JP3915626B2 - Fluorescence sample analyzer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for exciting a plurality of fluorescent substances present in a sample substantially simultaneously. By labeling a protein or the like in a biological sample such as a cell with a fluorescent substance and using this method, it becomes possible to simultaneously and independently track the movement, action, etc. of a plurality of proteins contained in the living body. .
[0002]
[Prior art]
When an ultrashort light pulse in the order of femtoseconds ( ^10-15 seconds) is spatially converged and irradiated onto a material, the energy density at that point becomes very high, and between the ultrashort light pulse and the material. Linear and nonlinear interactions occur. As a result, various phenomena occur, one of which is the generation of white continuous spectrum light. Since this white continuous spectrum light has a white continuous spectrum while being an ultrashort pulse similar to the original pulse, various uses are considered.
[0003]
Japanese Patent Application No. 2001-372570 discloses a method for exciting a plurality of fluorescent substances present in a sample almost simultaneously using the ultrashort light pulse. In this method, white continuous spectrum pulsed light generated by ultrashort pulsed light is dispersed, wavelength light necessary for excitation of a plurality of target fluorescent substances is selected, then they are synthesized by an inverse spectrograph, and are applied to a sample. Irradiating.
[0004]
[Problems to be solved by the invention]
Although the target wavelength can be arbitrarily selected in the above method, changing the target wavelength is not always easy. For example, when developing a new fluorescent material, when searching for the optimal excitation wavelength for the fluorescent material, the optimal excitation wavelength can be found by switching the expected excitation wavelength sequentially. If the switching is not easy, it takes a long time to search. In addition, when substances labeled with fluorescent substances are located in a complicated and overlapping manner, it is difficult to investigate the position of a specific substance in detail if the sample is irradiated with the excitation wavelength of each fluorescent substance simultaneously. .
The present invention has been made to solve such a problem, and provides a fluorescent sample analyzer capable of easily switching an excitation wavelength to be selected.
[0005]
[Means for Solving the Problems]
In order to solve the above problems, the first embodiment of the fluorescent sample analyzer according to the present invention is as follows:
a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) wavelength selection means for selecting only the target wavelength light in each of the dispersed wavelengths;
c) a shutter that can arbitrarily pass or block each selected target wavelength light; and
d) an inverse spectrometer that synthesizes the target wavelength light passing through the shutter to generate a synthesized light pulse;
It is characterized by providing.
[0006]
According to the second aspect of the fluorescent sample analyzer of the present invention,
a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) one or a plurality of wavelength selection means for selecting only the target wavelength light in each of the dispersed wavelengths;
c) Selected wavelength ON / OFF means for switching between valid / invalid of each wavelength selection means,
d) an inverse spectrometer that synthesizes the target wavelength light selected by the validated wavelength selection means and generates a synthesized light pulse;
It is characterized by providing.
[0007]
According to the third aspect of the fluorescent sample analyzer of the present invention,
a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) wavelength selection means for selecting only the target wavelength light in each of the dispersed wavelengths;
c) selection switching means for switching a plurality of wavelength selection means;
d) an inverse spectrometer that synthesizes the target wavelength light selected by the wavelength selection means selected by the selection switching means to generate a synthesized light pulse;
It is characterized by providing.
DETAILED DESCRIPTION OF THE INVENTION
[0008]
The fluorescent sample analyzer of the first aspect is used as follows. In advance, one or a plurality of fluorescent substances to be excited are determined, and the wavelength for exciting the fluorescent substances is examined. In the present invention, since excitation light is generated from ultrashort light pulses, multiphoton excitation such as two-photon excitation and three-photon excitation occurs. Therefore, the excitation wavelength is set to a value considering them.
[0009]
Only one or a plurality of excitation wavelengths (target wavelengths) are selected from the spectrum after the white continuous spectrum light pulse is separated by the wavelength selection means. As specific modes of the wavelength selection means, various considerations such as a reflector array (array) in which reflectors are arranged only at locations of each target wavelength, a reflector that covers an absorption mask at locations other than each target wavelength location, and the like are considered. be able to.
[0010]
Of the target wavelength light thus selected, only the target wavelength light that is actually applied to the sample is allowed to pass through the shutter. Of the selected target wavelength light, light that is not irradiated on the sample at this time is blocked by a shutter.
[0011]
As a shutter, a liquid crystal shutter can be used in addition to a mechanical shutter such as a rotary disk shutter provided with a notch or a plain shutter of a camera.
[0012]
Only the target wavelength light that has passed through the shutter is sent to the inverse spectrometer, where a combined light pulse is generated. The inverse spectrometer here may be provided separately from the spectrometer for generating the spectrum, or the spectrometer may be used as it is as an inverse spectrometer.
[0013]
In the synthesized light pulse generated in this way, only the excitation light pulse corresponding to the target fluorescent material is selected at the time of selection by the first wavelength selection means. Only a plurality of fluorescent materials can be excited. Furthermore, in the present invention, by switching the passage / blocking state of each shutter as appropriate, only the fluorescent material required at present can be excited among the target fluorescent materials. Since the shutter is used, switching can be performed very easily and in a short time. This makes it possible to search for the optimum excitation wavelength of a new fluorescent material, or to investigate the distribution and behavior by distinguishing a large number of complicated fluorescent materials. This makes it easier to conduct surveys.
[0014]
In the fluorescent substance analyzer of the second aspect, the wavelength selection means can be switched between valid / invalid by the wavelength selection ON / OFF means. When the wavelength selection means is enabled, the wavelength light is selected, sent to the inverse spectrometer, and included in the synthesized light pulse. On the other hand, when the wavelength selecting means is disabled, the light of that wavelength is not selected, and the light of that wavelength is not included in the synthesized light pulse. In this way, by controlling the wavelength selection means by the wavelength selection ON / OFF means, it is possible to arbitrarily switch the combination of wavelengths of light included in the synthesized light pulse.
[0015]
The fluorescent substance analyzing apparatus according to the third aspect includes a plurality of wavelength selection means, which can be switched by the selection switching means. Therefore, the combination of the selection wavelength light included in the synthesized light pulse can be arbitrarily and easily switched.
[0016]
In the apparatus according to the present invention, since the synthesized light pulse to be generated is an ultrashort light pulse like the original ultrashort light pulse, the fluorescent material in the sample is converted into a multiphoton excitation mode such as two-photon excitation or three-photon excitation. Excited with In normal linear absorption excitation (linear excitation), absorption occurs in all parts where light passes, but by using multi-photon excitation as in the present invention, nonlinear absorption can occur only in the focal part. , Information only in the vicinity of the focal point can be obtained. If this focal position is scanned two-dimensionally or three-dimensionally, a two-dimensional or three-dimensional analysis image can be obtained. Moreover, the movement etc. can also be observed by tracking temporally.
[0017]
【The invention's effect】
In the fluorescent substance analyzer according to the present invention, it is possible to easily and easily switch the combination of wavelength light included in the synthetic light pulse irradiated to the sample. Such switching is effective in the following cases.
First, when developing a new fluorescent material as described above, it is possible to search for an optimum excitation wavelength in a short time by sequentially switching the expected excitation wavelength with the apparatus according to the present invention. . In addition, when substances labeled with fluorescent substances are mixed and mixed in the sample, they can be emitted individually by sequentially switching the excitation wavelength of each fluorescent substance, and the position of each fluorescent substance can be accurately determined. It becomes possible to know.
[0018]
Next, when a sample is irradiated with a plurality of excitation wavelength lights at the same time, they may interfere and difference frequency component light may be generated due to nonlinear effects. Such difference frequency component light may excite fluorescent substances other than the intended one and cause noise in analysis. Thus, the apparatus according to the present invention excites the fluorescent substances in the sample one by one, individually excites and observes the fluorescence, and finally synthesizes all the observation results, thereby causing such interference. It is possible to observe only the target fluorescent material with less noise from which the difference frequency component light is removed.
[0019]
【Example】
FIG. 1 shows a fluorescent sample analyzer which is an embodiment of the first aspect of the present invention. Ultrashort light pulses generated by a light source 11 such as an Er-doped fiber laser, a mode-locked Ti: sapphire laser, or a mode-locked Cr: forsterite laser (see Japanese Patent Laid-Open No. 11-284260) are transmitted by the first optical system 12. The light is condensed at one point in an appropriate transparent material 13 such as water or crystal. A white continuous spectrum light pulse is generated by nonlinear interaction between the substance 13 and the ultrashort light pulse at this condensing point. The generated white continuous spectrum light pulse is applied to the spectroscope 15 by the second optical system 14 (including a lens, a reflecting mirror, and the like) and split there. The split light is sent to the wavelength selecting means 17 by the collimating optical system 16.
[0020]
In FIG. 1, reference numeral 17a denotes a wavelength selecting unit in which a small reflecting mirror is disposed at a location of the target wavelength, and 17b denotes a wavelength selecting unit using a reflecting mirror provided with a light absorbing portion at a location other than the target wavelength light.
[0021]
A shutter 21 is provided between the collimating optical system 16 and the wavelength selection means 17. As shown in FIG. 2, the shutter 21 is composed of a rotary shutter unit 22 provided in front of each small reflecting mirror of the wavelength selection means 17a (or the reflection portion of the wavelength selection means 17b). Each rotary shutter unit is arbitrarily rotated by an operation of a key or the like in an operation unit 23 provided near the operator, and can pass / block light in the optical path. Note that the operation unit 23 can be realized by a simple switch, as well as a control device (in many cases, a personal computer is used) that controls the operation of the entire fluorescence sample analyzer. It can also be realized by incorporating.
[0022]
Only the wavelength light selected by the wavelength selection means 17 and allowed to pass by the shutter 21 is returned to the spectroscope 15 by the collimating optical system 16, where it is synthesized into one ultrashort light pulse. By irradiating the sample 18 with this, only the target substance is excited, and the fluorescence is detected by the detector 19.
As described above, in the fluorescence sample analyzer of this embodiment, the wavelength included in the synthesized light pulse applied to the sample 18 can be easily switched by operating the operation unit 23 at the operator's hand.
[0023]
In addition, when a plurality of selection lights are allowed to pass through the shutter 21 and a plurality of wavelength lights are included in the synthesized light pulse irradiated to the sample, the component lights interfere with each other in the synthesized light pulse, and the difference is caused by a non-linear effect. Frequency component light may be generated. Such difference frequency component light may excite fluorescent substances other than the target one in the sample 18 and may cause analysis noise. Therefore, in the analyzer of the above embodiment, the optical path length of each target wavelength light may be different from each other. Accordingly, each component light pulse selected by the shutter 21 is included in the synthesized light pulse in a temporally separated manner, so that generation of the difference frequency component light is prevented and generation of the noise is suppressed ( For details, see Japanese Patent Application No. 2001-372570).
[0024]
Examples of such a configuration are shown in FIGS. In FIG. 5, the optical path difference is provided by moving the position of the small reflector 17c on the optical path back and forth. FIG. 6 shows the optical path by disposing a transparent optical medium (preferably a material having a high refractive index) 17d having a different thickness (dimension in the optical path direction) in the optical path using the selective reflector type wavelength selecting means 17b. The length is optically changed. In any case, the shutter 21 is configured by placing the rotary shutter unit 22 on each optical path.
[0025]
Next, an embodiment of the fluorescence sample analyzer according to the second aspect of the present invention will be described. The overall configuration of the present embodiment is as shown in FIG. 1, but the configuration after the collimating optical system 16 is as shown in FIG. In other words, the wavelength selection unit 31 stores a large number of small mirrors 32 corresponding to the optical paths of the respective wavelengths, and the small mirrors 32 are advanced into the optical paths of the respective wavelengths in response to a command signal from the operation unit 33. . At the location where the small mirror 32 has advanced, light of that wavelength returns to the collimating optical system 16 and is synthesized by the spectroscope 15 into ultrashort light pulses. In the part of the wavelength which is not so, since the split light does not return to the spectrometer 15, it is not included in the synthesized light pulse. Therefore, also in the apparatus of this embodiment, the wavelength included in the synthetic light pulse irradiated on the sample 18 can be arbitrarily selected by giving an instruction from the operation unit 33 as described above.
[0026]
Next, an embodiment of the fluorescent sample analyzer according to the third aspect of the present invention will be described. The overall configuration of the present embodiment is as shown in FIG. 1, but the configuration after the collimating optical system 16 is as shown in FIG. That is, a plurality of wavelength selection means 42 selectively provided with a light absorption part and a reflection part shown as 17b in FIG. 1, and a selection switching means 41 for selecting them are provided. In FIG. 4, the wavelength selection means 42 is switched to a rotary type, but this may be a parallel movement type. This switching instruction is given from the operation unit 43.
[Brief description of the drawings]
FIG. 1 is an overall configuration diagram of a fluorescent substance analyzer according to an embodiment of the first aspect of the present invention.
FIG. 2 is a configuration diagram of a part after the collimating optical system of the embodiment.
FIG. 3 is a configuration diagram of a portion subsequent to a collimating optical system of a fluorescent substance analyzer according to an embodiment of the second aspect of the present invention.
FIG. 4 is a configuration diagram of a part after a collimating optical system of a fluorescent substance analyzer according to an embodiment of the third aspect of the present invention.
FIG. 5 is a configuration diagram of a first modification of the embodiment of the first aspect.
FIG. 6 is a configuration diagram of a second modification of the embodiment of the first aspect.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 11 ... Light source 12 ... 1st optical system 13 ... Transparent substance 14 ... 2nd optical system 15 ... Spectroscope (reverse spectrometer)
16 ... Collimating optical system 17 ... Wavelength selection means 18 ... Sample 19 ... Detectors 23, 33, 43 ... Operation unit

Claims (8)

a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) a wavelength selection means consisting of a plurality of reflecting mirrors arranged at each spectral position of a plurality of target wavelength lights after spectroscopy, and selecting only the target wavelength light in each of the split wavelengths;
c) a shutter that can arbitrarily pass or block each selected target wavelength light; and
d) an inverse spectrometer that synthesizes the target wavelength light passing through the shutter to generate a synthesized light pulse;
A fluorescent sample analyzer comprising: a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) one or a plurality of wavelength selecting means for selecting only the target wavelength light in each of the divided wavelengths, comprising a plurality of reflecting mirrors respectively arranged at the respective spectral positions of the plurality of target wavelength lights after the spectrum ;
c) Selected wavelength ON / OFF means for switching between valid / invalid of each wavelength selection means,
d) an inverse spectrometer that synthesizes the target wavelength light selected by the validated wavelength selection means and generates a synthesized light pulse;
A fluorescent sample analyzer comprising: a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) a wavelength selection means consisting of a plurality of reflecting mirrors arranged at each spectral position of a plurality of target wavelength lights after spectroscopy, and selecting only the target wavelength light in each of the split wavelengths;
c) selection switching means for switching a plurality of wavelength selection means;
d) an inverse spectrometer that synthesizes the target wavelength light selected by the wavelength selection means selected by the selection switching means to generate a synthesized light pulse;
A fluorescent sample analyzer comprising: The fluorescence sample analyzer according to any one of claims 1 to 3, wherein an optical path difference is provided for each target wavelength light by moving the positions of the plurality of reflecting mirrors back and forth. a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) a wavelength selection means that consists of a reflecting mirror that covers a portion other than each target wavelength with an absorption mask, and that selects only the target wavelength light in each of the dispersed wavelengths;
c) a shutter that can arbitrarily pass or block each selected target wavelength light; and
d) an inverse spectrometer that synthesizes the target wavelength light passing through the shutter to generate a synthesized light pulse;
A fluorescent sample analyzer comprising: a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) one or a plurality of wavelength selection means consisting of a reflecting mirror that covers a portion other than each target wavelength with an absorption mask, and that selects only the target wavelength light in each of the dispersed wavelengths;
c) Selected wavelength ON / OFF means for switching between valid / invalid of each wavelength selection means,
d) an inverse spectrometer that synthesizes the target wavelength light selected by the validated wavelength selection means and generates a synthesized light pulse;
A fluorescent sample analyzer comprising: a) a spectroscope that separates white continuous spectrum light pulses generated from ultrashort light pulses;
b) a wavelength selection means that consists of a reflecting mirror that covers a portion other than each target wavelength with an absorption mask, and that selects only the target wavelength light in each of the dispersed wavelengths;
c) selection switching means for switching a plurality of wavelength selection means;
d) an inverse spectrometer that synthesizes the target wavelength light selected by the wavelength selection means selected by the selection switching means to generate a synthesized light pulse;
A fluorescent sample analyzer comprising: The fluorescent sample analyzer according to any one of claims 1 to 7, further comprising transparent optical media having different thicknesses arranged at respective spectral positions of the plurality of target wavelength lights after the spectroscopy. .

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