WO2006120770A1 - Vital cell-controlling apparatus and vital cell-controlling method - Google Patents

Vital cell-controlling apparatus and vital cell-controlling method Download PDF

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Publication number
WO2006120770A1
WO2006120770A1 PCT/JP2005/020685 JP2005020685W WO2006120770A1 WO 2006120770 A1 WO2006120770 A1 WO 2006120770A1 JP 2005020685 W JP2005020685 W JP 2005020685W WO 2006120770 A1 WO2006120770 A1 WO 2006120770A1
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Prior art keywords
irradiation
light
cell
organelle
step
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PCT/JP2005/020685
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French (fr)
Japanese (ja)
Inventor
Wataru Watanabe
Sachihiro Matsunaga
Kiichi Fukui
Kazuyoshi Itoh
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Osaka Industrial Promotion Organization
Osaka University
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Priority to JP2005134794A priority Critical patent/JP2006311807A/en
Priority to JP2005-134794 priority
Application filed by Osaka Industrial Promotion Organization, Osaka University filed Critical Osaka Industrial Promotion Organization
Publication of WO2006120770A1 publication Critical patent/WO2006120770A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/645Specially adapted constructive features of fluorimeters
    • G01N21/6456Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458Fluorescence microscopy

Abstract

It is intended to provide a vital cell-controlling apparatus whereby cell organelles in a fluorescent stained cell are irradiated with ultrashort light pulses in the near infrared region to induce an adequate dysfunction and a method thereof. The vital cell-controlling method is conducted by focus-irradiating a stained vital sample with a light having a wavelength shorter than or almost the same as visible lights, extracting the florescence thus emitted from an excited fluorescent substance, visualizing the location of a cell organelle in the vital cell by displaying the image depending on the extracted fluorescence and thus performing fluorescent monitoring. Based on the visualized image display, a target cell organelle is selected from among the cell organelles and then the target cell organelle is irradiated with ultrashort light pulses in the near infrared region. Further, the vital sample having been irradiated with the ultrashort light pulses is fluorescent monitored. Thus, the displayed images of the vital sample before and after the irradiation with the ultrashort light pulses are compared so as to determine appropriate energy conditions for the ultrashort light pulses corresponding to the dysfunction of the desired cell organelle.

Description

 Biological cell control apparatus and biological cell control method TECHNICAL FIELD

 The present invention relates to a biological cell control device and a biological cell control method capable of allowing a living cell to survive while inhibiting the function of only a part of the organelle in a living cell such as a mitocondrier in a visible state. About.

Background art

 In recent years, to understand the complex dynamics of living cells, to investigate the function of organelles of living cells, typically to investigate the functions while taking advantage of mitochondria that generate energy used in cells. Etc. are desired. In particular, elucidation of the pathogenesis of autoimmune diseases by elucidating apoptosis, which is a mode of cell death in contrast to cell necrosis (necrosis), has attracted attention. Here we refer to apoptosis. Apoptotic cells shrink and the nucleus concentrates and fragments. Fragmented nuclei are formed as apoptotic bodies encased in cell membranes and processed by phagocytes. This process is controlled by a series of genes, consuming energy and actively performing. Unlike necrosis, it is considered to be an important mechanism for maintaining homeostasis of the cellular environment in the living body because it does not cause inflammation in principle. This is also deeply involved in the development and regulation of the immune system, and is essential for the establishment of immunological tolerance, especially the elimination of autoreactive T cells in the thymus.

Therefore, research that examines how organelles that affect the basic activities of cells inhibit their functions in living cells is extremely important in recent medical research. Therefore, there is a need for means to artificially inhibit the function of organelles. Proper to perform a functional study of living organelles here When considering the method, attention has been focused on methods of destroying and incising organelles by utilizing the fact that observation, manipulation, and destruction of organelles can be performed without contact using light. As this method, a method in which continuous light or pulsed light in the ultraviolet or visible region is condensed with a lens and irradiated into a cell has been verified. This method is advantageous in that the rupture of organelles can be achieved with submicron spatial resolution.

 However, when ultraviolet light or visible light is used as the irradiation light, absorption corresponding to the light intensity occurs in all parts of the cell irradiated with light due to the fountain effect. Therefore, there are problems such as damage occurring outside the light collecting region and possible cell damage due to absorption. It has also been proposed to use ultrashort light pulses in the near-infrared region (such as femtosecond laser pulses) as irradiation light.

Specifically, it is a method in which ultrashort light pulses are focused on an organelle through an objective lens, and multiphoton ionization and tunnel ionization occur selectively near the focal point to generate plasma. This method is advantageous in that the generation of plasma can cause damage to intracellular structures with sub-micron spatial resolution, and there is no absorption outside the focusing region, and there is little influence on the surroundings of heat and shock waves. is there. In other words, by irradiating a target organelle with an ultrashort light pulse in the near infrared region, it is considered possible to damage only the target organelle. However, even with the irradiation inside the cell using such an ultrashort light pulse, there remains a problem in verifying the transition of the target organelle. In particular, there is a problem in visualizing organelles. Specifically, as in conventional fluorescence observation, when organelles are stained with fluorescent dyes or labeled with fluorescent proteins, ultrashort light pulses are emitted. When focused on the organelle, no fluorescence from the target organelle is observed, and the loss of fluorescence due to this is due to bleaching, or the organelle has moved or been removed. I couldn't make a judgment as to whether or not it was based. In other words, in the prior art, verification of the true organelle transition due to the irradiation of the ultrashort light pulse was insufficient. Furthermore, in the case of conventional intracellular irradiation using ultrashort light pulses, there is no means for verifying the functional inhibition of organelles according to the optical conditions, and there is a lack of concreteness for actual example verification. As a result, it was not possible to provide a basis for performing the above-mentioned artificial induction of apoptosis. For example, US Optical Society No. 26 No. 8 1 9 (Opt. Lett., 26 (2001) 819) and Nyaiya No. 4 1 8 No. 2 90 (Nature, 418 (2002) 290) are examples of the above prior art. Referenced. Disclosure of the invention

Problems to be solved by the invention

 In view of the above circumstances, the present invention has been provided, and in the visible state, the target organelles (target microregions) among the organelles of living cells remain in a state where the cells remain alive. The device and method for performing and verifying the desired functional inhibition according to the target organelle, optical system, etc., that is, the biological cell control device can be controlled. It is intended to provide.

Means for solving the problem

The biological cell control method of the present invention includes a staining step in which a cell organelle of a biological sample is stained with a fluorescent substance, and the biological sample stained in this staining step is condensed with light having a wavelength shorter than or equal to the visible light wavelength. A first irradiation step for irradiation, a fluorescence extraction step for extracting fluorescence emitted from the fluorescent material excited in the first irradiation step, and an image display of the position of organelles in the living cell by the extracted fluorescence A step of selecting a target organelle from among the organelles based on the visualization process, an image display from the visualization step, and irradiating the selected target organelle with an ultrashort light pulse in the near-infrared region. And a second irradiation step for irradiating. Further, the present biological cell control method is performed by performing the first irradiation step, the fluorescence extraction step, and the visualization step again on the biological sample irradiated with the ultrashort light pulse in the second irradiation step. Before irradiation with short light pulses A display image of a later biological sample is compared, and an energy condition of an ultrashort light pulse corresponding to the desired inhibition of organelle function is set by the comparison. In addition, in this biological cell control method, the organelle of the biological sample irradiated with the ultrashort light pulse in the second irradiation step is stained again with a fluorescent substance to perform the first irradiation step, the fluorescence extraction step, and the visualization step. Can be executed. The energy condition of the ultrashort light pulse is determined by comparing the display images of the biological sample before and after the irradiation with the ultrashort light pulse, and the target organelle after irradiation is different from the target organelle before irradiation. It is possible to set the energy of an ultrashort light pulse that is fragmented and does not kill the nucleus in the cell or damage the cell membrane.

 In addition, the biological cell control device of the present invention includes a first irradiating means for irradiating a biological cell in which a cell organelle is stained with a fluorescent material with light having a wavelength shorter than or equal to the visible light wavelength, and a first irradiation. Scanning means for scanning the focused point of the irradiated light by the means in the living cells, and converting the fluorescence emitted from the fluorescent material of the organelle excited by the irradiated light from the first irradiation means into an electrical signal Detector, a second irradiation means for irradiating an ultrashort light pulse in the near infrared region, and a superimposing means for superimposing the irradiation light from the second irradiation means on the optical path of the irradiation light from the first irradiation means. is doing.

Specifically, the first irradiating means that collects and irradiates light with a wavelength shorter than or equal to the visible light wavelength, the reflected light from the first irradiating means, and the fluorescence on the longer wavelength side than the irradiated light. The first splitter that is transmitted, the irradiation light from the first irradiation means reflected by the first splitter is reflected, the scanning mirror whose reflection angle fluctuates, and the light reflected by the scanning mirror is condensed to focus. A first objective lens arranged so as to be located in a living cell, a second objective lens for condensing the fluorescence transmitted by the first splitter, and a focal point of the fluorescence collected by the second objective lens. An opening provided on the optical path of the fluorescence so as to match, and a detector for converting the fluorescence that has passed through the opening into an electrical signal. Furthermore, the biological cell control apparatus includes a second irradiation unit that irradiates an ultrashort light pulse in the near-infrared region, and irradiation light from the second irradiation unit. 1 It has the superimposition means to superimpose on the optical path of the irradiation light from an irradiation means. This superimposing means is composed of a second splitter having a function of reflecting the irradiation light from the first irradiation means and transmitting it to the first splitter, and transmitting the irradiation light from the second irradiation means to the first splitter. It is preferred that

The invention's effect

 According to the present invention, only a target organelle such as mitochondria can be irradiated while observing fluorescence of the organelle in a living cell, and an ultrashort light pulse in the near infrared region (for example, femtosecond) Since the laser pulse has a high peak value and has a nonlinear optical effect, it can be damaged only in the vicinity of the focal point and not damaged in other parts. In addition, according to the present invention, the organelle can be visualized by an image, and the scanning means used for fluorescence observation can also be used as the target of the ultrashort light pulse. It is easy to plan. In addition, according to the present invention, even if fluorescence after irradiation cannot be obtained from a cell organelle irradiated with an ultrashort light pulse by re-staining, the viability of the organelle can be determined, and function inhibition is prevented. Demonstration is possible. Furthermore, according to the present invention, how much energy of the ultrashort light pulse is irradiated to the target organelle can cause fragmentation of the organelle without proceeding to kill the nucleus or destroy the cell membrane. It can also be used for artificial induction of apoptosis (human control of the interaction of intracellular substances, etc.), which is an important medical issue.

Brief Description of Drawings

 FIG. 1 is a schematic diagram showing an optical system of the biological cell control apparatus of the present invention.

 FIG. 2 is a flowchart showing the main configuration of the biological cell control method of the present invention.

Fig. 3 shows the actual cell fluorescence images when the biological cell control apparatus of the present invention intends to inhibit the function of organelles. Upper left figure, lower left figure, middle upper figure, middle lower figure, upper right figure (A), (b), (c), (d), (e), (f) are shown in the order in the lower right figure. BEST MODE FOR CARRYING OUT THE INVENTION

 First, the function control process executed in the biological cell control apparatus of the present invention will be mentioned with reference to FIG. In the biological cell control apparatus of the present invention, fluorescent organs are first labeled on the organelles in the biological cells desired to be observed, and the fluorescent images are acquired and visualized as three-dimensional images (STEP 10). For this process itself, a known technique for obtaining a one-photon fluorescence image can be used. Since this process alone is not the essential technique of the present invention, a detailed description thereof will be omitted.

 Next, one or more organelles desired to be irradiated with femtosecond laser pulses in the near-infrared region are selected from the visualized organelles (whole specific cells are visualized) (STEP 12). This is made possible by visualizing the organelle in the living cell in STEP 10, and the selected organelle becomes the target organelle described later. This organelle selection is performed by specifying a predetermined area (target area) from the visualized 3D image, and the target area may be specified by a point or an arbitrary area. .

Next, the focal point of the near-infrared humtosecond laser pulse is positioned at the selected organelle (actually the target area specified in STEP 1 2), and the target area is irradiated with the pulse. (STEP 14). Then, again, as in STEP 10, a fluorescent image of a living cell is acquired and visualized as a three-dimensional image (STEP 16). Through this process, the 3D image acquired in STEP 10 and the 3D image acquired in STEP 16 can be compared, that is, the fluorescence images before and after femtosecond laser pulse irradiation in the near infrared region can be compared. it can. Therefore, it is possible to verify how the target organelle and the biological cells that contain the target organelle change when the target organelle is irradiated with a femtosecond laser pulse in the near-infrared region. This makes it possible to control the operation of the desired target organelle and other organelles associated therewith in a visualized state. Next, an optical system for actually performing the function control process shown in FIG. 2 and preferred conditions thereof will be described. Referring to FIG. 1, an example of the biological cell control apparatus 1 0 0 of the present invention is shown. As described above, the biological cell control apparatus 100 of the present invention includes a step of performing an artificial operation by irradiating a cell organelle with an ultrashort light pulse, and a step of visualizing the organelle before and after the operation. Therefore, the optical system is also configured by integrally combining optical systems corresponding to both processes. Specifically, it has an optical system for irradiating a target organelle with an ultrashort light pulse, and an optical system for observing fluorescence of a cell organelle. First, the optical system for fluorescence observation of organelles will be explained. This optical system has the same structure as the optical system known as the confocal laser scanning fluorescence microscope. It acquires a one-photon fluorescence image of the organelle inside the cell, and the ultrashort light pulse described later. The target organelle is specified by the irradiation optical system.

More specifically, as shown in FIG. 1, in this embodiment, the first laser 1 that emits light in the medium wavelength region of an Ar + laser (wavelength 488 nm) or a He-Ne laser (wavelength 543 nm) as an irradiation light source 1 0 0 is used. The light emitted from the first laser 100 is reflected by the second dichroic mirror 1002, and the optical path is bent 90 °. Here, the dichroic mirror has a property of reflecting only light of a specific wavelength range and transmitting light of other wavelengths, and is generally referred to as a beam splitter. The second dichroic mirror 102 is arranged to reflect the light emitted from the first laser 100 but to transmit the light emitted from the second laser, which will be described later, and to superimpose it on the same optical path. Further, the irradiation light of the first laser 100 reflected by the second dichroic mirror 10 0 2 is further supplied to another dichroic mirror (second dichroic mirror 1 0 4) disposed in the optical path. The light path is bent by 90 °. The first dichroic mirror 10 04 reflects the irradiation light from the first laser 10 100 and a second laser 1 1 6 described later. It is arranged to reach the sample (biological cell) and transmit the fluorescence emitted from the biological sample to transmit to the detector 118 (details will be described later). The laser beam reflected by the first dichroic mirror 104 is reflected by a stray mirror (galvanomirror) 10 6 disposed at the end of the optical path and condensed by the objective lens 10 8, and the living body. Intracellular 1 1 0 pin spot 1 1 0 a is irradiated. At this time, since the laser beam is swung in the XY direction (a plane direction parallel to the living cell as shown in FIG. 1) by the scanning mirror 106, the pin spot 110a also moves in the XY direction. It is possible to crawl. In Fig. 1, for the sake of visual recognition, only one scanning mirror 10 06 is displayed, but it is actually composed of two sheets to perform the XY direction stroking. It will reach the objective lens 10 8 through these two mirrors. Although scanning in the Z direction (the depth direction of living cells) is not shown, it can be performed by sliding the stage on which the living cells are placed in the Z direction. The living cells 110, particularly the organelles, are excited by the light from the first laser 100 that has been focused and irradiated while scanning. The organelles irradiated at this time must be stained in advance with a fluorescent dye or labeled with a fluorescent protein. Reference is now made to fluorescent materials. When excited by an appropriate wavelength, fluorescent materials increase their energy levels and form unstable states. The substance then works to eliminate this unstable state and return to a stable state, releasing fluorescence. This fluorescence has a characteristic that the wavelength is shifted to the longer wavelength side by 20 to 50 nm than the excitation light. Returning to the description of the present embodiment again, the pinpoint of the irradiation light from the first laser 100 (condensing point) 110 1 a is stained, specifically, the fluorescence-stained mitochondria of the mitochondria. When in position, the fluorescent substance is excited along with the mitochondria, and emits fluorescence whose wavelength is shifted to the long wavelength side of the laser light. This fluorescence is emitted spatially in all directions, and the light passing through the objective lens 10 8 becomes parallel light and is reflected by the scanning mirror 1 0 6 to be the dichroic light described above. Transmitted to mirror 1 0 4. In addition, since a cover glass or the like is usually used for mounting biological cells, the light transmitted to the dichroic mirror 104 is also transmitted simultaneously with the laser light (excitation light) reflected by the cover glass or the like. Of these, the laser light is reflected by the dichroic mirror 104 (the wavelength of the light from the first laser 100 is reflected as described above), but the fluorescence whose wavelength is shifted to the longer wavelength side is dichroic. Mirror 1 0 4 is transmitted. Therefore, organelle 1 1 0

The light emitted from (and the cover glass or the like) passes through the dichroic mirror 104, and only the fluorescent component is extracted. The extracted fluorescence is condensed through the objective lens 1 1 2 and transmitted to the pinhole 1 1 4. The objective lens 112 preferably has a large numerical aperture so as to collect as many emitted fluorescent signals as possible.

 Furthermore, the pinhole 1 1 4 is arranged so that the fluorescence passes only when the focal point of the light collected by the objective lens 1 1 2 matches the pinhole position. Only the light excited and emitted by is allowed to pass. Therefore, the light after passing through the pinhole 1 1 4 is extracted only from the fluorescent material of the organelle excited at the focal point of the laser beam. The fluorescence is then transmitted to a detector (photomultiplier (PMT)) that converts the optical signal into an electronic signal. Further, although not shown, the fluorescence converted into the electronic signal is A / D converted and displayed as an image on the monitor. The whole living cell can be imaged by scanning with a scanning mirror 10 6 or a slide on the stage (described above). Therefore, the target organelle is detected by the ultrashort light pulse described below.

When irradiating (target mitochondria), the operator visually recognizes the position of the target organelle and performs pulse irradiation, that is, the target cell organelle shown above and shown in STEP12 of FIG. 2 can be selected. It becomes.

Next, the irradiation optical system for specific organelles using femtosecond laser pulses (ultrashort light pulses) will be described. In this embodiment, the first as an ultrashort optical pulse light source 2Laser 1 1 6 uses a femtosecond titanium sapphire laser amplifier (center wavelength 80 O nm, pulse time width about 150 fs, repetition frequency 1 kHz). The irradiation light from the second laser 1 1 16 is transmitted to the second dichroic mirror 10 2 and transmitted therethrough. Here, when referring to the second dichroic mirror 1002, the light emitted from the first laser 100 is longer than the light irradiated from the first laser 100. As described above, the light irradiated from the first laser 100 is Although reflected, the irradiation light from the second laser 1 16 is transmitted. Thus, after passing through the second dichroic mirror 10 0 2, the optical path of the second laser 1 16 overlaps with the optical path of the first laser 1 100. Therefore, after that, the irradiation light of the second laser 1 16 passes through the second dichroic mirror 10 4, the scanning mirror 1 0 6, and the object lens 1 0 8 in the same manner as the irradiation light of the first laser 1 100. It is transmitted to living cells. At this time, the second dichroic mirror 10 04 matches the focal point of the irradiation light of the second laser 1 1 6 with the focal point of the irradiation light of the first laser 1 1 0 0, and the target is determined based on the fluorescence image. Choosing the focal point for selecting the organelle is selecting the irradiation point of the ultrashort light pulse.

The above is the outline of the optical system according to the embodiment of the present invention. A demonstration example in which the functional inhibition of the organelle in the living cell is actually controlled will be described. First, refer to FIG. 3 as a first experimental example. This is an example of an experiment in the optical system exemplified in the above description, and the functional inhibition of mitochondria is verified as an organelle using HeLa cells, which are human eclampsia cancer cells, as an experimental sample. Also, as described above, a titanium sapphire laser regenerative amplifier is used as the light source for manipulating organelles (center wavelength: 800 nm; pulse time width: about 150 fs; repetition frequency: 1 kHz), and the number of pulses of irradiation light uses a shutter. Control. Furthermore, Ar + laser (wavelength 488 nm) or He-Ne laser (wavelength 543 nm) is used for fluorescence observation as one-photon fluorescence observation. Next, referring to the experimental results, Fig. 3 shows how HTFP (Enhanced Yellow Fluorescent Protein) fused with a mitochondrial targeting sequence is used to label mitochondrials in living HeLa cells. ing. Using an objective lens 10 8 with a numerical aperture of 1.4, 125 laser pulses 1 1 6 were incident and focused on one mitochondria in a living HeLa cell. The irradiation energy of laser pulse 1 1 6 is increased from 5.5 nj / pulse to 8 nj / pulse, and the target mitochondria and the dynamics of the whole cell are observed. As a result of this observation, Figs. 3 (a) and 3 (b) are confocal fluorescence images before and after irradiation with a femtosecond laser pulse of 5.5 nJ / pulse. As is clear from this figure, when the laser pulse is irradiated, one mitochondria is destroyed, but there is no effect on the organelles near the focal point. Figure 3 (c), (d) shows a 6. 4 nj / pulse confocal fluorescence images before and after the femtosecond laser pulses irradiation shines in. In this case, it has been observed that mitochondria split and fragment about 10 minutes after irradiation with femtosecond laser pulses. In addition, as shown in Fig. 3 (f), when the irradiation energy is irradiated at 7.4 nj / pulse or more, several mitcondriers near the condensing point are destroyed, and the mitcondriers of the whole cell are changed from elliptical to spherical. It can be seen that the shape changed to fragmented.

Furthermore, in order to investigate the damage to live cells by laser pulse intensity, we are conducting experiments using PI (propidium iodide). PI has the property of strongly staining the nuclei of dead cells and cells with damaged cell membranes, so it is used for cell viability discrimination. After mitochondrial intracellular irradiation with laser pulses, culture around the cells PI is added to the solution, and cell viability and cell membrane damage after laser pulse irradiation are confirmed. As shown in Figs. 3 (b) and 3 (d), when one mitochondria was disrupted, and when it split and fragmented, PI staining of the nucleus was not observed. As shown in (), when it becomes spherical, it turns out that the nucleus in which red fluorescence from the nucleus by PI was confirmed is dead. In addition to re-staining using PI, mitochondrial staining performed before fluorescence observation was performed. It may be re-stained by a color method such as MTR (MitoTrackerRed) .In this case, unlike in the case of PI, the ability of mitochondria to inhibit the function of laser pulses is demonstrated. be able to. In other words, if the target mitochondrial fluorescence loss is due to discoloration, it should be able to acquire fluorescence if it is re-stained, and functional inhibition can be demonstrated by the inability to acquire fluorescence.

In addition, although the fluorescence observation results are not shown in particular, functional inhibition, especially fragmentation, occurs when a titanium sapphire laser oscillator (wavelength 800 nm, repetition frequency 76 MHz, pulse width approx. LOOfs) is used as an ultrashort light source. As an empirical example of an energy condition that does not cause cell membrane destruction or nuclear death, it is possible to use 2.5 X 10 "6pulses (irradiation time 1/31 sec) (from 0.26 nJ / pulse (20 mW) to 0.39. n J / pul S e (30mW )) irradiation pulse number than (irradiation time) is Yabu壌the mitochondria of the condensing region is shorter, for long irradiation times than this (4. 8 X 1CT

In the case of 6 pulses (irradiation time 1/16 sec): From 0.20 n J / pulse (15 mW) to 0.53 η J / pulse (40 mW)), cell destruction (burst) has occurred. It has been verified that the present biological cell control apparatus can detect the proper energy condition of a laser pulse that induces the function inhibition of a desired organelle or induces the protoplasmic flow observed with this, in a visible state. I know I can.

 The above is an embodiment of the present invention and an experimental example using the same, but the present invention is not limited to this, and can be applied to all intracellular substances that can be observed with fluorescence regardless of animals and plant cells. By controlling ultrashort light pulses (such as femtosecond laser pulses), it becomes possible to develop interactions between organelles and elucidation of organelle functions.

Claims

The scope of the claims
1. a staining step of staining a biological organelle with a fluorescent substance;
 A first irradiation step of condensing and irradiating the biological sample stained by the staining step with light having a wavelength shorter than or equal to the visible light wavelength; and
 A fluorescence extraction step of extracting fluorescence emitted from the fluorescent material excited by the first irradiation step;
 A visualization step of displaying an image of the position of the organelle in the living cell by the extracted fluorescence;
 Selecting a target organelle from among the organelles based on the image display from the visualization step;
 A second irradiation step of irradiating the selected target organelle with an ultrashort light pulse in the near-infrared region,
 Further, by performing the first irradiation step, the fluorescence extraction step, and the visualization step again on the biological sample irradiated with the ultrashort light pulse in the second irradiation step, the ultrashort light pulse is executed. Comparing the display images of the biological sample before and after the irradiation of the sample, and setting the energy condition of the ultrashort light pulse corresponding to the desired function inhibition of the organelle by the comparison. Cell control method.
2. Cell organelles of a biological sample irradiated with an ultrashort light pulse in the second irradiation step are again stained with a fluorescent substance to perform the first irradiation step, the fluorescence extraction step, and the visualization step. The biological cell control method according to claim 1, wherein the biological cell control method is executed.
3. The energy condition of the ultrashort light pulse is determined by comparing the display images of the biological sample before and after irradiation with the ultrashort light pulse, and the target organelle after irradiation is a cell other than the target organelle before irradiation. The living cell according to claim 1 or 2, wherein the energy of the ultrashort light pulse is set so that the organelle is fragmented and the nucleus of the cell is not killed or the cell membrane is damaged. Control method.
4. a first irradiating means for condensing and irradiating a living cell in which a cell organelle is stained with a fluorescent substance with light having a wavelength shorter than or equal to the visible light wavelength;
 Scanning means for scanning the living cell with a condensing point of light irradiated by the first irradiation means;
 A detector that converts fluorescence emitted from a fluorescent material of an organelle excited by irradiation light from the first irradiation means into an electrical signal;
 A second irradiation means for irradiating an ultrashort light pulse in the near infrared region;
 A biological cell control apparatus comprising: a superimposing unit that superimposes the irradiation light from the second irradiation unit on the optical path of the irradiation light from the first irradiation unit.
5. a first irradiation means for condensing and irradiating light with a wavelength shorter than or equal to the visible light wavelength;
A first splitter that reflects the light emitted from the first irradiation means and transmits fluorescent light having a wavelength longer than that of the light;
 A scanning mirror that reflects the irradiation light from the first irradiation means reflected by the first splitter, and whose reflection angle varies;
 A first objective lens disposed so that the light reflected by the stirrer mirror is collected and the focal point is located in the living cell;
 A second objective lens for condensing the fluorescence transmitted by the first splitter; an opening provided on the optical path of the fluorescence so as to coincide with the focal point of the fluorescence collected by the second objective lens;
 A detector that converts the fluorescence that has passed through the opening into an electrical signal, and
 A second irradiation means for irradiating an ultrashort light pulse in the near infrared region;
 A biological cell control apparatus comprising: a superimposing unit that superimposes the irradiation light from the second irradiation unit on the optical path of the irradiation light from the first irradiation unit.
6. The superimposing means reflects the irradiation light from the first irradiating means to reflect the first light.
1 is transmitted to the splitter, and the irradiation light from the second irradiation means is transmitted to transmit the second irradiation means. 6. The biological cell control device according to claim 5, comprising a second splitter that transmits up to one splitter.
PCT/JP2005/020685 2005-05-06 2005-11-04 Vital cell-controlling apparatus and vital cell-controlling method WO2006120770A1 (en)

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