulse width has been extended by the pulse stretcher 12 that has passed through the dichroic mirror 13, the laser beam is collected on one end face of an optical fiber (hereinafter referred to as fiber) 15. A lens 14 is provided.
[0051]
  The other end of the fiber 15 is connected to a fiber joint portion of a probe assembly 17a, which will be described later. The other end of the fiber 15 receives laser light that has passed through the fiber 15 at a condensing position 18a in the specimen 18. A condensing optical system 16 that condenses the fluorescence, which is detection light emitted from the focal position 18 a, on the other end surface of the fiber 15 is provided.
[0052]
The condensing optical system 16 is slidably provided in a probe assembly 17a having a metal protective housing and a fiber joint.
[0053]
The probe assembly 17a is provided with a flange portion 17b having an extremely small holding portion as an attachment, for example, a screw hole that directly comes into contact with the head of a small animal that is a specimen. The flange portion 17b is brought into contact with the specimen. It is mechanically fixed with screws.
[0054]
On the other hand, on the optical path of the detection light (reflected light / fluorescence) from the specimen 18 that is emitted from the fiber 15 and reflected by the dichroic mirror 13, a filter 19 for cutting the excitation light and the filter 19 are passed. A condensing lens 20 for condensing the detection light on one end surface of an optical fiber (hereinafter referred to as a fiber) 21 is provided.
[0055]
The other end surface of the fiber 21 is provided with a lens 22 for converting the detection light that has passed through the fiber 21 into parallel light. The detection light converted into parallel light by the lens 22 is guided to the detection / measurement system 26. It is burned.
[0056]
The optical path system of the detection light guided to the detection / measurement system 26 is provided with a photometric filter 24a for cutting off reflected light and selecting only the photometric wavelength.
[0057]
A photoelectric conversion element 25a for measuring the detection light is provided on the optical path of the detection light that has passed through the photometric filter 24a.
[0058]
When the fluorescence emitted from the sample 18 is for calculating the ratio by two-wavelength simultaneous photometry, the wavelength is separated by providing a dichroic mirror 23 in the optical path.
[0059]
The detection light reflected by the dichroic mirror 23 selects a wavelength by the photometric filter 24b provided on the optical path of the detection light, and measures the detection light by the photoelectric conversion element 25b.
[0060]
  These photoelectric conversion elements 25a and 25b are connected to a computer (not shown) for accumulating and processing data from the photoelectric conversion elements 25a and 25b.
[0061]
Next, the operation of the fiber probe photodetector of the biological function measuring device according to the present embodiment will be described.
[0062]
  Laser light emitted from the laser light source 11 is incident on the pulse stretcher 12 via the mirrors 10a and 10b.
[0063]
  The pulse stretcher 12 extends the pulse width of the laser light emitted from the laser light source 11 and decreases the peak power per pulse.
[0064]
  Further, in the pulse stretcher 12, the incident laser beam is spectrally dispersed corresponding to the wavelength so that the shorter one of the wavelengths of the incident laser beam is output earlier and the longer one is output later. The optical path length is adjusted.
[0065]
  The laser beam output from the pulse stretcher 12 passes through the dichroic mirror 13, and is collected and incident on the end face of the fiber 15 by the condenser lens 14.
[0066]
  Laser light that has passed through the fiber 15 and exited from the probe assembly 17a having the condensing optical system 16 travels faster on the long wavelength side while passing through the fiber 15, so that the pulse width is emitted shorter than that at the time of incidence.
[0067]
  Laser light emitted from the fiber 15 is condensed at a focal position 18a in the specimen 18 by a probe assembly 17a having a condensing optical system 16, and the focal position 18a is excited by a two-photon absorption phenomenon.
[0068]
  The probe assembly 17a including the condensing optical system 16 is fixed so as to be slidable with respect to a flange portion 17b that can be fixed to the head of a small animal, for example.
[0069]
  In the fiber probe photodetector of the biological function measuring device of the present embodiment, the probe assembly 17a including the condensing optical system 16 is slidably provided in the flange portion 17b. Since the focus can be changed in the direction, depth information can be obtained.
[0070]
  Fluorescence emitted from the focal position 18 a is again collected on the end face of the fiber 15 by the condensing optical system 16, passes through the fiber 15, returns to the dichroic mirror 13, is reflected by the dichroic mirror 13, and is laser light (excitation) by the filter 19. Light) is cut and condensed on the end face of the fiber 21 by the condenser lens 20.
[0071]
  The detection light that has passed through the fiber 21 exits from its end face, is collimated by the lens 22, and is guided to the detection / measurement system 26.
[0072]
  The collimated light guided to the detection / measurement system 26 has a photometric wavelength selected by the photometric filter 24a, and the photodetection light is measured by the photoelectric conversion element 25a.
[0073]
  In the case where two wavelengths are measured simultaneously, and when the dichroic mirror 23, the photometric filter 24b, and the photoelectric conversion element 25b are arranged, the detection light having a specific wavelength out of the detection light converted into parallel light by the lens 22 Is reflected by the dichroic mirror 23, and detection light of other specific wavelengths passes through the dichroic mirror 23.
[0074]
  Then, as described above, the photometric wavelength of the detection light transmitted through the dichroic mirror 23 is selected by the photometric filter 24a, and the detection light is measured by the photoelectric conversion element 25a.
[0075]
  On the other hand, the detection light reflected by the dichroic mirror 23 has a photometric wavelength selected by the photometric filter 24b, and the detection light is measured by the photoelectric conversion element 25b.
[0076]
  In the present embodiment, the light emitted from the scanning device or the condensing optical system 16 that scans the probe assembly 17a including the condensing optical system 16 in the flange portion 17b in the direction perpendicular to the optical axis is used as the optical axis. It is also possible to obtain a two-dimensional image of the specimen by using an optical system (not shown) that deflects in a perpendicular direction.
[0077]
  Therefore, according to the fiber probe photodetector of the biological function measurement device of the present embodiment, the optical system is fixed to the specimen in the fiber probe photodetector of the biological function measurement device that uses near-infrared ultrashort pulses. Since it has the holding | maintenance part and fiber 5 which can do, observation of a comparatively large animal can be made easy.
[0078]
  In addition, by providing the above-described holding unit with an XY adjustment micrometer and a Z adjustment micrometer, it is possible to configure a system that can adjust a three-dimensional direction so that a three-dimensional image can be acquired.
[0079]
  In the system that can be adjusted in the three-dimensional direction, the micrometer for XY adjustment can adjust ± 100 μm on the XY plane, and the micrometer for Z adjustment preferably has a resolution of 2 μm in the Z-axis direction.
[0080]
  In addition, it is desirable that the adjusting means provided in the holding portion described above is stable against small vibrations of the probe assembly and fiber extension.
[0081]
  The dimensions of the adjusting means itself are preferably 7 × 7 mm at the bottom surface and 15 mm or less in height, and the bottom surface is fixed to the specimen (contact object) by a flange portion with a screw hole. The
[0082]
  FIG. 2 is a diagram illustrating an example of the condensing optical system 700 of the above-described fiber probe photodetector.
[0083]
  FIG. 2A is a diagram showing the optical path of the laser light passing through the condensing optical system and the condensing position, and FIG. 2B is a diagram for explaining the prism 81 in the condensing optical system. It is.
[0084]
  A condensing optical system 700 shown in FIG. 2 includes a lens 80 that makes laser light emitted from the distal end portion 15a of the fiber 15 substantially parallel, and a direction in which the optical path of the laser light from the lens 80 is orthogonal to the optical axis ( Optical path changing means that can be arbitrarily changed (deflected) in the XY directions in FIG. 2A, here a prism 81, and a lens 82 for condensing the laser light that has passed through the prism 81 onto the specimen 18. It consists of.
[0085]
  Further, the lens 80 is arranged between the fiber 15 and the prism 81 in the optical axis direction (Z direction in FIG. 2A) so that the condensing position of the lens 82 is arbitrarily changed in the optical axis direction with respect to the specimen 18. For example, the first lens position 80a and the second lens position 80b.
[0086]
  The lens moving mechanism for moving the lens 80 in the optical axis direction can be realized by, for example, manual mechanical control means, or electrical control means using a stepping motor, a piezoelectric element, or the like.
[0087]
  Further, the control of the focal position in the optical axis direction is not limited to the movement of the lens 80. For example, the lens 82 may be moved in the optical axis direction.
[0088]
  Next, an example of the configuration of the prism 81 illustrated in FIG. 2A will be described below with reference to FIG.
[0089]
  The prism 81 includes two parallel plane plates having transparency such as a glass member, here a parallel plane plate 73 having an incident surface 81d, a parallel plane plate 74 having an exit surface 81b (81a, 81c), The parallel flat plates 73 and 74 are provided between the parallel flat plates 73 and 74, for example, so as to be parallel to each other, and a silicon rubber 75 elastically deformable with a liquid 76 such as silicon oil sealed therein. The at least three piezoelectric elements 77 are provided between at least three parallel plane plates 73 and 74 and are configured to swing the parallel plane plate 74.
[0090]
  The piezoelectric element 77 is provided with a control unit (not shown), and the optical path of the laser beam passing through the prism 81 is arbitrarily changed in a direction orthogonal to the optical axis direction by the control of the control unit ( Be deflected).
[0091]
  Hereinafter, operations of the lens 80 and the prism 81 of the above-described condensing optical system 700 will be described.
[0092]
  Here, an example in which the lens moving mechanism of the lens 80 shown in FIG. 2A is controlled by an electric control means such as a stepping motor or a piezoelectric element will be described.
[0093]
  First, the lens moving mechanism moves the lens 80 back and forth in the optical axis direction of the laser light, and positions the lens 80 at the first lens position 80a shown in FIG.
[0094]
  In FIG. 2A, it is assumed that the lens 80 is illustrated with the first lens position 80a and the second lens position 80b overlapped.
[0095]
  Next, the prism 81 is composed of parallel plane plates 73 and 74 with an enclosed silicon oil 76 interposed therebetween. By controlling the piezoelectric element 77, the prism 81 is parallel to the incident surface 81 d of the plane parallel plate 73. The exit surface 81b when the plane parallel plate 74 is parallel to the plane parallel plate 73 or the exit surface 81a when the plane parallel plate 74 is inclined with respect to the plane parallel plate 73. (Or 81c), and the inclination of the laser beam incident from the incident surface 81d can be changed.
[0096]
  FIG. 2B shows an example of the above-described state. The piezoelectric element 77 of the prism 81 is controlled in the direction indicated by the arrow 81e, and the emission surface 81b is controlled like the emission surface 81c. The optical axis 90 of the laser beam incident perpendicularly to 81d is bent at the exit surface 81c, and the laser beam as the optical axis 91 is emitted.
[0097]
  Next, a specific operation of the condensing optical system 700 having the above-described configuration will be described.
[0098]
  When the lens 80 is at the first lens position 80a, the optical paths 70a and 70b of the laser light pass through the lens 80 and travel like optical paths 71a and 71b, and the exit surface has a prism as indicated by reference numeral 81b. 81, travels like the optical paths 71c and 71d shown by dotted lines, converges by the lens 82, travels from the optical paths 71e and 71f shown by the dotted lines, like optical paths 71g and 71h, and reaches the focal position 85 of the sample 18. Focused.
[0099]
  When the lens 80 is at the second lens position 80b, the optical paths 70a and 70b of the laser light pass through the lens 80 and travel like optical paths 72a and 72b, and the exit surface is as indicated by reference numeral 81b. The light travels through the prism 81 and travels as the optical paths 72c and 72d. The light is collected by the lens 82, travels from the light paths 72e and 72f as the light paths 72g and 72h, and converges at the focal position 86 of the sample 18. .
[0100]
  In this way, in the condensing optical system 700 shown in FIG. 2A, the focal position in the specimen 18 is adjusted to the laser beam by controlling the movement of the position of the lens 80 in the traveling direction (optical axis direction) of the laser beam. Can be moved in the direction of travel (optical axis direction).
[0101]
  Next, the movement of the focal position in the specimen 18 in a direction orthogonal to the traveling direction (optical axis direction) of the laser light will be described.
[0102]
  In FIG. 2A, when the lens 80 is at the second lens position 80b, the laser light traveling through the lens 80 is formed by setting the exit surface of the prism 81 to a surface inclined as indicated by reference numeral 81a. The optical paths 72a and 72b pass through the prism 81, travel as the optical paths 72c ′ and 72d ′, and are collected by the lens 82 and travel from the optical paths 72e ′ and 72f ′ to the optical paths 72g ′ and 72h ′. The light is collected at the focal position 87 of the sample 18.
[0103]
  In FIG. 2A, when the lens 80 is at the second lens position 80b, the exit surface of the prism 81 has a tilted surface as indicated by reference numeral 81c, so that the laser light traveling through the lens 80 is transmitted. The light paths 72a and 72b pass through the prism 81 whose exit surface is indicated by reference numeral 81c, travel as the light paths 72c '' and 72d '', and are condensed by the lens 82 and light path 72e ''. , 72f ″ and light paths 72g ″, 72h ″, and the light is collected at the focal position 88 of the specimen 18.
[0104]
  That is, by changing the inclination of the exit surface of the prism 81 with respect to the optical path to the surfaces indicated by reference numerals 81a, 81b, and 81c, the focal positions in the specimen 18 are changed to lasers as indicated by reference numerals 87, 86, and 88, respectively. It can be moved in a direction orthogonal to the light traveling direction (optical axis direction).
[0105]
  In this way, in the condensing optical system 700 shown in FIG. 2A, the focal position of the specimen 18 is changed to the laser beam by changing the emitting surface of the prism 81 and changing the traveling direction (optical axis direction) of the laser beam. Can be moved in a direction perpendicular to the traveling direction (optical axis direction).
[0106]
  Next, the pulse stretcher of the fiber probe photodetector of the biological function measuring device according to the first embodiment will be described with reference to FIG.
[0107]
  In the figure, incident light 101 is a near-infrared ultrashort pulse laser beam emitted from a laser light source 11.
[0108]
  The inside of the pulse stretcher 140 is configured by arranging four prisms 142, 144, 146, and 148 at predetermined positions, and the incident light 101 incident on the pulse stretcher 12 is reversely dispersed from the pulse stretcher 140. The emitted light 120 becomes incident on the fiber side.
[0109]
  Here, the prism pair 150 including the first and second prisms 142 and 144 will be described.
[0110]
  The first and second prisms 142 and 144 constituting the prism pair 150 are a first prism surface 1 and a second prism surface 4, a first prism surface 2 and a second prism surface 3. By arranging them in parallel, the laser light emitted from the second prism can be made parallel to the incident laser light regardless of the wavelength.
[0111]
  Here, since the laser light 101 incident on the first prism 142 constituting the prism pair 150 is refracted by the first and second prisms 142 and 144 as much as the short wavelength component S, the laser beam 101 in the second prism 144 The optical path length is shortened.
[0112]
  On the contrary, the long wavelength component L of the laser beam 101 has a longer optical path length in the second prism 144, and therefore the short wavelength component S is emitted from the second prism 144 earlier and given reverse dispersion. .
[0113]
  The laser beam emitted from the second prism 144 is shifted depending on the wavelength.
[0114]
  Although the optical paths of the long wavelength component L and the short wavelength component S do not match, the third and fourth prisms 146 and 148 are similar to the prism pair 150 including the first and second prisms 142 and 144. By configuring the prism pairs 152 to be symmetrically arranged, the laser beams are combined and then incident on a fiber (not shown).
[0115]
  The pulse stretcher 140 described here is configured such that the position of the second prism 144 and the third prism 146 can move in the direction of the arrow.
[0116]
  In this way, by configuring so that the positions of the second and third prisms 144 and 146 can be moved, the optical path length difference of each wavelength component of the laser light can be changed.
[0117]
  Therefore, according to the pulse stretcher 140 described above, it is not necessary to redo the entire alignment even if the length of the fiber is changed or the wavelength of the laser beam is changed, and the position adjustment of the second and third prisms 144 and 146 can be performed. It can accommodate fiber length and wavelength.
[0118]
  That is, as shown in FIG. 1, laser light from a laser light source 11 that oscillates near-infrared ultrashort pulse laser light is input as incident light 101 to a pulse stretcher 140 as shown in FIG. 3 through mirrors 10a and 10b. The position adjustment of the prism with respect to the optical path passing through the four prisms 142, 144, 146, 148 of the pulse stretcher 140 is determined according to the condition of the incident light 101 and the condition of the single mode fiber 15, and the specimen 18 is observed. By doing so, it is possible to irradiate the specimen 18 with a pulse width close to the pulse width of the original laser beam, to efficiently cause a multiphoton excitation phenomenon on the specimen surface, and to obtain a specimen image efficiently. it can.
[0119]
  The configuration of the pulse stretcher 140 is not limited to the combination of prisms as described above. For example, the pulse stretcher 140 may be a combination of diffraction gratings, a combination of prisms and diffraction gratings, or a combination of mirrors. Good.
[0120]
  FIG. 4 is a diagram showing a probe assembly 17a including a condensing optical system 16 used in the fiber probe photodetector of the biological function measuring apparatus according to the first embodiment.
[0121]
  The light emitted from the end face of the fiber 15 is converted into parallel light by the lens 31a in the probe assembly 17a, and condensed at the condensing position 18a in the sample 18 by the lens 31b.
[0122]
  Reflected light or fluorescence from the condensing position 18a is incident on the end face of the fiber 15 again by the lens 31b and the lens 31b.
[0123]
  The flange portion 17b is screwed to the adjustment ring 17c, and the probe assembly 17a together with the lenses 31a and 31b is always urged toward the adjustment ring 17a by the spring 17d.
[0124]
  By rotating the adjusting ring 17c, the probe assembly 17c together with the lenses 31a and 31b can be moved with respect to the specimen 18 in the optical axis direction.
[0125]
  That is, the focal position 18a in the sample 18 can be moved in the optical axis direction.
[0126]
  Further, since the flange portion 17b is fixed to the skull 18b with a screw, the focal position 18a does not change during measurement.
[0127]
  Therefore, according to the present embodiment, a laser light source that oscillates with near-infrared ultrashort pulses, an optical system that causes a light beam from the laser light source to enter the fiber, and laser light emitted from the tip of the fiber as a sample. An optical system for condensing and irradiating, means for fixing the optical system to the specimen, and a photodetector for collecting reflected light or fluorescence emitted from the specimen by the optical system and detecting the reflected light or fluorescence And a pulse stretcher having a prism or a grating having a mechanism capable of changing either or both of an angle and a position with respect to the optical path between the laser light source and the fiber. The specimen can be irradiated with a pulse width close to the pulse width of the light, the multi-photon excitation phenomenon on the specimen surface can be caused efficiently, and the specimen image can be acquired efficiently. Kill.
[0128]
  In addition, for example, information in the brain when the animal is originally active can be obtained.
[0129]
  Further, the present invention is not limited to the above-described measurement in the brain of an animal, and can be applied to, for example, a micro measurement of a uniform or non-uniform suspension, plaque, solid, or living tissue. .
[0130]
  In addition, regarding the single mode fiber used in the above-described embodiment, an appropriate standard connector is provided at the connection end with the laser light source side, and the fiber used has a length of 5 m or more and is curved. Therefore, it is desirable to have a tolerance that does not cause an instantaneous mode shift.
[0131]
  The probe assembly may include a protective stainless steel housing having a diameter not exceeding 3 mm and a length not exceeding 10 mm, and a lens capable of condensing the laser light from the laser light source at a distance of at least 1 mm from the surface thereof. desirable.
[0132]
  Further, it is desirable to fix the optical fiber with sufficient strength to withstand a tension of 4 kg, to have resistance to water, solvent, and mechanical impact so that the weight does not exceed 1 g.
[0133]
  The probe assembly may be a terminal assembly.
[0134]
  Further, the biological function measuring device of the present invention includes a probe assembly screwed to an incised part of an animal skull as a specimen, an adjustment unit having an adjustment capability capable of being probed obliquely with respect to different parts of the cerebral cortex, A focal length condensing lens capable of measuring fluorescence emitted from brain tissue at a distance of 1000 μm from the surface, and the length of the optical fiber allowing an animal to move to a radius of 5 m with the probe assembly fixed It is desirable that the biological function measuring device is applied to the study of the brain of an animal that freely moves around as a biological tissue based on the detected fluorescence. It can also be used to analyze the presence and concentration of various chemical substances, and to identify chemical substances that have not yet been quantitatively revealed. Kill.
[0135]
  Further, the biological function measuring device of the present invention is not limited to the measurement as in the above-described embodiment, and the obtained result can realize an expected application, for example, a laser from a laser light source. By reducing the light intensity so that even cells can avoid damage, even cells that are examined in bacterial colonies can confirm the presence of molecules based on the fluorescence collected from a site of about 1 μm, It can also be applied to detecting molecules such as GFP that emit green fluorescence in the brain, serotonin, or specific brain cells.
[0136]
  The intensity of the laser light from the laser light source is made small enough to avoid damage even in cells examined in bacterial colonies, and the presence of molecules can be confirmed based on fluorescence collected from a site of about 1 μm, It is possible to detect molecules such as GFP which emit green fluorescence in serotonin and specific brain cells.
[0137]
  The above-described condensing optical system 700 is not limited to the above example, and at least a condensing unit and an optical path changing unit are arranged in the optical path of the laser beam emitted from the tip of the fiber, and the light is emitted from the fiber. Various adjustments can be made as long as the focal position of the laser beam can be adjusted.
[0138]
  The condensing optical system 700 shown in FIG. 2 is merely an example, but is arranged between at least two convex lenses as condensing means and an optical path change that can bend the optical axis of laser light. It is desirable to have a means.
[0139]
  Then, the focal position of the laser light emitted from the fiber is determined in a three-dimensional direction by appropriately combining means capable of realizing the above-described operation and performing control of lens position control and bending of the optical axis of the laser light by the optical path changing means. Can be adjusted appropriately.
[0140]
  The probe assembly 17a described above may be made of any material as long as it can be mechanically protected such as stainless steel, gold, and ceramic and has rust prevention performance.
[0141]
  The condensing unit and the optical path changing unit described above are not limited to lenses and prisms, and various types can be used. For example, depending on the application, as the optical path changing unit, the prism may be a reflector. An optical path changing means other than the one that directly controls the focal position may be added, or the optical path may be changed with a reflective member without using a transmissive prism.
[0142]
  Further, since the above-described condensing optical system can be incorporated like a chip at the tip of the fiber due to its configuration, it is not necessary to prepare a very large holder especially for the outer diameter of the fiber.
[0143]
  This is very effective when using the fiber probe photodetector of the biological function measuring device of the present invention. For example, by inserting a condensing optical system part inside the living body, the in vivo information of the desired part can be obtained. In addition to being able to be obtained, the reliability of measurement is high, its practical application is wide, and it greatly contributes to the development of the field of light detection devices and microscopes using them.
[0144]
  The adjustment of the arrangement conditions of the condensing means and the optical path changing means can be performed using an electric adjusting means, a hydraulic pressure adjusting means, an optical adjusting means, or the like. At least a part of the means is disposed in the vicinity of the tip of the fiber, the light collecting means, and the optical path changing means.
[0145]
  Further, it is desirable that the adjusting means described above is integrated and integrated in the same housing together with the tip of the fiber, the condensing means, the optical path changing means, and the like. A transmission path for transmitting adjustment information to at least a part of the arrangement condition adjusting means incorporated in the same housing together with the optical means, the optical path changing means, etc. is integrated with the fiber. It can be expected to greatly expand the application.
[0146]
  In addition, as the adjustment information of the adjusting means, many types of information such as electrical information, information for performing hydraulic pressure control, optical information, and the like can be used.
[0147]
  In addition, the above-described condensing optical system is not limited to application to a biological function measurement device, and the effect obtained by using the light detection device of the present invention and the biological function measurement device using the same is greatly increased. It is possible to greatly increase the performance of the biological function measuring device.
[0148]
  Further, in the above-described fiber probe photodetector using the condensing optical system and the biological function measuring apparatus using the photodetector, an ultrashort pulse laser beam (excitation light) oscillated from a laser light source is used as a single mode fiber. To the sample or the like using the same, the configuration in which the reflected light and fluorescence from the sample are guided to the light detection means using the same single mode fiber as the excitation light, and further between the laser light source and the single mode fiber, By configuring each device by arranging a pulse stretcher that causes the laser beam to generate a group velocity delay opposite to the group velocity delay that is received when the single mode fiber is transmitted with the ultrashort pulse laser beam. It is possible to effectively measure fluorescence by multiphoton absorption such as photon absorption.
[0149]
Appendix
The following is a description corresponding to claims 9 to 14 added by amendment as the claims 9 to 14 at the beginning of the application are deleted.
According to a first aspect of the present embodiment, a laser light source that emits near-infrared ultrashort pulse laser light, a probe assembly that includes a protective housing and a fiber coupling portion, and a fiber coupling portion between the laser light source and the probe assembly. An optical fiber, a condensing lens in the probe assembly, a holding unit that mechanically holds the probe assembly with respect to the specimen, and at least one of the probe assembly and the focal position provided in the holding unit A biological function measuring device comprising: an adjustment unit capable of adjusting the three in a three-dimensional direction; and a light branching unit that separates laser light from the laser light source and light from the sample.
In a second aspect, in the first aspect, the light branching unit is an optical system using a dichroic filter or a dichroic mirror, and the biological function measurement device includes a light detection device that detects light from the specimen, A computer for storing and processing data from the photodetection device; and a second optical fiber for guiding the light from the specimen separated by the optical system to the photodetection measurement device. The system separates the laser light from the laser light source and the light from the sample, and guides the separated light from the sample to the light detection and measurement device through the second optical fiber.
In a third aspect, in the first or second aspect, the holding unit adjusts the focal position in the Z-axis direction with an XY adjustment micrometer capable of adjusting a focal position within a range of ± 100 μm on the XY plane. It consists of a shaft adjusting micrometer, and the adjusting portion is configured so that a flange having a screw hole formed on the bottom surface thereof is brought into contact with the specimen and can be fixed with screws.
In a fourth aspect, in any one of the first to third aspects, the optical fiber is a single mode fiber capable of supporting a length of up to 5 m that does not cause an instantaneous mode shift due to bending, and the laser light source The probe assembly includes a stainless steel housing and a lens capable of condensing the laser light from the laser light source at a distance of at least 1 mm from the surface thereof.
In a fifth aspect, according to any one of the first to fourth aspects, the probe assembly is screwed to the incised part of the animal skull as the specimen, and the adjustment unit is a cerebral cortex. The condensing lens has a focal length capable of measuring fluorescence emitted from brain tissue at a distance of 1000 μm from the surface, and the optical fiber is connected to the animal by the animal. It is possible to move to a radius of 5 m with the probe assembly fixed.
In a sixth aspect, in any one of the first to fifth aspects, the biological function measuring device is applied to a microscopic measurement of a suspension, a plaque, a solid, or a biological tissue.
[0150]
【The invention's effect】
  As described above in detail, according to the present invention, it is possible to provide a biological function measuring device capable of stable observation under conditions where an observation method using a normal microscope cannot be applied, for example, in a state where an animal is active. .
[Brief description of the drawings]
FIG. 1 is a diagram showing a fiber probe photodetector of a biological function measuring device according to a first embodiment of the present invention.
FIG. 2 is a diagram showing a condensing optical system of the biological function measuring device according to the first embodiment of the present invention.
FIG. 3 is a view showing a modification of the pulse stretcher according to the first embodiment of the present invention.
FIG. 4 is a diagram showing a condensing optical system used in the fiber probe photodetector of the biological function measuring device according to the first embodiment.
FIG. 5 is a diagram showing a scanning confocal microscope apparatus disclosed in a first prior example.
FIG. 6 is a diagram showing an ultrashort optical pulse transmission device disclosed in a second prior example.
[Explanation of symbols]
  1 ... Laser light source,
  2 ... Pulse stretcher,
  3 ... Fiber,
  4 ... Pulse compressor optical system,
  5 ... Optical device,
  10a, 10b ... mirror,
  11 ... Laser light source,
  12 ... Pulse stretcher,
  13 ... Dichroic mirror,
  14 ... Condensing lens,
  15 ... Fiber,
  16 ... Condensing optical system,
  17a ... probe assembly,
  17b ... flange part,
  17c ... adjustment ring,
  17d ... Spring,
  18 ... specimen,
  18a ... Focus position,
  18b ... skull,
  19 ... filter,
  20 ... Lens,
  21 ... Fiber,
  22 ... Lens,
  23 ... Dichroic mirror,
  24a, 24b ... photometric filter,
  25a, 25b ... photoelectric conversion elements,
  26 ... detection system,
  31a, 31b ... lenses,
  51 ... Laser light,
  52 ... Lens,
  53 ... Fiber,
  54 ... coupler,
  55 ... Fiber,
  56 ... Housing,
  57 ... retainer,
  58 ... Sample,
  58a: focal position,
  59 ... Piezoelectric element,
  60a, 60b ... lenses,
  61 ... Fiber,
  62 ... filter,
  63 ... photoelectric conversion element,
  64 ... computer,
  65 ... Driver,
  66 ... Monitor,
  67 ... Cable,
  101: Incident light,
  102 ... right angle prism,
  104: diffraction grating,
  106 ... right angle prism,
  108 ... Mirror,
  110 ... convex mirror,
  112 ... concave mirror,
  114 ... mirror,
  116 ... Mirror,
  118 ... Mirror,
  120 ... outgoing light,
  130 ... Arrow,
  140 ... pulse stretcher,
  142 ... first prism,
  144 ... second prism,
  146 ... the third prism,
  148 ... Fourth prism,
  150 ... Prism pair,
  152 ... Prism pair.

Claims (1)

  Emits ultrashort pulsed laser light that causes multiphoton excitation,
  Transmitting the ultrashort pulse laser light to a probe assembly fixed to contact the specimen using a single mode optical fiber;
  By the optical system of the probe assembly, the transmitted ultrashort pulse laser beam is focused on the specimen,
  Transmitting the fluorescence from a specimen generated by multi-photon excitation based on irradiation of the ultrashort pulse laser light to a photodetector through the probe assembly and the single mode optical fiber;
  By acquiring the information in the brain of the specimen by measuring the fluorescence with the photodetector,
  The optical system of the probe assembly has a focal length capable of measuring fluorescence emitted from a distance of up to 1000 μm from the surface of the specimen. In the biological function measuring apparatus used for observing moving animals (excluding humans) Biological function measurement method.

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