JP4628845B2 - Microscope observation device with automatic specimen tracking function - Google Patents

Description

  The present invention relates to a microscope observation apparatus that performs correlation spectral analysis (FCS) of fluorescence intensity fluctuations based on a confocal optical microscope.

  As a measuring device for analyzing statistical properties and molecular level functions from a specific part in a sample, for example, in Japanese Patent Publication No. 11-502608, a confocal optical microscope is used as a base, and laser light is fluorescently labeled through a microscope objective lens. A method and apparatus that irradiates a sample and analyzes fluorescence intensity fluctuations caused by fluorescent molecules in the sample (performs fluorescence correlation spectroscopy) to determine statistical properties such as translational diffusion coefficient of fluorescent molecules and interactions between molecules. Is disclosed.

  Regarding the confocal optical microscope, for example, “Confocal Microscopy” T. Wilson (ed.) Academic press (London), “Handbook of Biological confocal Microscopy” JB Pawley (ed.) Commentary by Plenum Press (New York), Takahiro Oide et al. ("Optics" Vol.18, Vol.8, pp392-398) and Commentary by Kei Kawada ("Optics" Vol.18, Vol.8, pp380 ~ 391). Regarding fluorescence correlation spectroscopy, for example, there is a description such as “Fluorescence correlation spectroscopy” R. Rigler, E. S. Elson (eds.) Springer (Berlin).

  In fluorescence correlation spectroscopy, proteins and carrier particles labeled with a fluorescent substance are suspended in a solution within the field of view of a laser scanning confocal microscope, and fluctuations in fluorescence intensity based on the Brownian motion of these fine particles are analyzed to self. A correlation function is obtained to estimate the number of target fine particles, translational diffusion rate, and the like. This technique is discussed in Masataka Kaneshiro “Protein Nucleic Acid Enzyme” (1999) Vol. 44, No. 9, p1431-1437.

In addition, several patents have been filed for this technology. The laser scanning confocal microscope is described in, for example, Japanese Patent Laid-Open No. 10-206742. Japanese Patent Application Laid-Open No. 10-206742 describes a method for obtaining dynamic characteristics of a specimen by irradiating a desired position on the specimen while observing the specimen image in a laser scanning confocal microscope.
Special table 11-502608 gazette Japanese Patent Laid-Open No. 10-206742 JP-A-7-253548 Japanese Patent Laid-Open No. 5-80255 JP-A-7-261097 “Confocal Microscopy” T. Wilson (ed.) Academic press (London) “Handbook of Biological confocal Microscopy” JB Pawley (ed.) Plenum Press (New York) Takahiro Oide et al. "Optics" Vol.18, Vol.8, pp392 ~ 398 Kaoru Kawada "Optics" Vol.18, Vol.8, pp380-391 “Fluorescence correlation spectroscopy” R. Rigler, ES Elson (eds.) Springer (Berlin) Masataka Kaneshiro “Protein Nucleic Acid Enzyme” (1999) Vol.44, No.9, p1431-1437

  In a conventional method for tracking a sample in an optical microscope, the observer himself operates the electric stage with a joystick or the like while observing the sample image, so that the sample image is almost at the center of the monitor screen.

  Alternatively, in Japanese Patent Laid-Open No. 7-253548, a sample image is taken with a video camera or the like, the image data is stored in a frame memory, and the movement of the specimen is detected by comparing the reference image data with the image data stored in the frame memory. The sample tracking is performed by controlling the position of the electric stage. According to this method, a video camera and a memory for tracking the sample are required in addition to the microscope observation apparatus. In this method, the sample is observed after the tracking of the movement of the sample. Therefore, the movement of the specimen sample must be measured before the specimen specimen is observed.

  In Japanese Patent Laid-Open No. 5-80255, a sample image is continuously obtained for a moving sample at regular intervals, binarized to obtain a binarized image, and then binarization of two adjacent images. While shifting the images one by one along the time axis, a logical product image is created, and based on the logical product image, the movement of the sample is measured, and automatic tracking is performed. In this method, a binary image of a sample is created, a logical product image is also created, and judgment based on the logical product is also required. For this reason, it requires a large amount of calculation, labor and a lot of memory.

  In Japanese Patent Laid-Open No. 7-261997, image data before and after movement is acquired, a plurality of measurement points (for example, three points) in a sample are designated in advance, and the values input here are used as auxiliary image data to move the image. The amount of movement of the sample is obtained by comparison, for example, by taking a difference from a value (auxiliary image data) input later. Based on this movement amount data, drive control of the stepping motor is performed to move to the center of the movable stage observation visual field. In this method, determination of sample movement is performed using image data, and a large amount of memory is required for determination of sample movement.

  The present invention has been made in consideration of such a situation, and the main purpose thereof is a microscope having an automatic tracking function of a specimen sample without requiring a video camera or a memory for tracking the sample. It is to provide an observation device.

Microscope apparatus of the present invention includes a sample holding means for holding a specimen sample, an image acquiring means for acquiring the microscopic image containing the object of measurement specimen sample, a plurality of measurement points within the measurement object microscope image Setting means for setting a plurality of movement detection points , correlation analysis means for obtaining a correlation function of fluorescence intensity fluctuations obtained from measurement points , and correlation calculation results for obtaining a correlation function of fluorescence intensity fluctuations obtained from movement detection points Based on the movement detection means for detecting the movement of the measurement object and update means for updating the movement detection point when the movement detection means detects the movement of the measurement object .

  According to the present invention, there is provided a microscope observation apparatus having an automatic tracking function of a specimen sample without requiring a video camera or a memory for tracking the sample.

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

First Embodiment The present embodiment is directed to a microscope observation apparatus that performs correlation spectral analysis (FCS) of fluorescence intensity fluctuations using a confocal optical microscope. More specifically, the present invention is directed to a microscope observation apparatus having an automatic sample tracking function that uses autocorrelation functions of fluorescence intensity fluctuations at four measurement points. FIG. 1 schematically shows a basic configuration of a microscope observation apparatus having an automatic sample tracking function in the first embodiment of the present invention.

  As shown in FIG. 1, the microscope observation apparatus 100 of the present embodiment includes a laser scanning confocal microscope 110. The laser scanning confocal microscope 110 enables a microscope main body 111, a sample stage 122 on which the sample 121 is placed, an objective lens 112 disposed below the sample stage 122, and a viewer to visually observe the sample 121. An eyepiece 118 and a CCD camera 128 for acquiring an optical image of the sample 121 are included. The objective lens 112, the eyepiece lens 118, the sample stage 122, and the CCD camera 128 are all attached to the microscope body 111.

  The laser scanning confocal microscope 110 further includes two stepping motors 123 and 124 for moving the sample stage 122. The microscope observation apparatus 100 further includes a sample stage controller 125 for controlling the stepping motors 123 and 124. And a computer 126 for controlling the sample stage controller 125.

  The stepping motors 123 and 124 are arranged so that their drive axes are orthogonal to each other, and can move the sample stage 122 in two directions orthogonal to each other, the so-called X direction and Y direction. The sample stage controller 125 is connected to the computer 126 and drives the stepping motors 123 and 124 according to a command from the computer 126 to move the sample stage 122 in the X direction and the Y direction.

  The microscope observation apparatus 100 further includes an optical scanning system 130 that supplies an excitation light beam for generating fluorescence to the laser scanning confocal microscope 110. FIG. 2 shows the overall configuration of the optical scanning system of the microscope observation apparatus shown in FIG.

  As shown in FIG. 2, the microscope observation apparatus 100 includes four systems of optical scanning systems 130A, 130B, 130C, and 130D for emitting and scanning the excitation light beam. The four optical scanning systems 130A to 130D all have the same configuration. In the following description, when it is not necessary to distinguish between them, the alphabetical character at the end of the reference symbol is omitted and simply referred to as the optical scanning system 130. Along with this, members constituting the optical scanning system are also described by omitting the alphabetic characters at the end of the reference numerals.

  The optical scanning system 130 includes a laser light source 131 that emits an excitation light beam, a lens 132 that collimates the excitation light beam from the laser light source 131, a mirror 133 that deflects the excitation light beam from the lens 132, and a mirror 133 A dichroic mirror 134 that reflects and deflects the excitation light beam and an XY scanning mechanism 135 that performs XY scanning on the excitation light beam from the dichroic mirror 134 are provided. The dichroic mirror 134 has a property of reflecting excitation light and selectively transmitting fluorescence generated by the excitation light.

  The XY scanning mechanism 135 is not limited to this. For example, the XY scanning mechanism 135 may be configured by combining two galvano scanners that can scan a light beam along one axis. The two galvanometer mirrors are preferably arranged so that their scanning axes are orthogonal to each other. Or you may comprise using an acousto-optic modulation element (AOM), a polygon mirror, a hologram scanner, etc.

  The optical scanning system 130 further has a lens 136 for converging the fluorescence generated by the excitation light beam emitted from the optical scanning system 130 and a convergence point of the excitation light beam emitted from the optical scanning system 130. A pinhole 137 having a focal position and a photodetector 138 for detecting fluorescence that has passed through the pinhole 137 are provided. Although not limited to this, the photodetector 138 may be configured by, for example, an avalanche photodiode (APD) (Avalanche Photo Diode). Or you may comprise with a photomultiplier tube.

  Inside the microscope main body 111, the excitation light beam from the light scanning system 130 is directed to the objective lens 112 with respect to each light scanning system 130, and the fluorescence beam generated by the excitation light beam is transmitted to the light. A deflection element 116 is provided to direct to the scanning system 130. The deflection element 116 may be constituted by a dichroic mirror, for example. The deflection element 116D arranged at the bottom does not need a selection function, and may be formed of a normal mirror.

  The sample 121 is previously labeled with four types of fluorescent substances. Each of the four optical scanning systems 130 emits a beam of excitation light having a wavelength suitable for the type of the fluorescent material.

  Examples of the fluorescent material include, but are not limited to, rhodamine green (RhG) (Rhodamine Green), TMR (Tetramethylrhodamine), 5-Tamra (5-carboxytetramethylrhodamine), and Cyfive (Cy5). In addition to these, FITC (Fluorescein-isothiocyanate), TOTO1, Acridine-Orange, Texas-Red, etc. may be applied as the fluorescent material.

  Corresponding to the fluorescent materials described above, for example, the laser light source 131A is an argon laser that emits light having a wavelength of 488 nm in order to excite rhodamine green (RhG), and the laser light source 131B excites TMR. Therefore, it is an argon laser that emits light with a wavelength of 514.5 nm, the laser light source 131C is a He / Ne laser that emits light with a wavelength of 543.5 nm to excite 5-Tamura, and the laser light source 131D is a laser light source 131D. In order to excite the Five (Cy5), this is a He / Ne laser that emits light having a wavelength of 632.8 nm.

  The excitation light beam emitted from the laser light source 131 is reflected by the mirror 133 and the dichroic mirror 134, enters the microscope body 111 through the XY scanning mechanism 135, and is directed to the objective lens 112 by the deflection element 116. The excitation light beam incident on the objective lens 112 is converged inside the sample 121 by the objective lens 112.

  The convergence point of the excitation light beam can be moved two-dimensionally by the XY scanning mechanism 135. The objective lens 112 has a large numerical aperture of about NA 1.0, and a substantially cylindrical confocal region having a diameter of about 0.6 μm and a length of about 2 μm is formed near the convergence point of the excitation light beam. Is done.

  The fluorescent substance existing in this region is excited by the excitation light and generates a fluorescence signal (photon pulse). Part of the fluorescence (signal light) generated from the fluorescent material is incident on the objective lens 112 and becomes a fluorescent beam (fluorescence signal) directed to the deflection element 116. The fluorescent beam incident on the deflecting element 116 is directed to the optical scanning system 130.

  The fluorescent beam returned to the optical scanning system 130 enters the dichroic mirror 134 through the XY scanning mechanism 135. As described above, since the dichroic mirror 134 has a characteristic of selectively transmitting the fluorescence generated by the excitation light reflected by the dichroic mirror 134, the fluorescent beam incident on the dichroic mirror 134 is transmitted therethrough. The fluorescent beam that has passed through the dichroic mirror 134 is converged by the lens 136, and only the component that has passed through the pinhole 137 is incident on the photodetector 138. The photodetector 138 outputs an electrical signal that reflects the intensity of the incident light.

  FIG. 3 shows the overall configuration of the fluorescence observation apparatus shown in FIG. As shown in FIG. 3, in addition to the above-described configuration, the optical scanning system 130 includes a driving device 141 that drives the XY scanning mechanism 135 and a convergence point of the excitation light beam based on the driving signal of the XY scanning mechanism 135. A position calculating device 142 for determining the position.

  In addition to the laser scanning confocal microscope 110 and the four optical scanning systems 130, the microscope observation apparatus 100 shapes the electrical signal output from the photodetector 138 of the optical scanning system 130 and shapes it on / off. Obtained by the signal processing device 151 for converting into a digitized pulse, the correlation analysis device 152 for obtaining a correlation function by performing a correlation operation on the binary pulse output from the signal processing device 151, and the correlation analysis device 152. And a computer 126 for obtaining a translational diffusion rate of the fluorescent substance and a change in the number of fluorescent molecules in the measurement region from the autocorrelation function. Alternatively, the correlation calculation may be performed by the computer 126 without using the correlation analyzer 152.

  The microscope observation apparatus 100 further processes the result obtained by the signal processing apparatus 151 together with the signal from the position calculation apparatus 142 to form a two-dimensional fluorescent image of the sample, and improves contrast and contour enhancement. An image processing device 155 that performs image processing and a TV monitor 156 that displays an image obtained by the image processing device 155 are provided.

  In FIG. 3, the electrical signal photoelectrically converted by the photodetector 138 is waveform-shaped by the signal processing device 151 and converted into a binary pulse signal. The binarized pulse signal is sent to the correlation analysis device 152, and the correlation analysis device 152 obtains, for example, an autocorrelation function. The autocorrelation function is sent to the computer 126, and is used to calculate the translational diffusion rate of the fluorescent material and the change in the number of fluorescent molecules in the measurement region.

  The electrical signal output from the photodetector 138 is a time series signal. In this case, AD conversion is performed by the signal processing device 151, waveform shaping is performed, and the signal is converted into a binary pulse signal as before. A correlation calculation is performed by introducing the binarized pulse signal to the computer 126, and an autocorrelation function is obtained.

  Further, by moving the objective lens 112 along the optical axis, the position of the convergence point of the excitation light can be moved along the optical axis. A three-dimensional fluorescence image can be obtained by performing two-dimensional light scanning at each convergence position of the excitation light and capturing a fluorescence signal.

  Next, a method for measuring the movement of the sample in real time based on the autocorrelation function of the fluorescence intensity fluctuation thus obtained will be described.

  1. A plurality of measurement points are defined in the sample 121, and the light spot of the excitation light beam is moved by the XY scanning mechanism so that the light spot of the excitation light beam hits these measurement points.

  2. The light spot position is fixed, the fluorescence intensity fluctuation is measured, and the autocorrelation function is obtained.

  3. The autocorrelation function of fluorescence intensity fluctuation is stored.

  4). Four measurement points are fixed, and the autocorrelation function of fluorescence intensity fluctuation is repeatedly obtained at regular time intervals for each measurement point.

  In the measurement of 5.4, if the autocorrelation function of the fluorescence intensity fluctuation changes with time, whether the autocorrelation function of the fluorescence intensity fluctuation due to the reaction or binding of molecules in the sample is a It is determined whether the autocorrelation function of the fluorescence intensity fluctuation due to the movement of the light changes.

  When the movement of the measurement object in the sample 121 is confirmed by analyzing the autocorrelation function of the fluorescence intensity fluctuation obtained in 6.5, the sample stage 122 and the objective lens 112 are relatively moved. The position of the measurement object in the sample 121 is adjusted so that the light spot of the excitation light beam hits the initially determined measurement point.

  7. When the reaction or binding of the molecules in the sample is confirmed along with the movement of the measurement object in the sample 121, the measurement result is stored in the memory in the computer 126 and displayed on the TV monitor.

  FIG. 4 shows an example of the positional relationship between the four light spots a, b, c, d and the measurement object in the sample 121, with the left side before the sample moving and the right side after the sample moving. In the example shown in FIG. 4, the four light spots a, b, c, and d are all located in the sample before the sample is moved, but after the sample is moved, the three light spots a, b, and Although d is located in the sample, the light spot c is off the sample.

  The light spot a is for exciting rhodamine green (RhG), the light spot b is for exciting TMR, and the light spot c is for exciting Tamra, The light spot d is for exciting sci-fi (Cy5).

  5 (a), FIG. 5 (b), FIG. 5 (c), and FIG. 5 (d) respectively show four light spots a, b, c, and d before the movement of the sample shown on the left side of FIG. The autocorrelation function of fluorescence intensity fluctuation is shown. 6 (a), 6 (b), 6 (c), and 6 (d) respectively show four light spots a, b, c, and d after the movement of the sample shown on the right side of FIG. The autocorrelation function of fluorescence intensity fluctuation is shown.

  FIGS. 7 (a), 7 (b), 7 (c) and 7 (d) respectively show the four light spots a, b, c and d before the movement of the sample shown on the left side of FIG. The change in the intensity fluctuation of the fluorescence is shown. 8 (a), 8 (b), 8 (c) and 8 (d) respectively show the four light spots a, b, c and d after the movement of the sample shown on the right side of FIG. The change in the intensity fluctuation of the fluorescence is shown.

  As shown in FIG. 4, the light spot c deviates from the sample after the sample is moved. For this reason, after the sample is moved, the autocorrelation function of the fluorescence intensity fluctuation becomes impossible to measure, and as shown in FIG. 6C, the autocorrelation function of the fluorescence intensity fluctuation cannot be obtained. Further, after the sample is moved, the fluorescence intensity fluctuation cannot be measured, and the fluorescence intensity fluctuation is a noise signal as shown in FIG. 8C.

  As described above, when the light spot deviates from the sample, an autocorrelation function of fluorescence intensity fluctuation corresponding to the light spot cannot be obtained. Therefore, it can be seen that the sample deviates from the light spot, that is, the sample has moved, based on the autocorrelation function of the fluorescence intensity fluctuation corresponding to each light spot. For example, when the autocorrelation function of the fluorescence intensity fluctuation corresponding to a specific light spot cannot be obtained, it can be seen that the sample has deviated from the light spot.

  The autocorrelation function of the fluorescence intensity fluctuation also changes depending on the reaction and binding of molecules in the sample, but the change is not to the extent that the autocorrelation function of the fluorescence intensity fluctuation can be obtained, but to the extent that the profile changes. Therefore, it is possible to easily discriminate whether the change is due to the reaction or binding of molecules in the sample or the change due to movement of the sample.

  FIG. 9 shows an arrangement example of four light spots a, b, c, and d. In the example shown in FIG. 9, four light spots a, b, c, and d are all located on concentric circles, two of which are located on the X axis, and the remaining two light spots. c and d are located on the Y-axis. Such an arrangement of the light spots a, b, c, and d has an advantage of facilitating determination of the moving direction of the sample. For example, if it is found that the sample deviates from the light spot a, it may be determined that the sample has moved approximately in the -X direction. If it is found that the sample deviates from the light spot d, the sample moves approximately in the + Y direction. You may judge that

  10 and 11 show flowcharts of detection of sample movement in the present embodiment. Here, a case where the number of measurement points is four and a case where the measurement points coincide with the detection points of sample movement will be described.

Step 1. First, a fluorescent microscope image of the sample is acquired, and then four measurement points are determined in the sample within the observation field. This is performed by the observer moving the light spot to a desired position by the XY drive mechanism 135 in each optical scanning system 130 while viewing the acquired microscope image. Further, the position coordinates of the measurement point are stored in a memory in the computer 126. At each measurement point, FCS measurement is performed, an autocorrelation function of fluorescence intensity fluctuation is obtained, and these values are stored in the memory of the computer 126. Further, these profiles are approximately obtained by curve fitting with a theoretical curve, and these are also stored together. The curve fitting may be obtained by the least square method or may be optimized by using the Levenberg Marquard method which is one of the nonlinear least square methods. ("Numerical Recipes in C", written by William H. Press et al., Translated by Tankeikatsu et al., Technical Critics, 1994)
Step 2. After a certain time or after a preset time, FCS measurement is performed again at exactly the same four measurement points, an autocorrelation function of fluorescence intensity fluctuation is obtained, and those values are stored in a memory in the computer 126.

  Step 3. If autocorrelation functions of fluorescence intensity fluctuations are obtained at all four measurement points, they are stored. These profiles are approximately obtained by curve fitting, and checked with the measurement result stored in step 1 by a computer to see if there is a change. As a result of the collation, when there is no change in any of the four autocorrelation functions of the fluorescence intensity fluctuations, it is determined that there is no sample movement and no state change. On the other hand, if there is a change in any of the autocorrelation functions of the fluorescence intensity fluctuations at the four measurement points, the sample is not moved, but it is determined that there is a state change, and the result is stored in the memory in the computer 126. And display on the TV monitor.

  Step 4. If the autocorrelation function of fluorescence intensity fluctuation cannot be obtained at any of the four measurement points, it is determined that the sample has moved, and all the measurement points are moved two-dimensionally or three-dimensionally at the same time. The measurement result stored in 1 is collated, a position where the difference is minimized is obtained while performing a comparison operation, and the position is arranged at that position. The measurement point is moved by moving the sample stage 122 by the sample stage controller 125 and, if necessary, by moving the objective lens 112 along the optical axis.

  Hereinafter, the autocorrelation function of the fluorescence intensity fluctuation will be described.

The autocorrelation function R (τ) of the fluorescence intensity fluctuation from the fluorescent material received by the photodetector is expressed by the following equation.

Where I is the intensity of the fluorescence from the fluorescent material received by the photodetector, t is the time, and τ is the correlation time. <> Represents an ensemble average.

The autocorrelation function of the fluorescence intensity fluctuation is normalized as follows.

Where G (τ) is the normalized autocorrelation function of the fluorescence intensity fluctuation, and N is the average number of particles existing in the confocal region. It is assumed that the presence of particles in the confocal region follows a Poisson distribution.

If the incident light is light having a Gaussian distribution (such as argon laser or helium neon laser), the autocorrelation function G (τ) of the light intensity fluctuation is as follows.

Where D is the translational diffusion coefficient of the fluorescent molecule, ω ₁ is ½ of the height when the confocal region is approximated to a cylinder, and ω ₂ is the radius.

At this time, when τ _D is the diffusion time of the fluorescent substance molecule, the following is obtained.

If the autocorrelation function of fluorescence intensity fluctuation is obtained by measurement, curve fitting of these profiles is performed to determine the average number of particles (N) existing in the confocal region and the diffusion time (τ _D ) of the fluorescent molecules. These time courses may be monitored at each measurement point.

  When various biological reactions and state changes occur at any measurement point of the sample with the lapse of time of observation, the profile of the autocorrelation function of the fluorescence intensity fluctuation changes at that measurement point. From this profile change, a change in diffusion time, a change in average number of fluorescent molecules in the observation region, and the like can be obtained. To examine the change in the autocorrelation function profile of the fluorescence intensity fluctuation, normalize the autocorrelation function, that is, divide the autocorrelation function of the fluorescence intensity fluctuation by the square of the time average value of the fluorescence intensity fluctuation and perform curve fitting. The diffusion time and the Y-axis intercept (y-axis coordinates of the point where the autocorrelation function intersects the y-axis) are obtained. Note that the value of the Y-axis intercept of the autocorrelation function of the fluorescence intensity fluctuation corresponds to the average number of fluorescent molecules existing in the confocal region, as can be seen from the equation (3).

  If only sample movement occurs, the profile of the autocorrelation function of fluorescence intensity fluctuations obtained at multiple measurement points in the sample does not basically change, or if the sample movement is large, the fluorescence intensity fluctuation Since it becomes impossible to obtain the autocorrelation function itself, this change can be captured.

  On the other hand, the profile of the autocorrelation function always changes when a biological reaction or state change occurs with time at a plurality of measurement points in the sample. Therefore, this can be grasped.

  If a biological reaction or state change occurs over time at multiple measurement points in the sample, and the sample is also moved at the same time, the biological reaction or state change in the sample occurs. The profile of the autocorrelation function of the fluorescence intensity fluctuation changes, and the profile of the autocorrelation function of the fluorescence intensity fluctuation basically does not change at the location where the sample has moved. When the sample is accompanied by the movement of the sample, the measurement of the autocorrelation function of the fluorescence intensity fluctuation is further accumulated several times to detect the moving amount and direction of the sample. Since measurements are actually performed at multiple locations, even if biological reactions and changes in state occur over time at multiple measurement points in the sample, and sample movement and deformation occur simultaneously, Can be captured. In this case, it is desirable that there are as many measurement points as possible.

  Note that the deformation of the sample can also be measured by increasing the number of measurement points and performing the same measurement.

  In addition, paying attention to changes in fluorescence intensity fluctuations, for example, the time average value and dispersion of fluorescence intensity fluctuations, the initial values and the values after a certain period of time have been obtained for all measurement points of these values, and these are compared. It is also possible to estimate the movement and direction of the sample. Furthermore, the sample stage may be moved by a necessary amount based on the estimation result.

  Although the case where the measurement point coincides with the detection point of the sample movement has been described this time, the measurement point and the detection point of the sample movement do not necessarily coincide with each other. In this case, when acquiring the measurement values at the four measurement points in the sample, there is a time lag between the measurement of the biological reaction and state change of the sample and the measurement to grasp the movement of the sample itself. The measurement results may be stored in a memory in the computer 126 and a fluorescent image may be displayed on the TV monitor.

  As can be seen from the above description, in the present embodiment, a microscope observation apparatus having an automatic tracking function of a specimen sample can be obtained without requiring a video camera or a memory for tracking the sample.

Second Embodiment This embodiment is directed to a microscope observation apparatus that performs correlation spectral analysis of fluorescence intensity fluctuations using a laser scanning confocal microscope. More specifically, the present invention is directed to a microscope observation apparatus having an automatic sample tracking function that uses a cross-correlation function of fluorescence intensity fluctuations at three measurement points.

  The microscope observation apparatus of this embodiment also basically has the same configuration as that of the first embodiment (that is, the configuration shown in FIG. 1).

  FIG. 12 shows the overall configuration of a microscope observation apparatus having an automatic sample tracking function in the second embodiment of the present invention.

  As shown in FIG. 12, the microscope observation apparatus 200 according to the present embodiment includes a laser scanning confocal microscope 110. The laser scanning confocal microscope 110 includes a microscope main body 111, a sample stage 122 on which a sample 121 is placed, and an objective lens 112 disposed below the sample stage 122.

  The microscope observation apparatus 200 further includes three systems of optical scanning systems 230A, 230B, and 230C that supply an excitation light beam for generating fluorescence to the laser scanning confocal microscope 110. The three optical scanning systems 230A to 230C have the same configuration. In the following description, when it is not necessary to distinguish between them, the alphabetical character at the end of the reference symbol is omitted and simply referred to as the optical scanning system 230. Along with this, members constituting the optical scanning system are also described by omitting the alphabetic characters at the end of the reference numerals.

  The optical scanning system 230 includes a first laser light source 231 that emits a first excitation light beam, a second laser light source 232 that emits a second excitation light beam, and a first laser light source 231. A mirror 233 that deflects the excitation light beam, a dichroic mirror 234 that reflects and deflects the excitation light beam from the mirror 233, a mirror 235 that deflects the excitation light beam from the second laser light source 232, and the mirror 235 It has a dichroic mirror 236 that reflects and deflects the excitation light beam, and an XY scanning mechanism 237 that performs XY scanning on both the first excitation light beam and the second excitation light beam.

  The dichroic mirror 234 has a characteristic of selectively transmitting the fluorescence generated by the first excitation light while reflecting the first excitation light. The dichroic mirror 236 reflects the second excitation light, and transmits the first excitation light, the fluorescence generated by the first excitation light, and the fluorescence generated by the second excitation light. Have.

  As in the first embodiment, the XY scanning mechanism 237 may be configured by combining two galvano scanners that can scan a light beam along one axis, for example. The two galvanometer mirrors are preferably arranged so that their scanning axes are orthogonal to each other. Or you may comprise using an acousto-optic modulation element (AOM), a polygon mirror, a hologram scanner, etc.

  The optical scanning system 230 further detects a first photodetector 238 for detecting the first fluorescence generated by the first excitation light, and a second fluorescence generated by the second excitation light. A second photodetector 239 for transmitting, a separation element 240 that transmits the first fluorescence and reflects the second fluorescence to separate the first fluorescence and the second fluorescence, and is reflected by the separation element 240 And a mirror 241 that reflects the second fluorescence.

  Although not shown in the drawing, a pinhole having a confocal positional relationship with respect to the convergence point of the beam of the first excitation light is disposed in front of the first photodetector 238, and the second photodetector is arranged. In front of 239, a pinhole having a confocal positional relationship with the convergence point of the second excitation light beam is disposed.

  The photodetector 238 and the photodetector 239 may be configured by, for example, an avalanche photodiode (APD) (Avalanche Photo Diode) as in the first embodiment. Or you may comprise with a photomultiplier tube.

  The optical scanning system 230 further includes a driving device 242 that drives the XY scanning mechanism 237 and an angle detection device 243 that obtains the position of the convergence point of the excitation light beam based on the driving signal of the XY scanning mechanism 237.

  Inside the microscope main body 111, the excitation light beam from the light scanning system 230 is directed to the objective lens 112 with respect to each light scanning system 230, and the fluorescence beam generated by the excitation light beam is transmitted to the light. A deflection element 216 is provided to direct to the scanning system 230.

  The microscope observation apparatus 200 further includes a signal processing device 251 that shapes the electrical signals output from the photodetector 238 and the photodetector 239 of the optical scanning system 230 and converts them into an on / off binary pulse, and a signal A correlation analysis device 252 and a computer 126 for performing a correlation operation on the binarized pulse output from the processing device 251 and obtaining a cross-correlation function of fluorescence intensity fluctuation are provided.

  The microscope observation device 200 further processes the result obtained by the signal processing device 251 together with the signal from the angle detection device 243 to form a two-dimensional fluorescent image of the sample, and improves contrast and contour enhancement. An image processing device 255 that performs image processing and a TV monitor 256 that displays an image obtained by the image processing device 255 are provided.

  The sample 121 is previously labeled with two types of fluorescent materials. Examples of the fluorescent material include, but are not limited to, rhodamine green (RhG) (Rhodamine Green) and sci-fi (Cy5). Correspondingly, for example, the laser light source 231 is an argon laser that emits light with a wavelength of 488 nm to excite rhodamine green (RhG), and the laser light source 232 excites sci-fi (Cy5). And a He / Ne laser emitting light having a wavelength of 632.8 nm.

  The first excitation light beam emitted from the first laser light source 231 is reflected by the mirror 233 and the dichroic mirror 234, passes through the dichroic mirror 236, and enters the microscope main body 111 through the XY scanning mechanism 237. The second excitation light beam emitted from the second laser light source 232 is reflected by the mirror 235 and the dichroic mirror 236 and enters the microscope main body 111 through the XY scanning mechanism 237. The first and second excitation light beams entering the microscope body 111 are directed to the objective lens 112 by the deflection element 216. The first and second excitation light beams incident on the objective lens 112 are converged inside the sample 121 by the objective lens 112.

  The convergence points of the first and second excitation light beams can be moved two-dimensionally by the XY scanning mechanism 237. Further, the objective lens 112 has a large numerical aperture of about NA 1.0, and has a substantially cylindrical shape having a diameter of about 0.6 μm and a length of about 2 μm in the vicinity of the convergence points of the first and second excitation light beams. The confocal region is formed.

  The convergence point of each excitation light beam is determined as a measurement point in the test substance by stopping the XY scanning mechanism 237. Then, the excitation light beam is two-dimensionally scanned by turning on and operating the XY scanning mechanism 237 again. When the XY scanning mechanism 237 is turned off again at a desired position, the convergence point of the excitation light beam is uniquely determined at a different position in the test substance. By repeating this operation, the convergence point, that is, the measurement point can be sequentially moved.

  The two types of fluorescent substances present in this region are each excited by corresponding excitation light to generate a fluorescence signal (photon pulse). Part of the fluorescence (signal light) generated from the two types of fluorescent materials is incident on the objective lens 112 and becomes a first fluorescent beam and a second fluorescent beam directed to the deflecting element 216, respectively. Both the first fluorescent beam and the second fluorescent beam incident on the deflecting element 216 are directed to the optical scanning system 230.

  The first fluorescent beam and the second fluorescent beam that have returned to the optical scanning system 230 pass through the XY scanning mechanism 237, pass through the dichroic mirror 236 and the dichroic mirror 234, and are separated by the separation element 240. In the first fluorescent beam transmitted through the separation element 240, only the component that has passed through a pinhole (not shown) is incident on the first photodetector 238. The second fluorescent beam reflected by the separation element 240 passes through the mirror 241, and only the component that has passed through a pinhole (not shown) enters the second photodetector 239. The photodetector 238 and the photodetector 239 each output an electrical signal reflecting the intensity of incident light.

  The electric signals output from the photodetector 238 and the photodetector 239 are waveform-shaped by the signal processing device 151 and converted into a binary pulse signal. The image processing device 255 processes the binarized pulse signal together with the signal from the angle detection device 243 to form a two-dimensional fluorescent image or a three-dimensional fluorescent image of the sample. The image processing device 255 further performs image processing such as contrast enhancement and edge enhancement. The microscope image is displayed on the TV monitor 256.

  The binarized pulse signal from the signal processing device 151 is also input to the computer via the signal processing device 251 via the correlation analysis device 252, and a cross-correlation function of fluorescence intensity fluctuation is obtained.

  In the microscope observation apparatus 200 of the present embodiment, the cross-correlation function of the fluorescence intensity fluctuation can be obtained while observing the fluorescence image, and the movement of the sample can be measured.

  The method of measuring the movement of the sample and moving the sample stage based on the measurement value can be performed in the same manner as in the first embodiment. Hereinafter, a case where the measurement point of the sample and the measurement point of the sample movement coincide with each other will be described.

  If the ratio of molecules such as proteins labeled with two fluorescent substances of interest at any measurement point of the sample has changed due to biological reaction, etc., as the time of measurement observation changes, The profile of the cross-correlation function of the fluorescence intensity fluctuation changes at the measurement point. From this change in profile, it is possible to determine the change in the average number of molecules present in the observation region of the conjugate of the two types of molecules.

To examine changes in the cross-correlation function profile of fluorescence intensity fluctuations, the cross-correlation function is normalized, that is, the cross-correlation function of fluorescence intensity fluctuations is the product of the time average values of the fluorescence intensity fluctuations received by the respective photodetectors. And curve fitting to determine the value of the Y-axis intercept. That is, the number of conjugates of two kinds of molecules can be obtained using the equation (6).

Where N _AB is the number of conjugates of two types of molecules, G _AB (0) is the value of the Y-axis intercept of the cross-correlation function of fluorescence intensity fluctuations due to the conjugates of two types of molecules, and G _A (0) is the fluorescence. The value of the Y-axis intercept of the autocorrelation function of the fluorescence intensity fluctuation due to the substance A, and G _B (0) is the value of the Y-axis intercept of the autocorrelation function of the fluorescence intensity fluctuation due to the fluorescent substance B.

  In the present embodiment, the number of measurement points is three, but the number of measurement points is not limited to this, and may be four or more. The greater the number of measurement points, the better the measurement accuracy.

  In the present embodiment, the amount and direction of movement of the sample can also be obtained by measuring the rotation angle of the galvano scanner using a rotation angle sensor such as an encoder. Therefore, it is possible to grasp the change in state and biological reaction at a plurality of observation locations in the sample in real time, and simultaneously know the amount of movement and the direction of movement of the sample itself.

  When only sample movement occurs, the cross-correlation function profile of fluorescence intensity fluctuations obtained at multiple measurement points in the sample does not basically change, or when the sample movement is large, the fluorescence intensity fluctuation Since it becomes impossible to obtain the cross-correlation function itself, this change can be captured.

  On the other hand, when the number of conjugates labeled with two kinds of fluorescent substances changes with time at a plurality of measurement points in the sample, the profile of the cross-correlation function always changes. Therefore, this can be grasped.

  If the number of conjugates labeled with two types of fluorescent substances changes over time at multiple measurement points in the sample, and the sample also moves simultaneously, the two types of fluorescent substances in the sample The cross-correlation function profile of the fluorescence intensity fluctuation changes at the place where the number of labeled conjugates changes, and the cross-correlation function profile of the fluorescence intensity fluctuation basically changes at the place where the sample movement occurs. It does not change. When the sample is accompanied by movement of the sample, the cross-correlation function of the fluorescence intensity fluctuation is further measured several times to accumulate data and detect the amount and direction of movement of the sample. Since measurements are actually performed at multiple locations, the number of conjugates labeled with two types of fluorescent substances changes over time at multiple measurement points within the sample, and sample movement and deformation also occur. You can catch this even if it happens at the same time. In this case, it is desirable that there are as many measurement points as possible.

  If the measurement point and the detection point of sample movement do not match, the number of conjugates labeled with the two types of fluorescent substances in the sample is determined when the measurement values are acquired at the three measurement points in the sample. Measurement of change and measurement for grasping movement and deformation of the sample itself are performed with a time lag, and each measurement result is stored in the memory in the computer 126 and a fluorescent image is displayed on the TV monitor. indicate.

  In the measurement of the cross-correlation function of the fluorescence intensity fluctuation, an autocorrelation function of the fluorescence intensity fluctuation can be obtained at the same time, so the movement of the sample is measured based on the data of both the cross-correlation function and the autocorrelation function of the fluorescence intensity fluctuation. May be.

  As can be seen from the above description, in the present embodiment, a microscope observation apparatus having an automatic tracking function of a specimen sample can be obtained without requiring a video camera or a memory for tracking the sample.

  Although the embodiments of the present invention have been described above with reference to the drawings, the present invention is not limited to these embodiments, and various modifications and changes can be made without departing from the scope of the present invention. May be.

1 schematically shows a basic configuration of a microscope observation apparatus having an automatic sample tracking function in a first embodiment of the present invention. The whole structure of the optical scanning system of the microscope observation apparatus shown by FIG. 1 is shown. The whole structure of the fluorescence observation apparatus shown by FIG. 1 is shown. An example of the positional relationship between the four light spots and the sample is shown, with the left side showing the sample before moving and the right side after the sample moving. FIG. 5 shows autocorrelation functions of fluorescence intensity fluctuations in the four light spots before moving the sample shown on the left side of FIG. FIG. 5 shows autocorrelation functions of fluorescence intensity fluctuations at four light spots after movement of the sample shown on the right side of FIG. FIG. 5 shows changes in fluorescence intensity fluctuations in the four light spots before the movement of the sample shown on the left side of FIG. 4. FIG. 5 shows changes in fluorescence intensity fluctuations in the four light spots after the movement of the sample shown on the right side of FIG. 4. An arrangement example of four light spots is shown. The front half part of the flowchart of the detection of the movement of the sample in this embodiment is shown. The latter half part of the flowchart of the detection of the movement of the sample in this embodiment is shown. The whole structure of the microscope observation apparatus which has the sample automatic tracking function in 2nd embodiment of this invention is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Microscope observation apparatus, 110 ... Laser scanning confocal microscope, 111 ... Microscope main body, 112 ... Objective lens, 116 ... Deflection element, 122 ... Sample stage, 123 ... Stepping motor, 125 ... Sample stage controller, 126 ... Computer, DESCRIPTION OF SYMBOLS 130 ... Optical scanning system, 131 ... Laser light source, 135 ... XY scanning mechanism, 137 ... Pinhole, 138 ... Photo detector, 141 ... Drive apparatus, 142 ... Position calculation apparatus, 151 ... Signal processing apparatus, 152 ... Correlation analysis apparatus 200 ... Microscope observation device, 216 ... Deflection element, 230A ... Optical scanning system, 230A to 230C ... Optical scanning system, 230 ... Optical scanning system, 231A ... Laser light source, 231 ... One laser light source, 231 ... Laser light source, 232A ... Laser light source, 232 ... Laser light source, 233 ... Mirror, 234 ... Ichroic mirror, 235 ... mirror, 236 ... dichroic mirror, 237 ... XY scanning mechanism, 238 ... photodetector, 239 ... photodetector, 240 ... separation element, 242 ... driving device, 243 ... angle detector, 251 ... signal Processing device, 252... Correlation analysis device.

Claims (9)

Sample holding means for holding a specimen sample;
An image capturing means which captures a microscopic image containing the measurement object of the specimen sample,
Setting means for setting a plurality of measuring points and a plurality of mobile detection point in the measurement target of the microscope image,
Correlation analysis means for obtaining a correlation function of fluorescence intensity fluctuation obtained from the measurement point;
A movement detecting means for detecting movement of the measurement object based on a result of correlation calculation for obtaining a correlation function of fluorescence intensity fluctuation obtained from the movement detection point;
A microscope observation apparatus , comprising: an updating unit that updates the movement detection point when the movement detection unit detects the movement of the measurement object . According to claim 1, wherein the image acquiring means comprises means for observable displaying the microscopic image, wherein the setting means for designating an arbitrary point of the microscope image displayed on the display means The microscope observation apparatus characterized by having said input means. According to claim 1, wherein the updating means, microscopic observation apparatus characterized by comprising moving means for relatively moving the said movement detection point and said specimen sample. According to claim 3, wherein the moving means is a microscope observation apparatus characterized by comprising means Before moving the sample holding means. 5. The image acquisition unit according to claim 1, wherein the image acquisition unit includes a plurality of optical scanning systems capable of scanning the light spot of the excitation light beam, and each optical scanning system includes each measurement point and each movement detection point. The movement detection means detects the movement of the measurement object when the correlation function is not obtained, and the update means hits the movement detection point. Thus, the microscope observation apparatus characterized by moving the holding means with respect to the light spot. 6. The update unit according to claim 5, wherein the update unit includes a storage unit that stores a result of the correlation calculation performed by the movement detection unit, and the correlation function is not obtained by the movement detection unit. Microscope observation, wherein all the movement detection points are moved simultaneously, a position where the difference from the result of the correlation calculation stored in the storage means is minimized is determined, and the movement detection point is arranged at the position. apparatus. The microscope observation apparatus according to claim 1, wherein the correlation function includes an autocorrelation function. The microscope observation apparatus according to claim 1, wherein the correlation function includes a cross-correlation function.   The microscope observation apparatus according to any one of claims 1 to 8, wherein the measurement point and the movement detection point coincide with each other.

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