JP2004361086A - Biomolecule analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biomolecule analyzer constituted so as to acquire the image of a specimen by a simple constitution to perform the correlation analysis of fluorescence. <P>SOLUTION: The biomolecule analyzer for analyzing the dynamic behavior of a biomolecule is equipped with a image acquiring means (11) for acquiring the image corresponding to at least one observation region of a biological specimen containing the biomolecule held to a measurable state, an arranging means (12) for arranging a measuring point at the desired point position in the image of the specimen acquired by the image acquiring means, a measuring means (102) for measuring the signal originating from the dynamic data of a substance to be measured from the measuring point arranged by the arranging means and an analyzing means (105) for analyzing the result measured by the measuring means. At least one of the image acquiring means and the arranging means comprises at least one variable focus optical element (11 or 12). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a biomolecule analyzing apparatus for determining statistical properties of one or more specific sites in a sample, interactions between the sites, and the like.
[0002]
[Prior art]
In recent years, a microscope using a confocal optical system has been used as an apparatus for observing or measuring a specific site in a sample with high resolution. Regarding the confocal optical microscope, see, for example, the document “Confocal Microscopy”, T.A. Wilson (ed.) Academic press (London) has commentary. Also, as a commentary mainly on biological samples, reference is made to the document “Handbook of Biological Confocal Microscopy”, J. Org. B. Pawley (ed.) Plenum Press (New York) and the like.
[0003]
In the 1990s, research on single molecule detection and imaging using fluorescence has been rapidly increasing. For example, as a method for detecting a single molecule, reference M. Goodwin etc. ACC. Chem. Res. (1996), Vol. 29, p607-613, and fluorescence correlation spectroscopy (FCS). In the fluorescence correlation spectroscopy, the autocorrelation function is obtained by analyzing the fluctuation of the fluorescence intensity based on the Brownian motion in the solution of the protein or carrier particles fluorescently labeled at a predetermined one focus in the field of view of the confocal microscope, Estimate the number and size of the target fine particles. This technique is described, for example, in Masataka Kaneshiro, “Protein Nucleic Acid Enzyme” (1999) Vol. 44, no. 9, pp 1431-1437.
[0004]
Conventionally, in order to acquire a microscope image of a sample using a point light source such as a laser beam, two-dimensional scanning has been performed by two galvanometer mirror scanning mechanisms in which the directions of rotation are set at right angles to each other. For the focus movement in the Z-axis direction, the sample stage is moved by a small amount in the Z-axis direction using a drive mechanism such as a motor, or a focus position adjustment mechanism including a fixed lens and a movable lens is inserted in the optical path. By moving the movable lens by a very small amount in the optical axis direction, the focus position is changed.
[0005]
[Problems to be solved by the invention]
On the other hand, several patent applications have been filed for the above technology. The technique disclosed in Japanese Patent Application Laid-Open No. 2001-194303 is a fluorescence correlation spectroscopy applied to a sample solution in which the density of fluorescent molecules to be measured is low, and two-dimensionally scans using a cylindrical lens. Thus, the entire sample is excited by irradiating the entire imaging region of the sample with the excitation light. Then, the fluorescence intensity values on the two-dimensional photodetector are collected to obtain one piece of overall fluctuation information.
[0006]
Further, in JP-A-08-43939 and JP-A-2000-98245, in a scanning optical microscope, fluorescence from a multi-stained sample is separated by a grating and a prism, and the component wavelength is detected by a plurality of photodetectors. Detection is performed every time. With this method, signals for each fluorescent dye can be extracted from the entire imaging area of the sample that is excited at one time, but the fluorescence signals emitted from a plurality of selected minute specific sites in the imaging area of the sample are respectively obtained. It cannot be taken out separately.
[0007]
In the conventional fluorescence correlation spectroscopy, fluctuations in fluorescence intensity from fluorescent molecules present in a visual field are observed, and a time-series signal is obtained based on the fluctuation to obtain an autocorrelation function. In this case, if only one type of fluorescent molecule exists in the visual field, information such as the translational diffusion speed of the fluorescent molecule can be obtained by directly analyzing the fluctuation of the obtained fluorescence intensity. Further, even if these fluorescent molecules move or change the movement speed, these changes can be statistically grasped. When two or more types of fluorescent molecules having different emission wavelengths are present in a sample, autocorrelation and cross-correlation of each fluorescent molecule can be obtained by performing wavelength separation. However, they are limited to the same field of view.
[0008]
When observing actual living cells, it is necessary to observe the behavior of desired molecules inside and outside the cell, inside and outside of the cell nucleus in real time, and to perform chronological analysis of events such as intracellular signaling, mass transport, and cell division. It is necessary to obtain change and localization information. In the conventional fluorescence correlation spectroscopy, it is possible to capture the state change and behavior as a group of molecules, but it is impossible to dynamically measure the state change of a desired site inside and outside a cell.
[0009]
In addition, in order to move the sample stage of the microscope in the Z-axis direction using a drive mechanism such as a motor, the motor must be fixed near the sample stage. Work was interrupted. Further, if the repetitive operation of the sample stage is continuously performed, there is a problem that the actual focus position and the calculated focus position are shifted due to backlash of the motor.
[0010]
In addition, when a focus position adjustment mechanism that is movable by adding a lens is used, the optical system becomes complicated, and if the adjustment of the lens movement mechanism is not performed accurately, the optical axis shifts due to lens movement or the lens is tilted. There were problems such as bending of the optical axis.
[0011]
That is, conventionally, laser light cannot be easily moved or scanned three-dimensionally by a simple mechanism.
[0012]
An object of the present invention is to provide a biomolecule analyzing apparatus capable of acquiring an image of a sample with a simple configuration and performing a fluorescence correlation analysis.
[0013]
[Means for Solving the Problems]
In order to solve the problem and achieve the object, the biomolecule analyzing apparatus of the present invention is configured as follows.
[0014]
(1) A biomolecule analyzer of the present invention is a biomolecule analyzer for analyzing dynamic behavior of a biomolecule, and observes at least one biological sample containing a biomolecule held in a measurable state. Image acquisition means for acquiring an image corresponding to an area, arrangement means for arranging measurement points at desired point positions in the sample image acquired by the image acquisition means, and measurement from the measurement points arranged by the arrangement means Measuring means for measuring a signal derived from dynamic information of a substance, and analyzing means for analyzing a result measured by the measuring means, wherein at least one of the image acquiring means and the arranging means is at least one variable Consists of a focusing optical element.
[0015]
(2) The biomolecule analyzing apparatus according to the present invention is the apparatus according to the above (1), wherein the image acquired by the image acquiring means is an image including a three-dimensional area, and Arrange at any position on the dimension.
[0016]
(3) The biomolecule analyzing apparatus of the present invention is the apparatus according to the above (1), and the at least one variable focus optical element performs at least one of scanning in a two-dimensional direction and Z-axis focus movement.
[0017]
(4) The biomolecule analyzing apparatus according to the present invention is the apparatus according to the above (1) or (3), and the at least one variable focus optical element includes at least one of a variable focus lens and a variable focus mirror.
[0018]
BEST MODE FOR CARRYING OUT THE INVENTION
(First Embodiment)
FIG. 1 is a diagram showing a basic configuration of an illumination optical system, a scanning optical system, and a detection optical system of a biomolecule analyzing apparatus according to a first embodiment of the present invention, and FIG. 2 is a partially enlarged view thereof. is there. 1 and 2, the same parts are denoted by the same reference numerals. In the first embodiment, as shown in FIGS. 1 and 2, a variable focus lens 11 is installed in the middle of the optical path, specifically, from the light source on the near side of the objective lens 5 (in the middle of the collimated light). I have.
[0019]
The laser light emitted from the laser light source 1 is focused by the first lens 2. An illumination light pinhole 3 is arranged at the in-focus position. The position of the illumination light pinhole 3 is adjusted to the focus position of the second lens 4. The second lens 4 guides the collimated light (parallel light) to the objective lens 5. Further, the sample surface is adjusted to the focal position of the objective lens 5.
[0020]
The laser light emitted from the laser light source 1 passes through the dichroic mirror 7 through the first lens 2 and the pinhole 3 for illumination light, and passes through the second lens 4 to the X-axis scanning scanner 61 and the Y-axis scanning scanner. Reach 62. The scanning directions of the X-axis scanning scanner 61 and the Y-axis scanning scanner 62 are orthogonal to each other. The laser beam is scanned in the X-axis and the Y-axis by the X-axis scanning scanner 61 and the Y-axis scanning scanner 62, respectively, and is two-dimensionally scanned in the plane of the sample S via the varifocal lens 11 and the objective lens 5.
[0021]
The fluorescent signal from the fluorescent molecule in the sample S passes through the same optical path as the irradiated laser light and is reflected by the dichroic mirror 7 disposed between the second lens 4 and the illumination light pinhole 3. The reflected light passes through the light receiving pinhole 8 arranged at the focal position of the second lens 4 and is guided to the light receiving lens 9. The dichroic mirror 7 has a spectral characteristic of transmitting irradiated laser light (excitation light) and reflecting fluorescence emitted from fluorescent molecules. The light receiving pinhole 8 is located at the focal position of the light receiving lens 9. The fluorescent light reaches the light detector (light receiver) 10 through the light receiving lens 9. As the photodetector 10, a two-dimensional photodetector such as a CCD camera for acquiring an image is used. An APD (avalanche photodiode), a photomultiplier, or the like is used to measure the intensity fluctuation of the fluorescence.
[0022]
The varifocal lens 11 is composed of two transparent glass substrates that are arranged facing each other, electrodes attached to the two glass substrates, and liquid crystal filled between the two glass substrates. The voltage applied to the liquid crystal is adjusted so that the liquid crystal molecules have an inclination in a certain direction, the light passing through the liquid crystal is subjected to wavefront modulation or phase modulation, and a lens function is provided to change the focal position. Since the focus position is changed by adjusting the voltage, it can be smoothly performed.
[0023]
Further, as disclosed in JP-A-5-100201, a liquid crystal is arranged between a common electrode and an annular electrode, and a voltage applied to the annular electrode is controlled to form a refractive index distribution in the liquid crystal. Alternatively, a varifocal lens configured so that the focal length can be freely changed may be used. Alternatively, as disclosed in JP-A-5-303011, a transparent film made of a synthetic resin film is filled with a transparent liquid such as silicon oil, and the capacity is changed to change the shape of the transparent film. May be used to change the focal length.
[0024]
FIG. 3 is a diagram showing a configuration of the variable focus mirror. For the focus movement of the laser beam in the optical axis direction, a variable focus mirror as shown in FIG. 3 may be used. The varifocal mirror 12 is configured by arranging electrodes on a deformable film 121, for example, a film (about 1 to 5 μm in thickness) of polyimide or the like, and attaching a metal thin film such as aluminum to the surface of the deformable film 121. Then, by applying a current to the counter electrode 122, an electrostatic attraction is generated, and the curvature of the film surface is changed to form a concave surface, thereby giving a lens effect.
[0025]
Also, by changing the voltage applied to the counter electrode 122, the curvature of the concave surface changes, and the focal position can be changed on the optical axis. In this case, as shown in FIG. 4, the varifocal mirror 12 mirror-reflects a parallel light beam from a laser serving as a light source, and at the same time, makes the reflected light into convergent light. By passing the reflected light (convergent light) through the objective lens 5, the focus position of the laser light can be arbitrarily changed in the Z-axis direction (depth direction) in the sample S. By using the variable focus mirror 12 in this way, the variable focus mechanism can be realized smoothly, the absorption loss of incident light can be reduced as much as possible, and light can be used effectively.
[0026]
As another configuration of the varifocal mirror, for example, a voltage is applied between a single crystal silicon thin film and a lower electrode film, and the shape of the single crystal silicon thin film is curved by electrostatic attraction to form a concave parabolic surface. It can be. By attaching a metal film such as aluminum to a thin film of single crystal silicon, a mirror surface can be formed. In this regard, Optronics Magazine No. 7, 1999, p. 161 to 166.
[0027]
FIG. 5 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to the first embodiment of the present invention.
[0028]
In order to obtain a two-dimensional microscope image, two-dimensional scanning of the laser light source 1a is required. For example, two galvanometer mirrors are arranged so that the scanning directions are orthogonal to each other to form an XY scanner 6a (consisting of an X-axis scanning scanner and a Y-axis scanning scanner), and the laser beam performs X-axis scanning and Y-axis scanning. This is defined as a first scanning system.
[0029]
The laser light emitted from the laser light source 1a is reflected by the dichroic mirror 71, scanned in the X-axis and Y-axis by the XY scanner 6a, reflected by the dichroic mirror 73, and passed through the varifocal lens 11 and the objective lens 5. Two-dimensional scanning is performed in the S plane, that is, in the cell. The reflected light and fluorescent light from the sample S pass through the same optical path as the irradiation light, and are received by the photodetector 101 via the dichroic mirror 71. The light intensity signal incident on the photodetector 101 is converted into an electric signal, the waveform is shaped by the signal processing device 103, and the signal is guided to the image processing device 106. The image processing device 106 generates an image signal, and generates a two-dimensional image of the sample S on the TV monitor 107.
[0030]
On the other hand, similarly to the first scanning system, for example, two galvanometer mirrors are arranged so that the scanning directions are orthogonal to each other to form an XY scanner 6b (consisting of an X-axis scanning scanner and a Y-axis scanning scanner). Are scanned in the X-axis and Y-axis directions. This is the second scanning system. The laser beam is scanned by the second scanning system, a fluorescence signal from a fluorescent molecule is received by the photodetector 102, and a correlation spectral analysis is performed by the computer 105.
[0031]
As a laser as the excitation light, an argon laser having a wavelength of 488 nm and a He.Ne laser having a wavelength of 633 nm are used. Rhodamine green (RhG) and sifive (Cy5) are used as fluorescent dyes for labeling a desired portion in a cell. Rhodamine green (RhG) is excited with an argon laser at a wavelength of 488 nm. Sifive (Cy5) is excited by a 633 nm He.Ne laser.
[0032]
The laser light (excitation light) emitted from the laser light source 1b is reflected by the dichroic mirror 72, further reflected by the mirror 74, scanned in the X-axis and Y-axis by the XY scanner 6b, and passed through the lens 75 and the dichroic mirror 73. Is two-dimensionally scanned in the plane of the sample S via the variable focus lens 11 and the objective lens 5.
[0033]
The fluorescence from the fluorescent dye molecule at a desired location in the sample S passes through the same optical path as the excitation light, and is separated from the incident optical path by the dichroic mirror 72 adjusted according to the emission wavelength of each dye. Is received at. The light intensity signal incident on the photodetector 102 is converted into an electric signal, waveform-shaped by a signal processing device 103, converted into an on-off binarized pulse, and guided to a computer 105. A correlation operation is performed on the binarized pulse signal input to the computer 105 to obtain an autocorrelation function. Further, from the obtained autocorrelation function, a translational diffusion speed of the fluorescent molecule, a change in the number of the fluorescent molecule in the measurement region, and the like are obtained.
[0034]
When the light intensity signal incident on the photodetector 102 is relatively large, the electric signal output from the photodetector 102 is a time-series signal. In this case, the electric signal is A / D-converted by the signal processing device 103 to be converted into a digital signal, and then the waveform is shaped and converted into a binarized pulse signal as before. A correlation operation is performed by introducing the binarized pulse signal to the computer 105, and an autocorrelation function is obtained.
[0035]
The X-axis scanning scanner and the Y-axis scanning scanner of the first scanning system and the second scanning system are arranged so as to scan in directions orthogonal to each other, and are driven by the XY scanner driving devices 111 and 111 controlled by the computer 105. The scanning movement is precisely controlled by 112. The XY scanner driving devices 111 and 112 are provided with a scanning position detection mechanism, and the scanning position is detected with high accuracy in real time, and is feedback-controlled to the computer 105. As a result, the scanning position and the image match.
[0036]
The Z-axis focus movement by the variable focus mechanism is performed by the variable focus lens driving device 113. The varifocal lens driving device 113 is connected to the computer 105, receives a drive control signal from the computer 105, adjusts the alignment state of the liquid crystal molecules of the varifocal lens 11 based on the control signal, and moves the Z-axis focus. Perform In this manner, the Z-axis focus movement is performed in conjunction with the two-dimensional scanning, and a three-dimensional image is generated on the TV monitor 107. Further, the irradiation position of the laser beam can be three-dimensionally moved to a desired position inside or outside the cell S simultaneously with the image acquisition.
[0037]
The scanning angles of the two independent X-axis scanning scanners and Y-axis scanning scanners performed by the above-described galvanometer mirror or the like are detected by an XY scanner angle detection device 114. The detection signal is guided to the image processing device 106, is combined with the image signal output from the image processing device 106, and matches the position of the microscope image of the sample S on the screen of the TV monitor 107.
[0038]
Designation of a scanning point on a two-dimensional microscope image is performed as follows. The observer designates a desired point using the designation means (keyboard, mouse pointer, etc.) of the computer 105 while viewing the observation image on the TV monitor 107. Then, the computer 105 adjusts the XY scanner driving device 112 so as to stop the scanning of the XY scanner 6b at one or more points designated on the screen. The point on the Z-axis is also specified based on the image in the Z-axis direction by the varifocal lens 11 driven in conjunction with the XY scanner 6b, whereby an arbitrary three-dimensional position of the sample S is obtained. The measurement point is located at. The scanning angle of the XY scanner 6b at this time is accurately detected by the XY scanner angle detecting device 114.
[0039]
In addition, by configuring a scanning system with a variable focus lens or a variable focus mirror instead of scanning with a galvanometer mirror, fluorescence correlation analysis of desired cells or molecules can be performed in a three-dimensional direction in a sample.
[0040]
In the first embodiment, an XY scanner (for example, using a galvanometer mirror) for acquiring a two-dimensional image using a variable focus lens, and an XY scanning optical system (for example, a galvanometer) for performing fluorescence correlation analysis. A mirror is used. In FIG. 1, a three-dimensional image of the inside and outside of the same sample located on the Z axis of the XY scanner can be obtained, and the fluorescence correlation analysis can be performed. That is, in the first embodiment, the Z-axis focus movement can be performed simultaneously for the optical system for acquiring the image and the optical system for performing the correlation analysis of the fluorescence. The angle of the XY scanner is detected by the XY scanner angle detection device, and the scan position can be surely matched with a desired part.
[0041]
(Second embodiment)
FIG. 6 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to the second embodiment of the present invention. 6, the same parts as those in FIG. 5 are denoted by the same reference numerals.
[0042]
In this case, a variable focus lens and a variable focus mirror are used for scanning with laser light. In the second embodiment, a two-dimensional image of a sample such as a cell is acquired by a first scanning system using a variable focus lens 11. The varifocal lens driving device 113 is connected to the computer 105 via a varifocal lens angle detecting device 116. Further, two-dimensional scanning is performed by the variable focus mirror 12 in the second scanning system, and fluorescence correlation analysis is performed on molecules at desired locations (for example, three locations) inside and outside the cell. The variable focus mirror 12 is driven by a variable focus mirror driving device 115. The variable focus mirror driving device 115 is connected to the computer 105 via a variable focus mirror angle detection device 117, and a drive control signal is sent from the computer 105.
[0043]
Of course, a variable focus mirror may be used as a scanning system for acquiring a two-dimensional image of the sample, and a variable focus lens may be used as a scanning system for performing fluorescence correlation analysis at a desired site inside or outside a cell. Furthermore, a scanning system for acquiring an image of a sample and a scanning system for performing fluorescence correlation analysis at a desired site inside and outside a cell may be all variable-focus mirrors or variable-focus lenses.
[0044]
The scanning angle and the Z-axis focus position of the varifocal lens 11 and the varifocal mirror 12 are detected by the varifocal lens angle detecting device 116 and the varifocal mirror angle detecting device 117, respectively. These detection signals are guided to the image processing device 106, combined with the image signals output from the image processing device 106, and matched with the position of the microscope image of the sample S on the screen of the TV monitor 107.
[0045]
(Third embodiment)
FIG. 7 is a diagram illustrating a configuration of a biomolecule analyzing apparatus according to the third embodiment of the present invention. FIG. 7 is a diagram showing a configuration example of a plurality of scanning systems for performing fluorescence correlation analysis of a plurality of desired cells and molecules in a three-dimensional direction in a sample and a scanning system for acquiring an image of the sample. is there.
[0046]
In FIG. 7, four illumination systems S11a, S12a, S22a, S32a, three scanning systems S11b, S12b, S13b, and four detection systems S11c, S12c, S22c, S32c are provided. The illumination systems S11a, S12a, S22a, S32a include laser light sources 11a, 11b, 21b, 31b and first lenses 12a, 12b, 22b, 32b, respectively. The scanning systems S11b, S12b, and S13b are composed of servo type galvano scanners (galvanometer mirrors) 16a, 16b, and 16c, which are XY scanners, respectively. Each of the galvano scanners 16a, 16b, and 16c includes an X-axis scanning scanner and a Y-axis scanning scanner. The detection systems S11c, S12c, S22c, and S32c include light receiving lenses 19a, 19b, 29b, 39b, light receiving pinholes 18a, 18b, 28b, 38b, and photodetectors 110a, 110b, 210b, 310b, respectively.
[0047]
First, in order to acquire an image of a biological sample (cell) S held in a state that can be measured on the stage ST, a first illumination system S11a, a first scanning system S11b, and a first detection system S11c is used. The laser light emitted from the laser light source 11a reaches the galvano scanner 16a via the first lens 12a and the dichroic mirror 17a. The laser beam is scanned XY by the galvano scanner 16a, reflected by the dichroic mirror 201, transmitted through the dichroic mirror 202, and illuminates the sample S on the stage ST via the objective lens 5.
[0048]
The reflected light and the fluorescent light from the sample S pass through the dichroic mirror 202 via the objective lens 5 and are reflected by the dichroic mirror 201, so that the galvano scanner 16b, the dichroic mirror 17a, the mirror 191a, the light receiving lens 19a, and the light receiving The light is received by the photodetector 110a via the pinhole 18a. The light detector 110a measures the intensity of the light signal. The optical signal is subjected to image processing such as enhancement of contrast and contour enhancement by an image processing device, and then guided to a computer to be converted into a two-dimensional image on a TV monitor.
[0049]
Next, in order to obtain the autocorrelation function of the fluorescent molecules in the sample S, for example, the second illumination system S12a, the second scanning system S12b, and the second detection system S12c are used. The laser light emitted from the laser light source 11b reaches the galvano scanner 16b via the first lens 12b, the dichroic mirror 17b, and the mirror 203. The laser light is scanned XY by the galvano scanner 16b, reflected by the dichroic mirror 202, and illuminates the sample S on the stage ST via the objective lens 5. Thus, similarly to the first embodiment, the laser light excites the fluorescent molecules in the sample S existing in the minute measurement region, and a fluorescent signal (photon pulse) is obtained.
[0050]
The obtained fluorescence signal, that is, the intensity fluctuation of the fluorescence from the fluorescent molecule, is reflected by the dichroic mirror 202 via the objective lens 5, and the galvano scanner 16b, the mirror 203, the dichroic mirror 17b, the mirror 191b, the light receiving lens 19b, and The light is received by the photodetector 110b via the light receiving pinhole 18b. The fluorescence signal is converted into a photocurrent pulse by the photodetector 110b, guided to a signal processing device to perform waveform shaping, binarization processing, and the like, and an autocorrelation function, a cross-correlation function, and the like are performed by a computer (correlation analysis device). Is required. From the autocorrelation function obtained here, statistical properties such as the speed of the translational diffusion movement of the fluorescent molecule are obtained.
[0051]
In the above example, a combination of the second illumination system S12a, the second scanning system S12b, and the second detection system S12c is used to obtain the correlation function of the fluorescent molecules. A combination of the system S22a, the second scanning system S12b, and the third detection system S22c, and a combination of the fourth illumination system S32a, the third scanning system S13b, and the fourth detection system S32c can be used. That is, each optical system can individually acquire an autocorrelation function or a cross-correlation function from fluorescent molecules present at desired different sites in the sample. Each of the galvanometer scanners 16a, 16b, and 16c operates independently of each other, and focuses laser light spots on a plurality of different portions in the sample. Alternatively, the galvanometer scanners 16a, 16b, and 16c may be linked with each other to simultaneously focus a laser beam spot on a desired portion in the sample.
[0052]
In the third embodiment, the first scanning system acquires a two-dimensional image of a sample such as a cell using an XY scanner. Each of the second to fourth scanning systems performs two-dimensional scanning by an XY scanner, and performs fluorescence correlation analysis of three or more desired molecules inside or outside a cell.
[0053]
For the first scanning system, a varifocal lens can be used as an XY scanner, and for the second to fourth scanning systems, a varifocal mirror can be used as an XY scanner. Of course, a varifocal mirror may be used as the XY scanner of the first scanning system, and a varifocal lens may be used as the XY scanners of the second to fourth scanning systems. Further, all of the first to fourth scanning systems may be varifocal mirrors, or all may be varifocal lenses.
[0054]
In the third embodiment, laser light can be simultaneously focused on three points present in three-dimensionally different portions, and fluorescence correlation analysis of desired cells or molecules can be performed. At this time, a two-dimensional image or a three-dimensional image of the sample is simultaneously acquired by the first scanning system. Further, the number of scanning systems for performing fluorescence correlation analysis of desired cells or molecules can be further increased in parallel. Similarly, a scanning system for acquiring an image of a sample may be similarly increased in parallel to acquire, display, and record microscope images of a plurality of discrete parts.
[0055]
(Fourth embodiment)
FIG. 8 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to a fourth embodiment of the present invention. 8, the same parts as those in FIGS. 5 and 6 are denoted by the same reference numerals.
[0056]
In the fourth embodiment, an XY scanner 6a composed of, for example, a galvanometer mirror is used as a two-dimensional scanning optical system for acquiring a two-dimensional microscope image. An XY scanner 6b composed of, for example, a galvanometer mirror is used as an XY scanning optical system for performing a fluorescence correlation analysis.
[0057]
In addition, the variable focus mirror 12 is used for the focus movement of the laser light in the Z-axis direction. That is, the curvature of the mirror surface of the variable focus mirror 12 is changed by the variable focus mirror driving device 115 to change the focus position of the laser light in the Z-axis direction.
[0058]
Thereby, the focus movement in the Z-axis direction can be simultaneously performed for the optical system for acquiring the microscope image and the optical system for performing the correlation analysis of the fluorescence. That is, it is possible to obtain a three-dimensional image of the inside and outside of the same sample located on the Z axis and to perform a fluorescence correlation analysis.
[0059]
The configuration of the varifocal mirror 12 is the same as that described in the first embodiment. The angles of the XY scanners 6a and 6b are detected by an XY scanner angle detecting device 114, and the XY scanner driving device 112 ensures that the scan position matches a desired portion.
[0060]
(Fifth embodiment)
FIG. 9 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to a fifth embodiment of the present invention. In FIG. 9, the same parts as those in FIGS. 5 and 6 are denoted by the same reference numerals.
[0061]
In the fifth embodiment, two-dimensional scanning for acquiring a two-dimensional microscope image and two-dimensional scanning for performing fluorescence correlation analysis are simultaneously performed by changing the curvature of the varifocal lens 11 or changing the refractive index. Perform by change. In addition, the focus position of the laser light in the Z-axis direction is changed by changing the curvature or the refractive index of the varifocal lens 11. At this time, the irradiating angle of the laser beam by the varifocal lens 11 is detected by the varifocal lens angle detecting device 116, and the focus position of the laser beam is surely matched to a desired portion.
[0062]
Thus, two-dimensional scanning and Z-axis focus movement can be simultaneously performed on the optical system for acquiring an image and the optical system for performing correlation analysis of fluorescence.
[0063]
(Sixth embodiment)
FIG. 10 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to a sixth embodiment of the present invention. In FIG. 10, the same parts as those in FIGS. 5 and 6 are denoted by the same reference numerals.
[0064]
In the sixth embodiment, an XY scanner 6a composed of, for example, a galvanometer mirror is used as a two-dimensional scanning optical system for acquiring a two-dimensional microscope image. Further, a variable focus mirror 12 is used as an XY scanning optical system for performing a fluorescence correlation analysis.
[0065]
The variable focus mirror 12 is also used for the focus movement of the laser light in the Z-axis direction. The change in the curvature of the mirror surface of the variable focus mirror 12 for changing the focus position of the laser light in the Z-axis direction and the change in shape for performing XY scanning are performed by the variable focus mirror driving device 115.
[0066]
Thus, the optical system for acquiring the microscope image and the optical system for performing the correlation analysis of the fluorescence can be independently operated. That is, the observation site of the sample and the site for performing the correlation analysis of the fluorescence can be separately selected. Further, the angles of the XY scanner 6a and the variable focus mirror 12 are detected by the XY scanner angle detection device 114 and the variable focus mirror angle detection device 117, respectively, so that the scan position can be surely matched with a desired portion.
[0067]
(Seventh embodiment)
FIG. 11 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a variable focus mechanism in the Z-axis direction according to a seventh embodiment of the present invention. 11, the same parts as those in FIGS. 5 and 6 are denoted by the same reference numerals.
[0068]
In the seventh embodiment, two-dimensional scanning for acquiring a two-dimensional microscope image is performed by scanning with the variable focus mirror 12a, and two-dimensional scanning for performing fluorescence correlation analysis is performed with the mirror of the variable focus mirror 12b. This is performed by changing the curvature of the surface. The focus movement of the laser beam in the Z-axis direction is separately performed by the optical system for acquiring a two-dimensional image and the optical system for performing a correlation analysis of the fluorescence by using the curvatures of the mirror surfaces of the variable focus mirrors 12a and 12b separately. Is changed. At this time, the irradiation angles of the laser light by the variable focus mirrors 12a and 12b are detected by the variable focus mirror angle detection devices 117a and 117b, and the desired designated position and the focus position of the laser light are surely matched.
[0069]
Thus, two-dimensional scanning and Z-axis focus movement can be simultaneously performed at different positions in the optical system for acquiring an image and the optical system for performing correlation analysis of fluorescence. As a result, a three-dimensional image of the sample can be obtained, and a correlation analysis of fluorescence at a position different from the position at which the image was obtained or at the same position can be performed.
[0070]
(Eighth embodiment)
FIG. 12 is a block diagram showing a configuration of a biomolecule analyzing apparatus having a variable focus mechanism in the Z-axis direction according to the eighth embodiment of the present invention. 12, the same parts as those in FIGS. 5 and 6 are denoted by the same reference numerals.
[0071]
In the eighth embodiment, two-dimensional scanning for acquiring a two-dimensional microscope image is performed by, for example, an XY scanner 6a including a galvanometer mirror. In the optical system for acquiring an image, the focus movement of the laser light in the Z-axis direction is performed by changing the refractive index of the variable focus lens 11 or changing the curvature of the shape. The change in the refractive index of the varifocal lens 11 or the change in the curvature of the shape is performed by the varifocal lens driving device 113. The varifocal lens driving device 113 is connected to the computer 105 via the varifocal lens angle detecting device 116, and controls the magnitude, direction, timing, and the like of the focus movement of the laser light in the Z-axis direction.
[0072]
On the other hand, two-dimensional scanning for performing a fluorescence correlation analysis is performed using, for example, an XY scanner 6b including a galvanometer mirror. In the optical system for performing the correlation analysis of the fluorescence, the focus movement of the laser beam in the Z-axis direction is performed by changing the curvature of the mirror surface of the variable focus mirror 12. The shape of the variable focus mirror 12 is changed by a variable focus mirror driving device 115. The variable focus mirror driving device 115 is connected to the computer 105 and controls a change in curvature of the mirror surface of the variable focus mirror 12.
[0073]
As described above, the change in the curvature of the mirror surface of the varifocal mirror 12 and the focus movement of the laser beam in the Z-axis direction are separately performed by an optical system for acquiring a two-dimensional image and an optical system for performing fluorescence correlation analysis. Or at the same time. At this time, the rotation angles of the two types of XY scanners are detected by the XY scanner angle detection device 114, and the desired designated position and the scanning angle of the laser beam are surely matched.
[0074]
Thus, two-dimensional scanning and Z-axis focus movement can be simultaneously performed at different positions in the optical system for acquiring an image and the optical system for performing correlation analysis of fluorescence. As a result, it is possible to acquire a three-dimensional image of the sample and to perform a correlation analysis of the fluorescence at a position different from the image acquisition position or at the same position. Note that a plurality of optical systems for acquiring an image and an optical system for performing correlation analysis of fluorescence may be arranged.
[0075]
According to the above-described embodiment, in the image acquisition device including the fluorescence correlation analysis device for living cells and biomolecules, the two-dimensional scanning of laser light and the Z-axis (depth direction) focus movement by the laser light are performed by the variable focus lens. Alternatively, a fluorescence correlation analysis of a plurality of three-dimensionally discrete molecules or cells is performed by a variable focus mirror. In addition, a three-dimensional laser beam is scanned using a variable focus optical system to obtain a three-dimensional image of a desired part.
[0076]
As described above, by applying the variable focus optical system to a fluorescence correlation analyzer for a plurality of desired living cells and biomolecules discretely existing, a Z-axis focus movement of a laser beam or a three-dimensional movement can be performed with a simple mechanism. Focus point can be moved. In addition, by using this mechanism in a scanning system for acquiring images, a three-dimensional image of a desired site inside and outside a cell can be recorded and observed, and a plurality of desired molecules distributed three-dimensionally inside and outside a cell. Can also be performed.
[0077]
Further, according to the above-described embodiment, since it is not necessary to use an XY scanning system such as a galvanometer mirror, the apparatus can be simplified, and there is no fear of mechanical abrasion and the like, and stable operation can be performed. It becomes strong against vibration. Further, the optical system for acquiring the image and the optical system for performing the correlation analysis of the fluorescence can be arranged spatially different, and a three-dimensional image at a desired position of the sample can be acquired simultaneously or with a time difference. In addition, correlation analysis of fluorescence at a position different from or the same as the position at which the image was obtained can be performed.
[0078]
Therefore, according to the present invention, the dynamic behavior of various biomolecules can be analyzed with stable accuracy from a sample in a three-dimensional region essential for a living natural state. For example, a protein composed of many proteins Complexes (such as chromatin structure conversion factor) can be analyzed. This makes it possible to elucidate the network mechanism controlling gene expression, elucidate the molecular mechanism of apoptosis, elucidate the mechanism of genome replication control, and observe and record changes in chromosome structure in real time. Through the elucidation of these life phenomena, it can be widely applied to the creation of new drugs, elucidation of canceration mechanisms, proteome analysis, etc. In addition, the dynamics of various organelle components and various ion channels in a biological sample such as a cell can be analyzed. Furthermore, the present invention can be applied to dynamic analysis of neurotransmitters and hormone receptors.
[0079]
Note that the present invention is not limited to the above embodiments, and can be appropriately modified and implemented without changing the gist.
[0080]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the biomolecule analyzer which can acquire the image of a sample with a simple structure and perform the correlation analysis of fluorescence can be provided.
[Brief description of the drawings]
FIG. 1 is a diagram showing a basic configuration of an illumination optical system, a scanning optical system, and a detection optical system of a biomolecule analyzing apparatus according to a first embodiment of the present invention.
FIG. 2 is a partially enlarged view of an illumination optical system, a scanning optical system, and a detection optical system of the biomolecule analyzing apparatus according to the first embodiment of the present invention.
FIG. 3 is a diagram showing a configuration of a variable focus mirror according to the first embodiment of the present invention.
FIG. 4 is a diagram showing the operation of the variable focus mirror according to the first embodiment of the present invention.
FIG. 5 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to the first embodiment of the present invention.
FIG. 6 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to a second embodiment of the present invention.
FIG. 7 is a diagram showing a configuration of a biomolecule analyzing apparatus according to a third embodiment of the present invention.
FIG. 8 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to a fourth embodiment of the present invention.
FIG. 9 is a block diagram showing a configuration of a biomolecule analyzing apparatus including a Z-axis direction variable focus mechanism according to a fifth embodiment of the present invention.
FIG. 10 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to a sixth embodiment of the present invention.
FIG. 11 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to a seventh embodiment of the present invention.
FIG. 12 is a block diagram showing a configuration of a biomolecule analyzing apparatus provided with a Z-axis direction variable focus mechanism according to an eighth embodiment of the present invention.
[Explanation of symbols]
1, 1a, 1b: laser light source 2: first lens 3: pinhole for illumination light 4: second lens 5: objective lens 61: X-axis scanning scanner 62: Y-axis scanning scanner 6a, 6b: XY scanner
7 dichroic mirror 8 light receiving pinhole 9 light receiving lens 10 photodetector 11 variable focus lens 12 variable focus mirror 71 dichroic mirror 72 dichroic mirror 73 dichroic mirror 74 mirror 75 lens 101 Reference Signs List 102 photodetector 103 signal processing device 105 computer 106 image processing device 107 TV monitor 111, 112 XY scanner driving device 113 variable focus lens driving device 114 XY scanner angle detection device 115 variable focus mirror driving Device 116: Variable focus lens angle detection device 117: Variable focus mirror angle detection device 121: Deformable film 122: Counter electrode 201, 202 ... Dichroic mirror 203, 204, 205 ... Mirror S: Sample

Claims (4)

A biomolecule analyzer for analyzing dynamic behavior of biomolecules,
Image acquisition means for acquiring an image corresponding to at least one observation region of a biological sample containing a biomolecule held in a measurable state,
Arranging means for arranging measurement points at desired point positions in the sample image acquired by the image acquiring means,
Measuring means for measuring a signal derived from the dynamic information of the substance to be measured from the measuring points arranged by the arranging means,
Analyzing means for analyzing the result measured by the measuring means,
A biomolecule analyzing apparatus, wherein at least one of the image acquisition unit and the arrangement unit includes at least one variable focus optical element. The image obtained by the image obtaining means is an image including a three-dimensional area,
2. The biomolecule analyzing apparatus according to claim 1, wherein the arranging unit arranges the measurement points at an arbitrary position in three dimensions. The biomolecule analyzer according to claim 1, wherein the at least one variable focus optical element performs at least one of scanning in a two-dimensional direction and Z-axis focus movement. 4. The biomolecule analyzing apparatus according to claim 1, wherein the at least one variable focus optical element includes at least one of a variable focus lens and a variable focus mirror.

Images