JP2015022070A - Optical microscope system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an optical microscope system capable of observing in a super-resolution manner how a plurality of molecules and nano-structures existing within an optical diffraction limit size move at a high speed equal to or more than a video rate.SOLUTION: The optical microscope system includes: a laser light source 3 for radiating an excitation light which causes a fluorescence probe to emit a light in excitation; an optical system 8 for obtaining an optical image of the fluorescence probe excited by the excitation light; a spectrometer 11 for subjecting an optical image of the fluorescence probe to an imaging spectral; and an imaging device 12 for obtaining an imaged image by imaging a spectral image obtained by the spectrometer; and analysis means for analyzing the imaged image and estimating respective positions of the fluorescence proves of a plurality of colors from the light point distribution.

Description

  The present disclosure relates to a super-resolution optical microscope system capable of acquiring an optical image with a resolution on the order of nanometers that is less than the diffraction limit size of light, and in particular, can simultaneously grasp the positions of fluorescent particles of multiple colors. It is related with the optical microscope system which can be performed.

  Optical microscopes and their peripheral devices have contributed to research in various fields.In recent years, optical microscopes and their peripheral devices have a function of capturing images of observation objects with an imaging device. It is sent to an external device such as a large monitor for image analysis and phenomenon analysis. In addition, by combining analysis means for analyzing images acquired with an optical microscope, it is impossible to grasp with an optical microscope alone, and it is possible to observe finely below the diffraction limit size of light that is in the order of several hundred nanometers in the visible light range. An optical microscope system that can observe an object has also been put into practical use.

  For example, in the field of life science research, a fluorescent substance is used as a label in order to analyze the changes in the fine structure of cells and cell differentiation patterns using a high-resolution optical microscope. Observing changes is being done. As an observation method using a high-resolution microscope in the life science research field, photo-activated localization microscopy, single-molecule fluorescence tracking technology, and the like are known.

  In photo-activated local microscopy, a molecule to be observed is labeled with a fluorescent probe that is activated when irradiated with ultraviolet light and emits light by excitation light, and the fluorescent probe in the molecule is sequentially activated to grasp its position. The work is repeated and the obtained information is reconstructed as an image. By controlling the intensity of the activation light and limiting the number of fluorescent probes activated at one time, the emission of each fluorescent probe can be regarded as discrete bright spots, and the resulting images are synthesized Thus, a molecular image can be obtained with a resolution of, for example, 10 nanometers, which is not more than the diffraction analysis size in the optical system of the optical microscope (see Non-Patent Document 1 and Patent Document 1).

  The single-molecule fluorescence tracking technique is a method for observing the dynamics of motor proteins and membrane proteins one molecule at a time. By observing a molecule to be observed with a fluorescent probe and observing it with a microscopic method with a small background light such as a total reflection illumination fluorescent microscope, the movement of the bright spot of the molecule can be observed as a moving image. Tracking the dynamics of molecules at the nanometer level with a resolution of several milliseconds to several tens of milliseconds by performing the task of identifying the location of a bright spot using a Gaussian fit method, etc., on each frame image. (See Non-Patent Document 2).

JP 2010-102332 A

Eric Betzig et al. "Imaging intracellular fluorescent proteins at nanometer resolution" SCIENCE vol.313 1642-1645 (2006) Yildiz A el al. "Myosin V walks hand-over-hand: single fluorescent imaging with 1.5-nm localization" SCIENCE vol.300 2061-5 (2003)

  According to the observation methods using the conventional optical microscope, it is possible to observe a living body with a high resolution equal to or smaller than the diffraction limit size of light.

  However, in the photo-activated localized microscopy, it is necessary to acquire a large number of images in order to obtain a single super-resolution image, and a time of several minutes to several hours is required. For this reason, the dynamics of biomolecules that move at high speed in the nanometer to micrometer space cannot be observed. In addition, the single-molecule fluorescence tracking technology can observe the dynamics of a single molecule, but cannot simultaneously observe the movement of a plurality of molecules that are close to each other within a distance within the diffraction limit of light.

  Therefore, the present application solves the above-mentioned conventional problems, and super-resolution observation of the movement of a plurality of molecules and nanostructures existing within the diffraction limit size of light at a high speed equivalent to or higher than the video rate. An object of the present invention is to obtain an optical microscope system.

  In order to solve the above-described problems, an optical microscope system disclosed in the present application is an optical microscope system that acquires an optical image of an object having a size equal to or smaller than the diffraction limit of light using a plurality of color fluorescent probes, A laser light source for irradiating excitation light for exciting the probe, an optical system for obtaining an optical image of the fluorescent probe excited by the excitation light, a spectroscope for imaging spectroscopy of the optical image obtained by the optical system, An optical microscope having an imaging device that captures a spectral image obtained by the spectroscope to obtain a captured image, and analyzes the captured image and estimates the positions of the fluorescent probes of the plurality of colors from the bright spot distribution And an analyzing means.

  The optical microscope system disclosed in the present application estimates the position of each fluorescent probe by analyzing a captured image obtained by spectrally dividing the light images of the fluorescent probes of a plurality of colors with a spectroscope. Therefore, it is possible to accurately estimate the position of multiple fluorescent probes existing in the region below the diffraction limit size of light based on the emission color, super-resolution of the observation object, and observation at high speed An image can be obtained.

The block diagram for demonstrating schematic structure of the optical microscope part in the optical microscope system concerning this embodiment. The schematic diagram for demonstrating the principle of the optical microscope concerning this indication. The figure which shows the myosin V of a measuring object and the spectral image obtained with the optical microscope concerning this embodiment. The graph which shows the relationship between the distance from the position of an observation start time, and time about two quantum dots. The block block diagram explaining the structure of the modification of the optical microscope system of this embodiment. The figure which shows typically the 1st light image which is not rotating, the rotation light image which rotated 90 degrees, and also the spectral image which carried out the spectroscopy of them. The figure which shows the specific example of the optical image rotated by the optical microscope system of this embodiment mutually rotated. The figure which shows the state which isolate | separated and observed the two-dimensional position of the three fluorescent probes arrange | positioned in the area | region within the diffraction limit size of light. The figure explaining a mode that the position change of the fluorescence probe of multiple colors was caught as a two-dimensional moving image. The figure explaining a mode that the position change of the fluorescence probe of multiple colors was caught as a two-dimensional moving image. The figure explaining the method to specify light emission from the same fluorescence probe in two optical images obtained by the separation optical system.

  The optical microscope system of the present disclosure is an optical microscope system that acquires an optical image of an object having a size that is less than the diffraction limit of light using a plurality of color fluorescent probes, and irradiates excitation light that excites the fluorescent probe to emit light. A laser light source, an optical system for obtaining an optical image of the fluorescent probe excited by the excitation light, a spectroscope for imaging spectroscopy of the optical image obtained by the optical system, and a spectral image obtained by the spectroscope And an optical microscope having an imaging device that obtains a captured image, and an analysis unit that analyzes the captured image and estimates the positions of the fluorescent probes of the plurality of colors from the bright spot distribution.

  By doing so, since it exists in a region smaller than the diffraction limit size of several hundreds of nanometers, the fluorescent probes of multiple colors that are observed in an optically overlapping state are emitted. Each position can be estimated by analyzing means and separated by color. For this reason, an object can be observed with high resolution of nanometer order. In addition, since observation and spatial coordinate acquisition can be performed simultaneously at the imaging speed of the camera, high-speed observation is possible.

  In the optical microscope system according to the present disclosure, the optical system includes a first partial optical system that obtains a first light image of the fluorescent probe, and a rotation that is rotated 90 ° to the left or right with respect to the first light image. A splitting optical system including a second partial optical system that obtains an optical image, and the spectroscope includes the first optical image obtained by the first partial optical system and the second partial optical system. It is preferable that the rotational light image obtained in step 1 is simultaneously imaged and the imaging device captures the spectral image of the first light image and the spectral image of the rotational light image as one captured image. By doing so, it is possible to acquire a spectral image based on two optical images rotated by 90 ° as one captured image.

  The two-dimensional arrangement of the fluorescent probes of the plurality of colors is made to correspond to the positions of the fluorescent probes of the plurality of colors obtained from the spectral image of the first light image and the spectral image of the rotated light image. It is preferable to specify the position. By doing so, it is possible to easily acquire the two-dimensional position information of the observation object.

  Furthermore, it is preferable to use quantum dots as the fluorescent probe. By using quantum dots that are emitted by a single excitation light and can be realized as an index of multiple colors, a maximum of about a dozen or so fluorescent probes can be separated and observed simultaneously.

  It is preferable that the analysis unit estimates the position of the fluorescent probe from the luminance distribution of the captured image by a Gaussian fit method. By using the Gaussian fit method using a Gaussian function, the position of each fluorescent probe can be estimated quickly and accurately.

  It is preferable that the imaging device captures the captured image at a video rate or higher, and obtains a moving image of the position change of the fluorescent probe from the obtained captured image. In this way, the dynamics of the observation object can be observed as a moving image.

  Moreover, it is preferable that the said optical microscope is further provided with the control part which performs operation | movement control of the said microscope apparatus, and the said analysis means is incorporated as a function of the said control part. By doing in this way, the optical microscope which implement | achieved high resolution is realizable independently.

(Embodiment)
Hereinafter, embodiments of an optical microscope system according to the present disclosure will be described with reference to the drawings.

  FIG. 1 is a block diagram for explaining a schematic configuration of an optical microscope portion in the optical microscope system according to the present embodiment.

  In the optical microscope shown in FIG. 1, the observation object 1 is placed on a stage (not shown in FIG. 1), and fine adjustment is possible in each of the x, y, and z directions by operating the stage.

  The observation object 1 is irradiated with the laser light 4 from the laser light source 3 through the objective lens 2. As an example, the laser light 4 emitted from the laser light source 3 can be a blue semiconductor laser having a wavelength of 488 nm.

  The laser light 4 irradiated from the laser light source 3 is converted into a parallel light beam having a predetermined thickness by the beam expander 5, reflected by the blue reflecting dichroic mirror 6, and irradiated to the observation object 1 through the objective lens 2. The For example, the objective lens 2 having a numerical aperture NA = 1.4 and a magnification of 150 times can be used.

  1 shows an example of the coaxial epi-illumination in which the laser beam 4 is irradiated through the objective lens 2, but the optical microscope according to the present embodiment illuminates the entire sample surface uniformly ( In addition to the coaxial epi-illumination shown in the drawing, other illumination methods such as side illumination, total reflection illumination, and transmission illumination can be employed. When total reflection illumination is used, the background light from the fluorescent substance floating in the solution can be suppressed, so that there is an advantage that the fluorescent probe in the sample can be easily detected.

  The laser light 4 irradiated to the observation object 1 excites the fluorescent probe labeled on the observation object 1 to emit light. In the present embodiment, quantum dots (QDs) are used as fluorescent probes. Details of the quantum dots will be described later.

  The optical image 7 of the fluorescent probe excited and emitted by the laser beam 4 is shifted from the green to red side, which is the long wavelength side, with respect to the blue laser beam 4. For this reason, the optical image 7 passes through the objective lens 2 and then passes through the blue reflecting dichroic mirror 6, and has a total magnification of several hundreds by the magnifying relay optical system 8 composed of optical members such as a lens, a mirror and a diaphragm. It is enlarged so as to be about double (in the case of this embodiment, 375 times as an example).

  The enlarged optical image 7 of the observation object 1 is collected by the condenser lens 9 and enters the spectroscope 11 through the slit 10 and is dispersed. In the present embodiment, a dispersive imaging spectroscope is used as the spectroscope 11. As the spectroscope 11, various dispersive spectroscopes using a reflective or transmissive diffraction grating, a reflective or transmissive prism, and the like can be used, and a light wave is split in an axial direction perpendicular to the slit 10. At the same time, a spectral image in which the spatial position information is directly imaged in the direction parallel to the slit 10 is obtained.

  The spectral image of the light emission 7 of the fluorescent probe dispersed by the spectroscope 11 is captured by an EM-CCD (Electron Multiplying CCD) which is an imaging device 12 attached to the spectroscope 11 to obtain a captured image.

  The captured image captured by the imaging device 12 is output to an analysis unit (not shown) as analysis means, and the analysis unit calculates the fluorescent probe position as the center position based on the bright spot distribution on the captured image. The optical microscope system according to the present embodiment uses a Gaussian fitting method that performs fitting using a Gaussian function, which is the most excellent method for estimating the center position of a fluorescent probe from captured image data. In the optical microscope system of the present embodiment, the Gaussian fit method using an elliptic Gaussian function is used in consideration of the distribution shape of bright spots in the captured image. In addition to the Gaussian fit method, various numerical analysis methods such as a pattern fitting method based on cross correlation and a centroid position calculation method using light intensity as a weight can be used.

  In addition, the analysis part in the optical microscope system of this embodiment should just perform a fixed calculation process with respect to the acquired captured image data, grasp | ascertain the position of a fluorescent probe, and can output the position data. For this reason, an analysis part is realizable as a computer into which the captured image imaged with the imaging device 12 was input. In addition, when the optical microscope includes a control unit that performs various controls of the microscope apparatus itself, such as movement control of the stage on which the laser light source 3 and the observation object 1 are arranged, and autofocus control, the analysis means controls As one function incorporated in the microcomputer unit in the unit, it can be realized integrally with the optical microscope.

  Further, a display of an external computer that functions as an analysis unit is used as a monitor that displays an image captured by the imaging device 12 and / or an image indicating the position of the fluorescent probe as a result of analysis by the analysis unit. be able to. Furthermore, the optical microscope may include an image display panel, and the image display panel may be used as a monitor that displays an image indicating the position of the fluorescent probe as a result of analysis by the analysis unit.

  FIG. 2 shows an optical microscope system according to the present disclosure that separates and distinguishes a plurality of color fluorescent probes arranged in a region of several tens of nanometers that are equal to or smaller than the diffraction limit size of light as an observation target, It is a schematic diagram for demonstrating the principle which can grasp | ascertain the position of a probe.

  In FIG. 2, FIG. 2 (a) shows an arrangement state of fluorescent probes as observation objects, and FIG. 2 (b) shows an observation object optically grasped by an optical microscope whose structure has been described with reference to FIG. FIG. 2C shows a spectral image obtained by spectrally dividing the optical image.

  As shown in FIG. 2A, it is assumed that the fluorescent probes of four colors are arranged in a dense state in the observation object. The fluorescent probe shown in FIG. 2A has four colors arranged in the order of green 21, orange 22, yellow 23, and red 24 from the top. In the optical microscope system of the present embodiment, it is assumed that each color quantum dot is used as a fluorescent probe. Quantum dots used as fluorescent probes are semiconductor fine particles having a polymer-coated particle diameter of about several nanometers to 20 nanometers, and commercially available products having a particle diameter of 500 nm to 800 nm are widely distributed.

  Since the particle size of the quantum dot, which is a fluorescent probe, is less than several hundred nanometers, which is the diffraction limit of light, an optical image obtained by an optical microscope shown in FIG. Then, all of the bright spots of the four quantum dots 21 to 24 are blurred and overlapped with each other, and this cannot be distinguished.

  On the other hand, in the spectroscopic image shown in FIG. 2C in which the optical image shown in FIG. 2B is spectrally separated by a spectroscope, four quantum dots 21 to 24 are green 21, yellow 23, orange 22 from the left. , And red 24 in order. Since each bright spot is smaller than the diffraction limit of light, it is blurred in the spectroscopic image, but it is displayed separately in the horizontal direction in the figure according to the spectrum. It is possible to grasp. As described above, numerical analysis by the Gaussian fit method is performed on each of the four bright spots spectrally separated by the spectroscope, and the center position can be estimated from the brightness distribution shape of the bright spots. As a result, as shown in FIG. 2C, the Y-direction positions (Y coordinate values) y1 to y4 of the four quantum dots 21 to 24 can be specified.

  In the optical microscope system according to the present embodiment, using the image data of the captured image obtained by capturing the spectral image, the analysis unit calculates the center position of each bright spot appearing as the spectral image by the Gaussian fit method. . In particular, as shown in FIG. 2 (c), the bright spot in the spectral image spectrally dispersed by the spectroscope corresponds to a horizontally long oval shape instead of a perfect circle because of the spectral analysis. As described above, analysis is performed by the Gaussian fit method using an elliptic Gaussian function, and fitting parameters are used assuming that the width is different between the spectral direction and the vertical direction. As described above, the region to be analyzed by the Gaussian fit method for one luminescent spot is not an exact circle but an ellipse, and the data to be analyzed is limited in advance. Processing time required until the center position of the bright spot is estimated from the image data can be shortened.

FIGS. 3 and 4 show how the myosin V, which is a motor protein, moves on an actin filament using the optical microscope system of the present embodiment, and each of the two motor domains is represented by quantum dots of different colors. The observation result observed by labeling is shown.

As shown in the schematic diagram on the left side of FIG. 3, one of the two motor domains (head portions) of myosin 32 moving on the actin filament 31, one is a green quantum dot 33 (emission wavelength 565 nm), and the other is Are labeled with two-color quantum dots of red quantum dots 34 (emission wavelength 655 nm). Specifically, the myosin motor domain was modified with biotin, and the streptavidin-modified quantum dots were labeled with biotin-avidin binding. A photograph on the right side of FIG. 3 shows the luminescent spot position of each quantum dot analyzed using the Gaussian fit method. In the photograph on the right side of FIG. 3, the left bright spot indicates the green quantum dot 33, and the right bright spot indicates the red quantum dot 34.

  FIG. 4 is a graph showing the relationship between the distance from the base point that is the position of the observation start time of the two quantum dots 33 and 34 and time.

  From FIG. 4, it is possible to clearly grasp how the two quantum dots 33 and 34 move from the base point position while walking alternately while sometimes changing the front and rear positions. It is also possible to observe the movement of the two quantum dots 33 and 34 as a moving image.

  As described above, according to the optical microscope system of the present embodiment, it is possible to accurately specify the positions of a plurality of fluorescent probes in a very small region that is equal to or smaller than the diffraction limit size of light even if they are close to each other. Furthermore, the change in the position of the fluorescent probe can be expressed as a moving image at a video rate. For this reason, it is possible to easily observe protein dynamics and the like that have been difficult to observe directly in the past.

  Next, as a modification of the optical microscope system of the present embodiment, a configuration of an optical microscope capable of specifying the two-dimensional position of the fluorescent probe and a method for specifying the position of the fluorescent probe in the analysis unit will be described.

  FIG. 5 is a block configuration diagram illustrating a configuration of a modification of the optical microscope system of the present embodiment.

  In the modified optical microscope system shown in FIG. 5, in addition to the configuration of the optical microscope portion described with reference to FIG. 1, the optical image of the observation object magnified by the magnifying relay optical system is used as it is in the first direction. The difference is that a splitting optical system is provided that splits the light image into a rotated light image rotated by 90 °.

  In the configuration of the optical microscope portion of the modified optical microscope system shown in FIG. 5, the laser light source 3, the objective lens 2, the spectroscope 11, and the imaging device 12 are the same as those shown in FIG. It can be used as it is. For this reason, in FIG. 5, the same reference numerals are given to members common to the configuration of the optical microscope described in FIG. 1, and the detailed description thereof is omitted.

  In the configuration of the optical microscope shown in FIG. 5, the optical image 7 of the observation object 1 that is transmitted through the objective lens 2 and the blue-reflecting dichroic mirror 6 and is magnified 375 times in total by the magnification relay optical system 8 is divided optically. The two light images 43 and 44 are separated by the non-polarization type half mirror 42 of the system 41.

  A first optical image 43, which is one of these optical images, is a first partial optical system comprising a first mirror 45, a second mirror 46, a third mirror 47 and a first condenser lens 48. After passing through 49, the light is condensed and incident on the slit 10 of the spectroscope 11. Since the first mirror 45 and the second mirror 46 that form the first partial optical system 49 are arranged so that the reflecting surfaces thereof face each other, the first mirror passing through the first partial optical system 49 In the optical image 43, the rotation from the optical image 7 of the observation object 1 obtained by the objective lens does not occur.

  A rotating light image 44 that is the other light image divided by the half mirror 42 is a second partial optical system 54 including a fourth mirror 50, a fifth mirror 51, a sixth mirror 52, and a condenser lens 53. Then, the light is condensed and incident on the slit 10 of the spectroscope 11. Note that, in the slit 10, the first light image 43 that has passed through the first partial optical system 49 and the rotated light image 44 that has passed through the second partial optical system 54 are vertically moved so that their incident positions do not overlap. Incidently shifted. In the case of the example shown in FIG. 5, the first optical image 43 that has passed through the first partial optical system 49 is incident on the upper side.

  In the second partial optical system 54, the reflecting surface of the fourth mirror 50 is arranged in a direction shifted by 90 ° with respect to the reflecting surface of the half mirror 42, so that the rotated light that has passed through the second partial optical system 54. The image 44 is a light image rotated by 90 ° as compared with the first light image 43 that has passed through the first partial optical system 49.

  FIG. 6 shows a first non-rotated light image and a rotated light image rotated by 90 ° incident on the spectroscope through the split optical system described in FIG. The spectral image is schematically shown.

  In FIG. 6, FIG. 6 (a) shows the relationship of the arrangement position of the fluorescent probe as the observation object, and FIG. 6 (b) shows the first light image not rotated through the first partial optical system. 6 (c) shows spectral images obtained by the spectroscope from the first optical image shown in FIG. 6 (b). FIG. 6D shows a rotated light image rotated by 90 ° with respect to the first light image that has passed through the second partial optical system, and FIG. 6E shows a spectroscope from the rotated light image. The spectral image obtained by is shown.

  In FIG. 6, as shown in FIG. 6A, a state is assumed in which the fluorescent probes of four colors of green 61, yellow 62, orange 63, and red 64 are two-dimensionally spread. In addition, as in the case of FIG. 2, it is assumed that quantum dots are used as fluorescent probes.

  In FIG. 6, in the first optical image shown as FIG. 6B, the bright spots of the four quantum dots 61 to 64 are all blurred and overlapped with each other as in FIG. 2A. In the spectral image shown in FIG. 6C obtained by spectrally separating the first light image with the spectroscope, the bright spots are separated and displayed in the order of green 61, yellow 63, orange 62, and red 64 from the left. It is possible to grasp the bright spots of the four quantum dots 61 to 64 separately. For each of the four bright spots in the captured image spectrally separated by the spectroscope, the center position is estimated from the bright spot distribution shape of the bright spots by performing numerical analysis by the Gaussian fit method. 64 y-coordinates can be specified.

  FIG. 6E shows a spectral separation of the rotated light image shown in FIG. 6D that is rotated by 90 ° with respect to the first optical image shown in FIG. 6B. Even in the spectral image, the bright spots of the four quantum dots 61 to 64 are displayed separately as green 61, yellow 63, orange 62, and red 64, so it is possible to grasp the four bright spots separately. The center position can be estimated by performing numerical analysis by the Gaussian fit method for each. Since the rotated light image is rotated by 90 ° with respect to the first light image, the center position of the quantum dot estimated from the spectral image of the rotated light image specifies the x coordinate of each quantum dot. It will be a thing.

  In the spectroscope used in the optical microscope system of the present embodiment using a diffraction grating, a prism, and the like, the spectrum was analyzed in one direction of the spectroscopic image (the horizontal axis direction in FIGS. 2 and 6), and thus the spectrum was split. As the bright spot position information, only one direction (the vertical direction in the case of FIGS. 2 and 6) which is the extending direction of the slit is obtained.

  Therefore, in the optical microscope system of the present embodiment, as shown in FIG. 5, as the optical system of the optical microscope, the light image at the stage of being focused and incident on the spectroscope 11 is two lights rotated by 90 ° with respect to each other. A two-dimensional arrangement position of the fluorescent probe to be observed is provided by correlating the biaxial position information obtained from the two spectral images with respect to each optical image. It is possible to identify.

  FIG. 7 shows a specific example of optical images rotated with respect to each other acquired by the optical microscope system of the present embodiment. FIG. 7 shows, as an observation object, a sample in which four fluorescent probes are arranged on a glass substrate, in order to verify that the two optical images obtained by the splitting optical system are correctly rotated. Yes.

  FIG. 7A shows the first optical image 43 and the second partial optical system 54 that have passed through the first partial optical system 49 rotated with respect to each other in a state of being incident on the spectroscope 11 through the slit 10. A rotated light image 44 is shown. The first optical image 43 obtained through the first partial optical system 49 cut out as shown in FIG. 7B has an x-axis in the left-right direction and a y-axis in the up-down direction as shown in the figure. The arrangement of the observation object as it is is shown.

  On the other hand, the rotated light image 44 that has been cut out as shown in FIG. 7C and passed through the second partial optical system 54 has a y-axis in the left-right direction and an x-axis in the up-down direction, as shown in FIG. It can be seen that the first optical image 43 that directly represents is rotated by 90 °. Since this is an optical image obtained by the splitting optical system 41 shown in FIG. 5 as a specific example, the rotated optical image 44 rotated 90 ° as shown in FIG. 7C is shown in FIG. 7B. The first non-rotating first light image 43 is reversed. As shown in FIG. 7D, the rotated image 44 ′ shown in FIG. 7C is turned upside down and the image 44 ′ is rotated as shown in FIG. 7B. It can be seen that it matches the optical image 43. Thus, by passing through the split optical system 41 shown in FIG. 5, it is possible to obtain two optical images 43 and 44 that are rotated by 90 ° with respect to each other.

  In the optical microscope system of the present embodiment, the first light image that is not rotated and the rotated light image that is rotated by 90 ° are each subjected to spectral analysis, and the position information of each bright spot is obtained from the obtained spectral image. A two-dimensional arrangement position is specified by obtaining a certain coordinate value and associating it. For this reason, if the correct x-coordinate is obtained from the rotated light image, it is not a problem that the image is further inverted with respect to the Y-axis due to the addition of the axis target operation. If two light images that are rotated by 90 ° with respect to the extending direction of the slit in the spectroscope can be obtained, the arrangement positions of the fluorescent probes in the one-dimensional direction orthogonal to each other can be specified and combined. The position of the fluorescent probe can be specified in two dimensions. In this case, the direction of 90 ° rotation may be either the right direction (clockwise) or the left direction (counterclockwise).

  FIG. 8 shows a state where two-dimensional positions of three fluorescent probes arranged in a region within the diffraction limit size of light are separately observed by the optical microscope system of the present embodiment.

  FIG. 8A shows a light image incident on the spectroscope, and FIG. 8B shows a spectral image spectrally separated by the spectroscope. 8 (a) and 8 (b), the upper half is the first optical image in the arrangement state of the observation object as it is and the spectral image obtained from the first optical image, and the lower half. Are a rotated light image obtained by rotating the first light image by 90 ° and a spectral image obtained from the rotated light image. Therefore, the y-axis coordinate of each fluorescent probe is determined from the upper half of FIG. 8B, and the x-axis coordinate of each fluorescent probe is determined from the lower half of FIG. 8B.

  In FIG. 8A, the luminescent spot indicated by the arrow in the drawing is seen as one luminescent spot both in the luminescent spot 71 in the upper half of the first light image and in the lower half of the rotated light image. . However, according to FIG. 8B showing a spectral image, the bright spot 71 and the bright spot 72 are both blue-green quantum dots 73 (emission wavelength 525 nm), green quantum dots 74 (emission wavelength 565 nm), It can be seen that the bright spots of the three quantum dots 73 to 75 of the red quantum dot 75 (emission wavelength: 655 nm) are overlapped.

  The center positions of the bright spots are estimated by analyzing the bright spots of the three-color quantum dots 73 to 75 dispersed in FIG. 8B by the Gaussian fit method, and the respective quantum dots 73 to 75 are estimated. The numerical values of the x coordinate and the y coordinate were specified. FIG. 8C shows the three quantum dots 73 to 75 two-dimensionally represented on the plane of the xy coordinates based on the estimated coordinate values.

  In the display in FIG. 8C, as shown in FIGS. 8A and 8B, each quantum dot 73 to 75 is based on each frame image of a moving image captured at a video rate of 30 frames / second. The x coordinate value and the y coordinate value of were estimated and plotted as they were. Although the numerical value of the estimated position of the quantum dot obtained from each frame image fluctuates due to the physical vibration of the microscope, the brightness of the quantum dot, and the minute change in the emission wavelength, etc., as shown in FIG. It can be seen that the positions of the three quantum dots 73 to 75 can be specified on the order of several nanometers to 10 nanometers.

  FIG. 8D shows a two-dimensional light image obtained with an optical microscope as it is for reference. For reference, in FIG. 8D, two-dimensional positions of the three quantum dots 73 to 75 grasped in FIG. As shown in FIG. 8D, since the three quantum dots 73 to 75 are arranged at a distance much smaller than the diffraction limit size of light, in the obtained light image, one small blurred spot is obtained. I can only see it. On the other hand, as shown in FIG. 8C, it is understood that the analysis image by the optical microscope system of the present disclosure that can specify the two-dimensional positions of the three quantum dots 73 to 75 has high resolution. it can.

  In the optical microscope system according to the present embodiment, it is possible to obtain the arrangement position information of the two-dimensional fluorescent probe acquired using the split optical system as a moving image.

Hereinafter, using FIG. 9 and FIG. 10, the results of two-dimensional moving image analysis using the fluorescence microscope system of the present disclosure of the state in which myosin V, which is a motor protein, moves on an actin filament will be shown. In FIGS. 9 and 10, the motor domain of myosin V is labeled with four fluorescent probes as in the case of using FIGS. 3 and 4, and the fluorescent probe moves. In the examples shown in FIGS. 9 and 10, only one of the two motor domains of myosin is labeled by keeping the quantum dot concentration lower than in the cases of FIGS. .

FIG. 9A is a spectral image obtained by a spectroscope based on an optical image of myosin V in the initial state. Also in FIG. 9A, as in FIG. 8B, the upper half specifies the spectral image obtained from the unrotated first optical image that determines the y coordinate position, and the lower half specifies the x-axis direction. It is the spectroscopic image obtained from the rotation light image rotated 90 degrees.

  As described with reference to FIG. 8, the center position is estimated by performing an analysis based on the Gaussian fit method on each of the upper and lower luminescent spots in FIG. 9A, and the x coordinate of each fluorescent probe and FIG. 9B shows the y-coordinate obtained and shown two-dimensionally. That is, FIG. 9B shows two-dimensional positions of the four quantum dots 91 to 94 in the initial state at the observation start time.

  Hereinafter, FIG. 9C is 10 seconds after the start of observation, FIG. 9D is 20 seconds later, FIG. 10A is 30 seconds later, FIG. 10B is 40 seconds later, FIG. After 50 seconds, FIG. 10 (d) after 60 seconds, FIG. 10 (e) after 70 seconds, FIG. 10 (f) after 80 seconds, two quantum dots 91 to 94 every 10 seconds. Indicate the location.

  It can be seen from FIGS. 9B to 10F that the positions of the four quantum dots change with the movement of myosin. 9 and 10, the positions of the four quantum dots 91 to 94 are shown every 10 seconds from the start of observation while omitting the image on the way, but in the optical microscope system of the present embodiment, the four quantum dots 91 are shown. It is possible to shoot and reproduce the two-dimensional position change of ~ 94 at the video rate, so that the movement of the four quantum dots 91 to 94 each indicating the motor domain of myosin can be observed as a moving image. it can.

  In particular, even when a plurality of quantum dots exist at extremely close positions, such as the quantum dots 91 and 92 in FIGS. 9 and 10, the optical microscope system according to the present embodiment is based on the emission wavelength. Can be clearly distinguished.

  When the two-dimensional fluorescent probe position described with reference to FIGS. 8, 9, and 10 is specified, the first light image not rotated and the rotated light image rotated by 90 ° are used. Thus, it is necessary to specify which two bright spots emit light from the same quantum dot and to correctly correspond the x coordinate value and the y coordinate value obtained separately. In particular, when the slit width is measured to be larger than the size of the bright spot as in the example of FIG. 7, the wavelength of each bright spot on the dispersed light image cannot be known. It is difficult to specify a combination of bright spots.

  In such a case, the optical microscope system according to the present embodiment distinguishes whether light emission is from the same quantum dot or light emission from a different quantum dot using the spectral fluctuation of the quantum dot in the spectral image. .

  In the optical microscope system of the present embodiment, the quantum dot used as the fluorescent probe is the fluorescence emission of the semiconductor nanoparticles, and therefore the emission intensity and the emission wavelength slightly change (fluctuate) with time. Have. As described above, in the optical microscope system of the present embodiment, one optical image obtained by the objective lens is optically separated by the separation optical system, and rotated by 90 ° with the first optical image that has not been rotated. Since the rotated light image is obtained, the first light image and the rotated light image are obtained at the same timing, so if the light emission of the quantum dots fluctuates, the bright spot in the two light images is At the same timing, the light emission brightness and the light emission wavelength change minutely. Therefore, by comparing fluctuations in emission luminance and emission wavelength, it is possible to easily determine whether two bright spots are emitted from the same quantum dot or not.

  FIG. 11 shows a bright spot in the spectral image obtained from the first non-rotated light image and a bright image in the spectral image from the rotated light image rotated by 90 ° based on the fluctuations in the emission intensity of the two bright spots. The result of associating points is shown.

  In FIG. 11, the state in which the four-color quantum dots 91 to 94 are present within the diffraction limit size is observed, and the upper half based on the first light image is determined from the change in emission luminance of each bright spot in the captured image. The four bright spots 91a, 92a, 93a, and 94a appearing in the image are associated with the four bright spots 91b, 92b, 93b, and 94b appearing in the lower half image based on the rotating light image.

  The table shown on the right side of FIG. 11 shows the correlation values in the Pearson product-moment correlation coefficient when the correlation of the emission luminance fluctuation is determined for the four corresponding bright spots 91 to 94 in a brute force manner. is there. As shown in the correspondence table shown on the right side of FIG. 11, the fluctuations in the emission brightness of the bright spots obtained from the two light images are different from those between the bright spots assumed to correspond to each other and other bright spots. Also shows a very high degree of correlation. From this result, it can be determined that the four bright spots are correctly associated.

  In the optical microscope system of the present embodiment, two bright spots based on the two light images obtained by the separation optical system can be correctly associated with each other, so that the two-dimensional positions of a plurality of fluorescent probes can be specified accurately. Can be done.

  As described above, according to the optical microscope system of the present disclosure, the optical image obtained by the optical microscope can be obtained from the spectroscope even when a plurality of fluorescent probes exist in a narrow region that is smaller than the diffraction limit size. By performing imaging spectroscopy with, it is possible to easily separate and estimate the position of each fluorescent probe.

  In addition, by obtaining two optical images rotated 90 ° relative to each other as optical images of the fluorescent probe, the x-coordinate and the y-coordinate can be specified from the spectral image of the fluorescent probe. Can be expressed in two dimensions.

  Furthermore, the optical microscope system according to the present disclosure is a method for acquiring spectra one by one while performing spatial beam scanning, an optical filter, and the like as a means for acquiring spectral information and two-dimensional spatial information at the same time. Unlike the method that takes time to acquire data, such as the method of sweeping the wavelength by sequentially switching, the spectrum and spatial coordinates are acquired simultaneously at the imaging speed of the camera without performing spatial scanning or wavelength sweep. be able to. For this reason, observation at high speed is possible, and changes in the positions of the fluorescent probes of a plurality of colors can be represented as moving images. Furthermore, it can be assumed that moving images can be displayed in real time in the future by shortening the analysis time.

  In the above embodiment, an example in which quantum dots are used as the fluorescent probe has been described. Each quantum dot emits light with a specific wavelength by one excitation light, and is easily separated by spectroscopy because the spectrum width of fluorescence emission is narrow. In addition, by devising materials and particle sizes, it is highly possible to obtain products of various wavelengths in a wide range from visible light to near infrared light, in addition to those currently on the market. For this reason, it is preferable as an index for observation in the optical microscope system of the present disclosure that can observe fluorescent probes of a plurality of colors separated by their emission wavelengths. In particular, when a molecule is labeled with a fluorescent probe, which molecule is labeled with which fluorescent probe is determined probabilistically, so that more fluorescent probes than the number of molecules to be separated and observed are required. For this reason, the quantum dot which can implement | achieve many fluorescence spectra is very effective as a fluorescence probe used for observation in the life science field with the optical microscope system concerning this indication.

  In the optical microscope system according to the present disclosure, fluorescent probes having different wavelengths are separated from each other by a spectral image, and each of them is discriminated. Various fluorescent probes can also be used. When a fluorescent probe other than quantum dots is used, it is preferable that the excitation light emission is maintained for a long time, and all the fluorescent probes emit light when irradiated with a single excitation light.

  Moreover, in the said embodiment, the example which enlarged the magnification by an optical system about 400 times was shown. In the optical microscope according to the present disclosure, an optical image obtained optically is obtained as a spectral image via a spectroscope, and this is analyzed by an analysis unit based on a captured image captured by an imaging device, and the center of a bright spot is obtained. From the relationship of estimating the position, if there are too few elements to capture one bright spot in the relationship between the size of the image sensor in the imaging device and the light image obtained as a captured image, the contour of the bright spot is measured. There is a risk that accuracy may be reduced and accurate center position estimation may not be possible. For this reason, the magnification of the optical microscope should be determined as appropriate in relation to the resolution determined by the size of the imaging element of the imaging device. As an example, the optical state is such that at least one bright spot is imaged by an image sensor having about 7 to 10 sides, and the entire bright spot can be imaged by 50 to 100 or more image sensors. It is preferable to ensure the magnification.

  Moreover, the structure of the optical microscope part demonstrated in the said embodiment is only an illustration. For example, the optical system such as the number and arrangement position of lenses and mirrors can be appropriately changed by design. Furthermore, in the method of measuring the two-dimensional arrangement of the fluorescent probe, the example provided with the split optical system has been described with reference to FIG. 5, but the method of obtaining two optical images rotated by 90 ° relative to each other is a method using the split optical system. Is not limited. The optical microscope system according to the present disclosure is not limited to the above-exemplified method, and various methods that can obtain the position of the bright spot in the x-axis direction and the y-axis direction can be employed. In addition, the optical microscope may be configured to include two spectroscopes and imaging devices, each of which acquires a separate spectral image and captured image.

  Mathematically, an arbitrary position on the two-dimensional plane can be expressed by a linear combination of any two vectors that are not parallel, so the rotation angle does not have to be 90 degrees, and two different mutually Various optical and analytical methods that can obtain two optical images whose positions in the axial direction can be detected can be employed.

  According to the optical microscope according to the present disclosure, it is possible to estimate the positions of several to dozens of fluorescent probes having different emission colors that are arranged in a region that is equal to or smaller than the diffraction limit size of light. For this reason, especially in the field of life science research, the simultaneous observation of many myosin molecules that make up muscles and receptors on cell membranes, which have been discussed only in combination with the observation results of dynamics and fixed samples. It is expected to have a very high effect, such as the observation of protein segregation / concentration, which enables a straightforward approach to elucidating the mechanism of biological nanosystems that generate functions through cooperative movement of molecules.

  The optical microscope system disclosed in the present application has a high resolution capable of separating and grasping the positions of fluorescent probes of a plurality of types of luminescent colors, with an extremely fine region below the diffraction limit of light being observed, and acquisition of moving images. As a high-speed optical microscope system that enables it, it can be expected to contribute to research in various fields including life science.

1 Observation object 3 Laser light source 8 Magnification optical system (optical system)
11 Spectrometer 12 EM-CCD (imaging device)

Claims (7)

An optical microscope system that acquires an optical image of an object having a size less than or equal to the diffraction limit of light using a fluorescent probe of multiple colors,
A laser light source that emits excitation light that causes the fluorescent probe to emit light; and
An optical system for obtaining an optical image of the fluorescent probe excited by the excitation light;
A spectroscope for imaging spectroscopy of an optical image obtained by the optical system;
An optical microscope having an imaging device that captures a spectral image obtained by the spectroscope to obtain a captured image;
An optical microscope system comprising: an analyzing unit that analyzes the captured image and estimates the positions of the fluorescent probes of the plurality of colors from the bright spot distribution. A first partial optical system that obtains a first light image of the fluorescent probe; and a second part that obtains a rotated light image rotated by 90 ° to the left or right with respect to the first light image. A splitting optical system composed of an optical system,
The spectroscope simultaneously performs imaging spectroscopy of the first light image obtained by the first partial optical system and the rotating light image obtained by the second partial optical system,
The optical microscope system according to claim 1, wherein the imaging device captures the spectral image of the first light image and the spectral image of the rotating light image as one captured image.   By associating the positions of the fluorescent probes of the plurality of colors obtained from the spectral image of the first light image and the spectral image of the rotating light image, the two-dimensional arrangement position of the fluorescent probes of the plurality of colors is determined. The optical microscope system according to claim 2 to be specified.   The optical microscope system according to claim 1, wherein a quantum dot is used as the fluorescent probe.   The optical microscope system according to claim 1, wherein the analysis unit estimates the position of the fluorescent probe from a luminance distribution of the captured image by a Gaussian fit method.   The optical microscope system according to claim 1, wherein the imaging device captures the captured image at a video rate or higher, and obtains a moving image of a change in position of the fluorescent probe from the obtained captured image.   The optical microscope system according to claim 1, wherein the optical microscope further includes a control unit that performs operation control of the microscope apparatus, and the analysis unit is incorporated as a function of the control unit.