JP2011196873A - Fluorescence detection method and fluorescence detection device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescence detection device capable of detecting fluorescence generated from an observation object with a high SN ratio, without enlargement, complication and cost increase of the device.SOLUTION: In this fluorescence detection device 1 for irradiating the observation object 115 with excitation light emitted from an excitation light source 102, and amplifying optically fluorescence generated from the observation object 115 by an optical amplifier 110 to detect it, the excitation light emitted from the excitation light source 102 is allowed to enter the optical amplifier 110, and the fluorescence generated from the observation object 115 is optically amplified.

Description

  The present invention relates to a fluorescence detection method for detecting fluorescence emitted from an observation target such as a living body, and a fluorescence detection apparatus for implementing the method.

  Conventionally, fluorescence detection apparatuses such as a fluorescence microscope for observing a fluorescence phenomenon from a living body or a non-biological material, and a fluorescence endoscope for observing fluorescence emitted from a fluorescent material in a living tissue are known. However, the fluorescence emitted from a substance is much weaker than the excitation light irradiated to generate the fluorescence. For this reason, an image obtained with a fluorescence microscope or a fluorescence endoscope is extremely dark when compared with an image obtained by another illumination method, for example, a transmitted image of Koehler illumination in a microscope or a reflected image of white light illumination in an endoscope, The observation is generally more difficult.

  As one of the solutions, it is conceivable to obtain a bright fluorescent image by strengthening the excitation light applied to the observation target. As another solution, it is conceivable to amplify an electric signal from a photodetector that photoelectrically converts fluorescence with a high gain amplifier.

  However, when the excitation light is irradiated to the observation target as in the former case, the observation target, that is, the specimen in the microscope or the patient's body in the endoscope is damaged. Also, as in the latter case, when an electric signal from a photodetector that photoelectrically converts fluorescence is amplified by a high gain amplifier, a certain amount of noise is inevitably generated at the stage of photoelectric conversion, and added to the signal. Even if amplified by a high gain amplifier at the subsequent stage, a signal that is so small that it is buried in the noise added by photoelectric conversion cannot be extracted.

  On the other hand, as a conventional microscope, for example, a microscope in which scattered light emitted from an observation target is amplified and detected by an optical amplifier (see, for example, Patent Document 1). According to such a microscope, even if the scattered light is smaller than the noise generated by the photoelectric conversion, the scattered light is optically amplified by an optical amplifier to a level higher than the noise and then photoelectrically converted. The ratio can be improved and smaller scattered light can be extracted.

  Therefore, if such an optical amplifier is applied to a fluorescence detection apparatus so that weak fluorescence generated from the observation target is detected after being amplified by the optical amplifier, the intensity of the excitation light applied to the observation target can be increased. It is possible to obtain a brighter and better fluorescent image by taking out the weak fluorescence generated by it as an electrical signal with a high signal-to-noise ratio without burying it in noise, while suppressing the observation target to a level that does not damage the object. become.

US Pat. No. 6,351,325

  However, in order to amplify fluorescence with an optical amplifier, a light source that excites a gain medium that is the main component of optical amplification is required. As a typical light source, use of a laser oscillator is assumed. Moreover, many fluorescence detection apparatuses are provided with a laser oscillator as an excitation light source to be observed, for example, as represented by a laser scanning fluorescence microscope.

  For this reason, when the technology disclosed in Patent Document 1 is simply applied to a conventional fluorescence detection apparatus so as to optically amplify the fluorescence, a laser oscillator that excites the observation target and a gain medium of the optical amplifier are excited. This requires two excitation light sources with the laser oscillator, which leads to an increase in size, complexity and cost of the entire apparatus.

  This invention solves this subject and provides the fluorescence detection method and fluorescence detection apparatus which can detect the fluorescence which generate | occur | produces from an observation object by a high S / N ratio, without causing the enlargement of an apparatus, complexity, and a cost increase. is there.

In order to solve the above-described problem, the fluorescence detection method according to the present invention irradiates an observation target with excitation light emitted from an excitation light source, and detects the fluorescence generated from the observation target by optical amplification using an optical amplifier.
Excitation light emitted from the excitation light source is made incident on the optical amplifier, and fluorescence generated from the observation target is optically amplified.

  According to the present invention, a part of the excitation light emitted from one excitation light source contributes to the excitation of the observation target, and the remaining part contributes to the optical amplification. Therefore, since one excitation light source can be shared for illumination and light amplification for observation, compared with the case where they are used as separate excitation light sources, downsizing of the apparatus, simplification of configuration, It is possible to detect the fluorescence generated from the observation target with a high S / N ratio while reducing the cost.

  In the fluorescence detection method according to an embodiment of the present invention, the observation target is irradiated with excitation light emitted from the excitation light source via the optical amplifier.

  According to the present invention, a part of the excitation light emitted from the excitation light source is absorbed by the optical amplifier, the gain medium constituting the optical amplifier is excited, and observation is performed by the remaining excitation light that is not absorbed by the gain medium. The object is excited. Then, the fluorescence generated from the observation target enters the optical amplifier and is optically amplified.

  As described above, irradiating the observation target with the excitation light through the optical amplifier means that the optical amplifier is arranged and used in the optical path of the excitation light with respect to the observation target. This method has the advantage of avoiding an increase in the optical path diameter since the excitation optical path to be observed and the excitation optical path of the gain medium are made common, and can be suitably used for an endoscope, for example. .

  In the fluorescence detection method according to an embodiment of the present invention, the excitation light emitted from the excitation light source is separated into at least two excitation lights, and the observation target is irradiated with the separated excitation light. The other separated pumping light is incident on the optical amplifier.

  According to the present invention, the observation object is excited by one of the separated excitation lights, the gain medium constituting the optical amplifier is excited by the other excitation light, and the fluorescence generated from the observation object is incident on the optical amplifier to generate light. Amplified.

  In this way, by separating the excitation light emitted from the excitation light source for observation object and for light amplification, the separated excitation light is individually controlled, for example, with respect to intensity, beam diameter, irradiation direction, etc. It is possible to improve the degree of freedom in setting the illumination conditions.

Furthermore, in order to solve the above-described problem, the fluorescence detection apparatus according to the present invention irradiates an observation target with excitation light emitted from an excitation light source, and detects the fluorescence generated from the observation target by optical amplification using an optical amplifier In the fluorescence detection device,
Excitation light emitted from the excitation light source is incident on the optical amplifier, and fluorescence generated from the observation target is optically amplified.

  According to the present invention, the excitation light source functions as a common light source for the illumination of the observation target and the light amplification of the fluorescence generated from the observation target, and a part of the excitation light emitted from the excitation light source is the observation target. The remaining part of the light contributes to the optical amplification.

  Thus, by sharing a single excitation light source for illumination and light amplification for observation, compared to the case where they are used as separate excitation light sources, the apparatus can be downsized and the configuration simplified. It is possible to detect the fluorescence generated from the observation target with a high S / N ratio while reducing the cost.

  In the fluorescence detection apparatus according to an embodiment of the present invention, a light amount adjustment unit that adjusts a light amount ratio between the excitation light irradiated to the observation target and the excitation light for optically amplifying the fluorescence by the optical amplifier. Prepare.

  According to the present invention, the light amount adjustment unit can optimize the light amount ratio between the excitation light applied to the observation target and the excitation light for optically amplifying the fluorescence by the optical amplifier. Thus, for example, relatively weak excitation illumination is used so as not to damage the observation target, and relatively strong excitation illumination is used so that sufficient gain and SN ratio can be obtained for the optical amplifier. it can. In addition, the light quantity ratio can be set optimally according to the purpose of observation and the type of observation target.

  In the fluorescence detection apparatus according to an embodiment of the present invention, the optical amplifier is configured to include an optical fiber to which a gain medium is added.

  With this configuration, for example, an optical fiber to which a gain medium is added is optically connected between an objective lens located near the observation target and an apparatus main body having an excitation light source located far from the observation target. By connecting, the optical fiber has three functions: transmission of excitation light from the apparatus main body to the objective lens, transmission of fluorescence from the objective lens to the apparatus main body, and light amplification of the fluorescence. It becomes possible.

  In the fluorescence detection device according to an embodiment of the present invention, the optical fiber to which the gain medium is added extends to the insertion portion of the endoscope.

  If constituted in this way, it becomes possible to constitute easily an endoscope type fluorescence microscope, a fluorescence endoscope, a fiberscope type fluorescence endoscope, etc., for example.

  In the fluorescence detection apparatus according to an embodiment of the present invention, the optical amplifier includes a bulk optical element to which a gain medium is added.

  If comprised in this way, since a bulk-shaped optical element can be manufactured easily, the further cost reduction can be aimed at.

  According to the present invention, it is possible to detect fluorescence generated from an observation object with a high S / N ratio without increasing the size, complexity, and cost of the apparatus.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows schematic structure of the endoscope type fluorescence microscope which concerns on 1st Embodiment of this invention. It is a figure for demonstrating the optical characteristic of the excitation light intensity balance filter shown in FIG. It is a figure which shows schematic structure of the fluorescence endoscope which concerns on 2nd Embodiment of this invention. It is a figure which shows schematic structure of the fiberscope type | mold fluorescence endoscope which concerns on 3rd Embodiment of this invention. It is a figure which shows schematic structure of the fluorescence microscope which concerns on 4th Embodiment of this invention. It is a figure for demonstrating the optical characteristic of the dichroic mirror shown in FIG. It is a figure which shows schematic structure of the total reflection fluorescence microscope which concerns on 5th Embodiment of this invention. It is a figure which shows schematic structure of the Niipou disc type | mold confocal fluorescence microscope which concerns on 6th Embodiment of this invention. It is a figure which shows schematic structure of the fluorescence macro observation apparatus for experimental small animals which concerns on 7th Embodiment of this invention.

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

(First embodiment)
FIG. 1 is a diagram showing a schematic configuration of the fluorescence detection apparatus according to the first embodiment of the present invention. The fluorescence detection apparatus is configured as an endoscope type fluorescence microscope 1 and includes a main body 100 and a probe unit 101 which is an insertion unit. In the main body 100, an excitation light source 102, a collimating lens 103, a dichroic mirror 104, a first relay lens 105, an excitation light cut filter 106, an imaging lens 107, and a photodetector 108 are arranged. The probe portion 101 includes a flexible portion 101a and a tip portion 101b. The flexible portion 101a is provided with an optical fiber 110 that constitutes an optical amplifier extending, and the tip portion 101b includes a first portion. The second relay lens 111, the excitation light intensity balance filter 112 constituting the light amount adjustment unit, the scanner 113, and the microscope objective lens 114 are arranged.

  In FIG. 1, the excitation light emitted from the excitation light source 102 of the main body 100 is collimated by the collimator lens 103 and reflected by the dichroic mirror 104, and then passes through the first relay lens 105 and the optical fiber 110 of the probe unit 101. Is incident on. A gain medium for amplifying fluorescence is added to the optical fiber 110, and a part of the excitation light incident on the optical fiber 110 is absorbed by the optical fiber 110 to excite the gain medium. The remaining excitation light that has not been absorbed is emitted from the optical fiber 110 and enters the excitation light intensity balance filter 112 via the second relay lens 111.

  The excitation light intensity balance filter 112 transmits incident excitation light at a predetermined rate. Then, the excitation light transmitted through the excitation light intensity balance filter 112 is condensed on the observation object 115 by the microscope objective lens 114 through the scanner 113. Thereby, the molecules constituting the condensing site of the observation target 115 or the fluorescent dye contained in the condensing site are excited to generate fluorescence.

  The fluorescence generated from the observation target 115 enters the optical fiber 110 through the microscope objective lens 114, the scanner 113, the excitation light intensity balance filter 112, and the second relay lens 111. Here, since the gain medium is already excited in the forward path, the optical fiber 110 causes stimulated emission when fluorescence enters, and amplifies and emits the incident fluorescence.

  Fluorescence emitted after being amplified by the optical fiber 110 is incident on the dichroic mirror 104 via the first relay lens 105. The fluorescence that has passed through the dichroic mirror 104 enters the excitation light cut filter 106, where unnecessary excitation light included in the incident light is cut, and is condensed on the photodetector 108 by the imaging lens 107 and subjected to photoelectric conversion. Is done. Then, on the probe unit 101 side, as the excitation light scans the observation target 115 by the action of the scanner 113, a signal reflecting the fluorescence intensity distribution of the observation target 115 is obtained.

In the above configuration, the gain medium added to the optical fiber 110 may be various elements such as praseodymium (Pr), neodymium (Nd), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb). Terbium (Tb) and the like can be used, but praseodymium, holmium, erbium, thulium and terbium are particularly effective when amplifying fluorescence in the visible region. The scanner 113 only needs to be capable of optical scanning. For example, the scanner 113 can be configured using a MEMS (micro electro mechanical systems) type galvanomirror, and the end of the optical fiber 110 on the objective lens side is connected by an actuator. It can also be configured to drive in a direction orthogonal to the optical axis. In FIG. 1, each of the optical paths to be scanned by the scanner 113, the on-axis optical path indicated by reference numeral 116 _CT, shows Jikugaihikariro by reference numeral 116 _PR.

FIG. 2 is a diagram for explaining the optical characteristics of the excitation light intensity balance filter 112. FIG. 2A shows the spectral distribution of excitation light and fluorescence incident on the excitation light intensity balance filter 112, and FIG. ) Shows the spectral transmittance characteristics of the excitation light intensity balance filter 112. In FIG. 2 (a), the spectral intensity of the fluorescence is highlighted for the sake of clarity. As shown in FIG. 2, the excitation light intensity balance filter 112 has a relatively low transmittance T _EX at the excitation light wavelength λ _EX (for example, 488 nm), and conversely, the fluorescence wavelength band λ _{FL1 to} λ _FL2 (for example, , 500 nm to 600 nm), the transmittance T _FL is configured to be relatively high. Such an excitation light intensity balance filter 112 can be composed of, for example, a dielectric multilayer filter such as a dichroic filter, a tunable filter, a Fabry-Perot interferometer, or the like.

In this way, by configuring the excitation light intensity balance filter 112 and disposing the excitation light intensity balance filter 112 between the optical fiber 110 and the observation target 115, of the excitation light emitted from the excitation light source 102. While exciting a relatively high ratio by being incident on the optical fiber 110, a relatively low ratio of excitation light limited by the transmittance T _EX of the excitation light transmitted through the optical fiber 110 is applied to the observation target 115. Can be irradiated. Further, most of the fluorescence emitted from the observation object 115 can be incident on the optical fiber 110 by the transmittance T _FL in the excitation light intensity balance filter 118.

  Accordingly, the excitation light that irradiates the observation target 115 is moderately weakened and the damage to the observation target 115 due to the excitation light (for example, fading of the fluorescent dye) is suppressed without substantially hindering the excitation and fluorescence amplification functions of the optical fiber 110. Can be reduced. That is, it is possible to optimize the light quantity ratio between the excitation light irradiated on the observation object 115 and the excitation light that is incident on the optical fiber 110 and optically amplifies the fluorescence.

  As described above, according to the endoscope fluorescent microscope 1 according to the present embodiment, the excitation light emitted from one excitation light source 102 is used for illuminating the observation object 115 and for amplifying light by the optical fiber 110. Therefore, even when the fluorescence generated from the observation object 115 is weak, the optical fiber 110 is used to amplify the signal-to-noise ratio without increasing the size, complexity, and cost of the apparatus. A good image can be obtained. In addition, the bending action of the flexible part 101a can arbitrarily change the position and direction of the distal end part 101b without moving the main body part 100, and the probe part 101 can be used in a narrowed part of a living body or a lumen / inner body. Since it can be put into the cavity, it can be easily inserted into the body of a laboratory animal or a small experimental animal for fluorescence observation, for example. Therefore, according to the present embodiment, it is possible to provide an endoscopic fluorescence microscope 1 that can observe fluorescence microscope images of tissues and cells in a living body with high sensitivity, and in particular, it is used for medical and biological research. be able to.

(Second Embodiment)
FIG. 3 is a diagram showing a schematic configuration of a fluorescence detection apparatus according to the second embodiment of the present invention. This fluorescence detection apparatus is configured as a fluorescence endoscope 2. The fluorescence endoscope 2 is a medical device that performs diagnosis by observing autofluorescence of a region to be examined or fluorescence emitted from a fluorescent dye that specifically binds to a lesion. An endoscope objective lens is used as an objective lens. Except for using 201, the overall configuration and the operation of each part are basically the same as those of the endoscope type fluorescence microscope 1 shown in FIG. Therefore, the same reference numerals are given to the components having the same action as the components shown in FIG.

  Also in the fluorescence endoscope 2 according to the present embodiment, as in the first embodiment, the excitation light emitted from one excitation light source 102 is used to increase the size, complexity, and cost of the apparatus. Without inviting, even if the fluorescence generated from the observation target 202 is weak, it can be amplified by the optical fiber 110 to obtain an image with a good SN ratio.

(Third embodiment)
FIG. 4 is a diagram showing a schematic configuration of a fluorescence detection apparatus according to the third embodiment of the present invention. This fluorescence detection apparatus is configured as a fiberscope type fluorescence endoscope 3 and includes an endoscope main body part 300 and a probe part 301 as an insertion part. In the endoscope body 300, an excitation light source 302, an excitation light condensing lens 303, a dichroic mirror 304, a relay lens 305, an excitation light cut filter 306, an imaging lens 307, and an image sensor 308 are arranged. The probe portion 301 includes a flexible portion 301a and a tip portion 301b. An optical fiber bundle 310 formed by bundling a number of optical fibers is extended and arranged on the flexible portion 301a. The excitation light intensity balance filter 312 and the endoscope objective lens 314 are arranged.

  In FIG. 4, the excitation light emitted from the excitation light source 302 of the endoscope main body 300 is collected by the excitation light condensing lens 303 and reflected by the dichroic mirror 304, and then passes through the relay lens 305 and the probe unit 301. The optical fiber bundle 310 is incident on the entire end surface of the rear end portion 310a. In the optical fiber bundle 310, each optical fiber constitutes an optical amplifier, and a gain medium for amplifying fluorescence similar to that of the optical fiber 110 in the first embodiment is added to each optical fiber. . A part of the pumping light incident on the optical fiber bundle 310 is absorbed by each optical fiber to pump the gain medium, and the remaining pumping light that has not been absorbed passes through the tip 310b of the optical fiber bundle 310. The light exits from the end face and enters the excitation light intensity balance filter 312.

  The excitation light intensity balance filter 312 constitutes a light amount adjustment unit, is configured in the same manner as the excitation light intensity balance filter 112 in the first embodiment, and transmits incident excitation light at a predetermined ratio. Then, the excitation light transmitted through the excitation light intensity balance filter 312 illuminates an illumination region (field of view) 316 corresponding to the image observation region of the observation target 315 by the endoscope objective lens 314. As a result, the molecules in the illumination area 316 of the observation object 315 or the fluorescent dye contained in the illumination area is excited to generate fluorescence.

  Fluorescence generated from the illumination region 316 of the observation target 315 is condensed by the endoscope objective lens 314 through the excitation light intensity balance filter 312 onto the end surface of the distal end portion 310b of the optical fiber bundle 310, and is irradiated onto the distal end surface. 316 fluorescent images are formed. Then, the fluorescent image formed on the front end surface is optically amplified and transmitted through the optical fiber bundle 310, whereby a light amplified fluorescent image is formed on the end surface of the rear end portion 310a.

  The light-amplified fluorescent image formed on the end surface of the rear end portion 310a is incident on the excitation light cut filter 306 via the relay lens 305 and the dichroic mirror 304, and unnecessary excitation light included in the fluorescence image is cut off here. Then, an image is formed on the image sensor 308 by the imaging lens 307 and subjected to photoelectric conversion.

  Therefore, in this embodiment as well, in the same way as in the above embodiment, by using excitation light emitted from one excitation light source 302, an observation target can be obtained without increasing the size, complexity, and cost of the apparatus. Even when the fluorescent image 315 is weak, it is possible to obtain an image with a good S / N ratio by optical amplification with the optical fiber bundle 310.

(Fourth embodiment)
FIG. 5 is a diagram showing a schematic configuration of a fluorescence detection apparatus according to the fourth embodiment of the present invention. This fluorescence detection apparatus is configured as a fluorescence microscope 4 and has one excitation light source 401 as in the above-described embodiment. In FIG. 5, the excitation light emitted from the excitation light source 401 is collimated by the first collimating lens 402, enters the dichroic mirror 403, is transmitted and reflected by the dichroic mirror 403, and is separated into two excitation lights. .

  One excitation light transmitted through the dichroic mirror 403 is reflected by the reflection mirror 404 and is incident on the dichroic mirror 403 again. The excitation light reflected here passes through the scanner 405 and the pupil relay lens 406 by the microscope objective lens 407. Focused on the observation object 408. As a result, the molecules constituting the condensing site of the observation target 408 or the fluorescent dye contained in the condensing site are excited to generate fluorescence.

  The fluorescence generated from the observation object 408 is collected by the microscope objective lens 407 and enters the dichroic mirror 403 through the pupil relay lens 406 and the scanner 405. Then, the fluorescence that has entered the dichroic mirror 403 passes through the dichroic mirror 403, passes through the first imaging lens 410, the first confocal pinhole 411, and the second collimating lens 412, and is added with a gain medium. The light enters the bulk optical element 413 thus formed. Note that the first confocal pinhole 411 is a pinhole for performing three-dimensional measurement of the observation target 408 by blocking light from other than the focus of the microscope objective lens 407.

  On the other hand, the other excitation light emitted from the excitation light source 401 and reflected by the dichroic mirror 403 passes through the first imaging lens 410, the first confocal pinhole 411, and the second collimating lens 412, and the bulk optical The light enters the element 413 and excites the gain medium of the bulk optical element 413. As a result, the fluorescence from the observation object 408 that has entered the bulk optical element 413 is amplified and emitted from the bulk optical element 413.

The fluorescence emitted from the bulk-like optical element 413 is condensed by the second imaging lens 415 to the second confocal pinhole 416 after unnecessary excitation light is cut by the excitation light cut filter 414, Undesired spontaneously emitted light generated from the bulk optical element 413 is shielded, and the fluorescence transmitted through the second confocal pinhole 416 is condensed on the photodetector 418 by the third imaging lens 417 and photoelectrically detected. Converted. As the excitation light irradiated onto the observation object 408 is scanned by the scanner 405, a signal reflecting the fluorescence intensity distribution of the observation object 408 is obtained. In FIG. 5, among the optical paths scanned by the scanner 405, the on-axis optical path is indicated by reference numeral 419 _CT , and the off-axis optical path is indicated by reference numeral 419 _PR .

  Here, the dichroic mirror 403 constitutes a light amount adjustment unit that optimizes the light amount ratio between the excitation light irradiated on the observation target 408 and the excitation light that optically amplifies the fluorescence by the bulk optical element 413. FIG. 6 is a diagram for explaining the optical characteristics of the dichroic mirror 403. FIG. 6 (a) shows the spectral distribution of excitation light and fluorescence incident on the dichroic mirror 403, and FIGS. 6 (b) and 6 (c). Indicates the spectral reflectance characteristics and spectral transmittance characteristics of the dichroic mirror 403, respectively. In FIG. 6 (a), the spectral intensity of the fluorescence is highlighted for the sake of clarity.

As is apparent from FIG. 6, the dichroic mirror 403 has a relatively high reflectance R _EX at the wavelength λ _EX of the excitation light, and conversely, the reflectance R _FL at the fluorescence wavelength band λ _{FL1 to} λ _FL2 is as low as possible. Configure to be As a result, the transmittance T _{EX at} the wavelength λ _EX of the excitation light is relatively low, and conversely, the transmittance T _FL in the fluorescence wavelength band λ _{FL1 to} λ _FL2 is high.

With such a configuration, among the excitation light emitted from one excitation light source 401, dichroic mirror 403 that reflects once, i.e. used as a light amplifying R _EX equivalent to the ratio, also the dichroic mirror What is transmitted once at 403 and reflected once, that is, the portion equivalent to T _EX × R _EX can be used for illuminating the observation object 408. This makes it possible to optimize the light quantity ratio between the excitation light irradiated to the observation target and the excitation light for optically amplifying the fluorescence by the optical amplifier. Therefore, similarly to the above-described embodiment, the bulk optical element 413 amplifies the signal to reduce the SN ratio even when the fluorescence from the observation target 408 is weak, without increasing the size, complexity, and cost of the apparatus. Good images can be obtained.

(Fifth embodiment)
FIG. 7 is a diagram showing a schematic configuration of a fluorescence detection apparatus according to the fifth embodiment of the present invention. This fluorescence detection apparatus is configured as a total reflection fluorescence microscope 5 and has one excitation light source 501 as in the above-described embodiment. In FIG. 7, the excitation light emitted from the excitation light source 501 is collimated by the first collimator lens 502 and enters the beam splitter 503, and is transmitted and reflected by the beam splitter 503 to be separated into two excitation lights. .

  One excitation light that has passed through the beam splitter 503 passes through the reflection mirror 504, the condenser lens system 505, and the reflection mirror 506, and is irradiated to the sample container 509 that accommodates the observation target 508 from the peripheral edge of the pupil of the microscope objective lens 507. . At this time, the illumination light satisfies a condition for causing total reflection between the sample container 509 and the observation object 508. The observation object 508 is illuminated by the evanescent light generated by the total reflection of the illumination light, and the fluorescence is excited. Then, the fluorescence generated from the observation object 508 is collected by the microscope objective lens 507 and is collected by the condenser lens 510 in the bulk optical element 511 to which the gain medium is added.

  On the other hand, the other excitation light emitted from the excitation light source 501 and reflected by the beam splitter 503 enters the bulk optical element 511 and excites the gain medium of the bulk optical element 511. As a result, the fluorescence from the observation target 508 that has entered the bulk optical element 511 is optically amplified and emitted from the bulk optical element 511. Then, the fluorescence emitted from the bulk optical element 511 is imaged on the imaging element 515 by the imaging lens 514 via the second collimating lens 512 and the excitation light cut filter 513.

  Here, the beam splitter 503 constitutes a light amount adjusting unit that optimizes the light amount ratio between the excitation light that totally illuminates the observation target 508 and the excitation light that amplifies the fluorescence by the bulk optical element 511. Thereby, also in this Embodiment, the effect similar to the said embodiment can be acquired.

(Sixth embodiment)
FIG. 8 is a diagram showing a schematic configuration of a fluorescence detection apparatus according to the sixth embodiment of the present invention. This fluorescence detection apparatus is configured as a Nipkow disk type confocal fluorescence microscope 6 and has one excitation light source 601 as in the above-described embodiment. In FIG. 8, the excitation light emitted from the excitation light source 601 is collimated by the first collimating lens 602, and the gain medium is added through the microlens array disk 603, the dichroic mirror 604, and the pinhole array disk 605. The light enters the bulk optical element 606.

  A number of microlenses are arranged in a spiral on the microlens array disk 603. The pinhole array disk 605 is the same as a so-called nippo disk, in which a large number of pinholes paired with a large number of microlenses of the microlens array disk 603 are similarly arranged in a spiral shape. . These microlens array disk 603 and pinhole array disk 605 are rotated together by a motor 607.

  A part of the excitation light that has entered the bulk optical element 606 is absorbed by the bulk optical element 606 to excite the gain medium, and the remaining excitation light that has not been absorbed from the bulk optical element 606. The light is emitted and enters the excitation light intensity balance filter 609 through the second collimating lens 608. The excitation light intensity balance filter 609 constitutes a light amount adjustment unit, and transmits incident excitation light at a predetermined ratio, similarly to the excitation light intensity balance filters of the first to third embodiments. Then, the excitation light transmitted through the excitation light intensity balance filter 609 is condensed on the observation object 611 by the microscope objective lens 610. As a result, the molecules constituting the condensing site of the observation target 611 or the fluorescent dye contained in the condensing site are excited to generate fluorescence.

  The fluorescence generated from the observation object 611 is incident on the bulk optical element 606 through the microscope objective lens 610, the excitation light intensity balance filter 609, and the second collimating lens 608, and is amplified. The light-amplified fluorescence is reflected by the dichroic mirror 604 via the pinhole array disk 605 and incident on the excitation light cut filter 613 via the third collimating lens 612, where unnecessary excitation light is cut off. The image is formed on the image sensor 615 by the imaging lens 614. Then, as the observation object 611 is scanned by the rotation of the microlens array disk 603 and the pinhole array disk 605 by the motor 607, a fluorescence image reflecting the fluorescence intensity distribution of the observation object 611 is obtained.

  Therefore, also in this embodiment, the excitation light emitted from one excitation light source 601 is used for illuminating the observation object 611 and for amplifying the fluorescence generated from the observation object 611 as in the above embodiment. Therefore, even if the fluorescence from the observation object 611 is weak, the bulk optical element 606 can perform optical amplification to obtain an image with a good S / N ratio without increasing the size, complexity, and cost of the apparatus. be able to.

(Seventh embodiment)
FIG. 9 is a diagram showing a schematic configuration of a fluorescence detection apparatus according to the seventh embodiment of the present invention. This fluorescence detection apparatus is configured as a fluorescence macro observation apparatus 7 for experimental small animals, and has one excitation light source 701 as in the above-described embodiment. In FIG. 9, the excitation light emitted from the excitation light source 701 is collected by the excitation light condensing lens 702, reflected by the dichroic mirror 703, and then incident on the bulk optical element 704 to which a gain medium is added. .

  A part of the excitation light incident on the bulk optical element 704 is absorbed by the bulk optical element 704 to excite the gain medium, and the remaining excitation light that has not been absorbed is transmitted from the bulk optical element 704. It is emitted and enters the excitation light intensity balance filter 705. The excitation light intensity balance filter 705 constitutes a light amount adjustment unit, and transmits the incident excitation light at a predetermined ratio, like the excitation light intensity balance filter 609 of the sixth embodiment. Then, the excitation light transmitted through the excitation light intensity balance filter 705 illuminates the image observation region of the observation object 707 that is an experimental small animal by the objective lens 706. Thereby, the observation object 707 is excited and fluorescence is generated.

  The fluorescence generated from the observation object 707 enters the bulk optical element 704 through the objective lens 706 and the excitation light intensity balance filter 705, and is optically amplified. The optically amplified fluorescence passes through the dichroic mirror 703, and After unnecessary excitation light is cut by the excitation light cut filter 708, it is imaged on the image sensor 710 by the imaging lens 709 and subjected to photoelectric conversion.

  As described above, in this embodiment as well, the excitation light emitted from one excitation light source 701 is used for illuminating the observation target 707 and light amplification of the fluorescence generated from the observation target 707 as in the above embodiment. Therefore, even when the fluorescence from the observation object 707 is weak, the bulk optical element 704 amplifies the image without causing an increase in size, complexity and cost of the apparatus, and an image with a good SN ratio. Can be obtained.

  In addition, this invention is not limited only to the said embodiment, Many deformation | transformation or a change is possible. For example, the excitation light intensity balance filter and dichroic mirror constituting the light amount adjustment unit are exchanged or switched depending on the purpose of observation, the type of observation target, the wavelength of excitation light to be used, the wavelength of fluorescence to be detected, etc. It can be configured to be possible.

DESCRIPTION OF SYMBOLS 1 Endoscopic fluorescence microscope 2,3 Fluorescence endoscope 4 Fluorescence microscope 5 Total reflection fluorescence microscope 6 Nipo disk type | mold confocal fluorescence microscope 7 Fluorescence macro observation apparatus for experimental small animals 100,300 Main body part 101,301 Probe part 102,302 , 401, 501, 601, 701 Excitation light source 108, 418 Photo detector 110 Optical fiber 112, 312, 609, 705 Excitation light intensity balance filter 115, 202, 315, 408, 508, 611, 707 Observation object 308, 515 615, 710 Image sensor 310 Optical fiber bundle 104, 304, 403, 604, 703 Dichroic mirror 503 Beam splitter 413, 511, 606, 704 Bulk optical element

Claims (8)

In irradiating the observation object with the excitation light emitted from the excitation light source, the fluorescence generated from the observation object is detected by optical amplification with an optical amplifier.
A fluorescence detection method, characterized in that excitation light emitted from the excitation light source is incident on the optical amplifier to amplify fluorescence generated from the observation target.   The fluorescence detection method according to claim 1, wherein the observation object is irradiated with excitation light emitted from the excitation light source through the optical amplifier.   The excitation light emitted from the excitation light source is separated into at least two excitation lights, the one of the separated excitation lights is irradiated on the observation target, and the other separated excitation light is incident on the optical amplifier The fluorescence detection method according to claim 1, wherein: In a fluorescence detection apparatus that irradiates an observation object with excitation light emitted from an excitation light source, and detects the fluorescence generated from the observation object by optical amplification with an optical amplifier,
A fluorescence detection apparatus configured to cause excitation light emitted from the excitation light source to enter the optical amplifier to optically amplify fluorescence generated from the observation target.   5. The fluorescence detection according to claim 4, further comprising: a light amount adjustment unit that adjusts a light amount ratio between excitation light applied to the observation target and excitation light for optically amplifying the fluorescence by the optical amplifier. apparatus.   The fluorescence detection apparatus according to claim 4, wherein the optical amplifier includes an optical fiber to which a gain medium is added.   The fluorescence detection apparatus according to claim 6, wherein the optical fiber is arranged to extend to an insertion portion of an endoscope.   The fluorescence detection apparatus according to claim 4, wherein the optical amplifier includes a bulk optical element to which a gain medium is added.

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