JP2005300278A - Living body observation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To acquire a clear image not only in the stationary state but also in the active state from a living body showing dynamic behavior. <P>SOLUTION: This living body observation device is equipped with an imaging means 3 for imaging an observation target portion of the living body A showing dynamic behavior, an imaging optical system 19 arranged between the imaging means 3 and the observation target portion, a focal point adjusting means 17 for adjusting the focal position C of the imaging optical system 19, a behavior detection means 16 for detecting the dynamic behavior of the living body A, and a control device 7 for controlling the focal point adjusting means 17 so that the focal position C agrees with the observation target portion based on the dynamic behavior of the living body A detected by the behavior detection means 16. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a living body observation apparatus that observes a living body in a living state (in vivo), for example.

In recent years, visualization of ion concentration, membrane potential, etc. using a fluorescent probe has been performed using an optical microscope. For example, biological functions such as nerve cells can be observed as specimens, especially dynamic behavior. ing.
As a device for observing such a dynamic behavior, a microphotographing device is known (for example, see Patent Document 1).
JP 2000-275539 A

  However, such a conventional microphotographing apparatus takes a photograph in accordance with the dynamic behavior of the specimen. However, while keeping the focal length of the camera constant, the dynamic behavior of the specimen is within the range. Since the in-focus still state is selectively photographed, the obtained image is fragmented, and in particular, there is a problem that the state of the moving sample cannot be observed.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a microscopic image capturing apparatus that can obtain a clear image from a living body exhibiting dynamic behavior.

In order to achieve the above object, the present invention provides the following means.
The present invention relates to an imaging unit that images a biological observation target region that exhibits a dynamic behavior, an imaging optical system disposed between the imaging unit and the observation target region, and a focal position of the imaging optical system is adjusted. Focus adjusting means, behavior detecting means for detecting the dynamic behavior of the living body, and based on the dynamic behavior of the living body detected by the behavior detecting means, the focus position matches the observation target site. An observation apparatus comprising a control device for controlling the focus adjusting means is provided.

  According to this invention, the dynamic behavior due to physiological phenomena such as pulsation and pulsation of the living body or peristalsis is detected by the operation of the behavior detecting means. When the living body exhibits dynamic behavior, if the focus position of the imaging optical system is kept constant, the image may be blurred or the observation position in the depth direction may fluctuate. Since the control means controls the focus adjustment means based on the dynamic behavior of the living body detected by the detection means, the focus position of the imaging optical system can be maintained in a state where it matches the observation target part of the living body. . As a result, not only an image in which the living body is stationary but also a clear image in the operating state can be obtained.

  In the above invention, the behavior detecting means may be a sensor that detects a surface position of a living body. By detecting the surface position by the sensor, the displacement amount due to the dynamic behavior of the living body can be directly obtained. Therefore, complicated calculation is not required for controlling the focal position of the imaging optical system, and the focal position of the imaging optical system can follow the dynamic behavior of the living body without delay.

  Moreover, in the said invention, it is preferable that the said focus adjustment means is provided with the variable focus lens which changes a focal distance based on the control signal from the said control apparatus. According to the variable focus lens, it is possible to sufficiently secure the adjustment speed of the focus position of the imaging optical system with a simple configuration.

Moreover, in the said invention, the said focus adjustment means is good also as comprising a linear actuator which moves the focus position of the said imaging optical system.
In the invention described above, a stage on which a living body is placed may be provided, and the focus adjustment unit may be a linear actuator that displaces the stage based on a control signal from the control device.
As the linear actuator, there are high-speed actuators such as a piezo motor and a voice coil motor, and a sufficient adjustment speed of the focal position of the imaging optical system can be secured with a simple configuration.

In the above invention, the control means controls the focus adjustment means to maintain the focus of the imaging optical system at a position in the depth direction that is deviated by a predetermined distance from the surface position of the living body detected by the sensor. Also good.
When the observation target site is below the surface of the living body, the focus adjustment means is controlled to maintain the focal position at a position shifted by a predetermined distance in the depth direction from the detection position by the sensor. It is possible to acquire an image while maintaining the focused state on the observation target portion that follows and follows.

In the above invention, the control means stores a history storage means for storing a history of the dynamic behavior of the living body detected by the behavior detection means, and a history of the living body based on the history stored in the history storage means. It is good also as a behavior estimation means which estimates a dynamic behavior, and controlling the said focus adjustment means based on the estimated dynamic behavior.
For example, in the case of vibration that occurs at a predetermined cycle such as a pulsation, the focal position of the imaging optical system can be tracked at a higher speed by estimating based on the history stored in the history storage means. Thus, a clearer image can be acquired.

  According to the present invention, when observing a living body exhibiting dynamic behavior while alive, it is possible to obtain an observation result including more information by imaging with the focal position following the dynamic behavior of the living body. There is an effect that can be done.

Hereinafter, a living body observation apparatus according to a first embodiment of the present invention will be described with reference to FIGS. 1 and 2.
As shown in FIG. 1, the living body observation apparatus 1 according to the present embodiment includes an optical unit 4 including a laser light source 2 and a light detector (imaging means) 3, a laser light from the laser light source 2, and a light detector. An optical fiber 5 that propagates fluorescence to 3 and a laser beam propagated through the optical fiber 5 are scanned onto a sample A such as an experimental small animal, and the fluorescence emitted from the sample A is received and guided to the optical fiber 5. A measuring head 6 and a control device 7 for controlling the focal position of the measuring head 6 are provided.

  The optical unit 4 includes a collimating lens 8 and a dichroic mirror 9. The laser light emitted from the laser light source 2 is once collimated by the collimating lens 8, then transmitted through the dichroic mirror 9, and condensed again on the one end 5 a of the optical fiber 5 by the collimating lens 8. It has become. On the other hand, the fluorescence emitted from the one end 5 a of the optical fiber 5 is reflected by the dichroic mirror 9 and is condensed and detected by the condenser lens 10 on the photodetector 3.

  The measurement head 6 includes a collimating optical system 11 that converts laser light propagated through the optical fiber 5 into parallel light, an optical scanning unit 12 that deflects the parallel light and scans it in a two-dimensional direction, and the optical scanning unit. The pupil projection optical system 13 that forms the light emitted from 12 at the intermediate image position B, the imaging optical system 14 that converts the light that formed the intermediate image into parallel light again, and the intermediate image as the observation object of the sample A An objective optical system 15 that re-images the part and a distance sensor 16 that measures the distance between the measuring head 6 and the surface of the sample A are provided. The collimating optical system 11 is provided with a linear actuator 17 that moves part or all of the lenses constituting the collimating optical system 11 in the optical axis direction. The optical scanning unit 12 includes, for example, two galvanometer mirrors 12a and 12b that can rotate around two axes orthogonal to each other.

The linear actuator 17 is constituted by, for example, a piezo motor.
The photodetector 3 is, for example, a photomultiplier tube.
The photodetector 3 is connected to a monitor 18 and displays a captured fluorescent image.

The control device 7 receives the detection signal from the distance sensor 16, calculates the distance between the measuring head 6 and the surface of the sample A in real time, and outputs a displacement command of the linear actuator 17 to the linear actuator 17. It has become.
The control device 7 is provided with an offset function for offsetting the focal position C of the imaging optical system 19 including the collimating optical system 11 to the objective optical system 15 by a predetermined distance.

The operation of the biological observation apparatus 1 according to the present embodiment configured as described above will be described below.
Laser light emitted from the laser light source 2 is propagated through the optical fiber 5 and enters the measuring head 6, converted into parallel light by the collimating optical system 11, deflected by the optical scanning unit 12, and pupil projection optics. An image is formed on the sample A through the system 13, the imaging optical system 14, and the objective optical system 15, and fluorescence is generated there. The fluorescence generated in the sample A returns to the optical unit 4 in the optical fiber 5 through the objective optical system 15, the imaging optical system 14, the pupil projection optical system 13, the optical scanning unit 12, and the collimating optical system 11, and the dichroic mirror 9. Thus, the light beam is separated from the optical axis toward the laser light source 2 and detected by the photodetector 3 and displayed on the monitor 18.

  In this case, in order to start observation of the sample A such as an experimental small animal, first, the sample A is irradiated with laser light, and the reflected light on the surface of the sample A is detected and displayed on the monitor 18. The operator operates the monitor 18 so that the focal position C of the imaging optical system 19 coincides with the surface of the sample A. Since the surface of the sample A is pulsating, for example, the focal position C may be made coincident with the surface of the sample A in a substantially stationary state during the pulsation. Then, when the focal position C coincides with the surface of the sample A, control by the control device 7 is started.

  Since the distance sensor 16 measures the distance between the measuring head 6 and the surface of the sample A, the control device 7 measures the measuring head 6 in a state where the focal position C coincides with the surface of the sample A (in the present embodiment, the measurement is performed). With reference to the distance L between the front surface of the distance sensor 16 fixed to the head 6 and the surface of the sample A, a displacement amount ΔL due to the pulsation of the surface of the sample A therefrom can be obtained. Then, by operating the linear actuator 17 to move the collimating optical system 11 so as to displace the focal position C in the same direction as the displacement direction of the surface of the sample A by this ΔL, the focal position C matches the surface of the sample A. It can be maintained in a state of being allowed to enter. In particular, since the high-speed piezo motor is used as the linear actuator 17 in the living body observation apparatus 1 according to the present embodiment, the focal position C follows the surface of the sample A with high speed and accuracy regardless of fluctuation due to pulsation. be able to.

  Actually, since the site to be observed is arranged at a position deeper than the surface of the sample A by a predetermined distance D in the depth direction, the collimating optical system 11 is moved by using the offset function, and FIG. The focal position is shifted by D as indicated by the chain line. Accordingly, when the control device 7 calculates that the surface of the sample A is displaced by ΔL due to the pulsation of the sample A, the focal position C is also ΔL by the operation of the linear actuator 17 as shown in FIG. Since it is displaced, the focal position C is maintained at a distance D below the surface of the sample A.

  That is, according to the living body observation apparatus 1 according to the present embodiment, the dynamic behavior of the sample A is detected by the distance sensor 16, and the focal position C of the imaging optical system 19 is positioned below the surface of the sample A at the distance D. Since it adjusts in real time so that it may correspond to the observation object site | part distribute | arranged to, the clear image which suppressed blurring can be obtained. In addition, since the image in the operating state as well as the stationary state of the sample A showing the dynamic behavior can be obtained, the information obtained from the sample A can be acquired without waste.

In the living body observation apparatus 1 according to the present embodiment, a piezo motor is employed as the linear actuator 17 that moves the collimating optical system 11, but instead, any other high-speed linear actuator such as a voice coil motor is employed. May be. In addition, the focal position C is adjusted by the collimating optical system 11, but instead, the focal position C is adjusted by moving the objective optical system 15 by the linear actuator 17, as shown in FIG. You may decide.
Further, the tip of the optical fiber 5 may be moved in the optical axis direction by the linear actuator 17.

  Further, in place of the method of moving the collimating optical system 11 and the objective optical system 15 by the linear actuator 17, as shown in FIG. The variable focus lens 27 that changes the focal position C by changing the shape of the lens body surface by changing the pressure of the liquid filled in the body may be adopted. In this case, a linear actuator such as a piezo element 28 connected to the variable focus lens 27 is provided, and the focal position C is changed by controlling the pressure applied to the variable focus lens 27 by a movement command from the control device 7. It may be.

  When a reflective objective optical system or the like is employed, a variable focus mirror (not shown) may be employed. Furthermore, although the example which arrange | positions the distance sensor 16 outside the objective optical system 15 was demonstrated, you may incorporate in the housing same as the objective optical system 15. FIG.

In addition, the focus is visually adjusted at the start of the observation. For example, the contrast of the detected image is calculated, and the linear actuator 17 is displaced to the position where the contrast is the highest, thereby performing the automatic focusing. May be.
Further, the distance sensor 16 is adopted as means for detecting the dynamic behavior of the sample A, but instead of this, other pulsation detection means such as an electrocardiogram detector, an ultrasonic detector, a sound sensor (electred red) Condenser microphone: ECM), Optical coherence tomography (Optical)
Coherence Tomography (OCT), an out-of-plane displacement measuring device using speckles, etc. may be employed.

Next, a living body observation apparatus according to a second embodiment of the present invention will be described with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to the portions having the same configuration as the biological observation apparatus 1 according to the first embodiment described above, and the description will be simplified.

  The biological observation apparatus 20 according to the present embodiment includes a base 21 that is horizontally disposed, a column 22 that extends in the vertical direction from the base 21, an arm 23 that is attached to the column 22 and supports the measurement head 6, And a stage 24 on which the sample A is mounted. The stage 24 includes an XY table 25 that moves the sample A in two horizontal directions, and an elevating mechanism 26 that moves the XY table 25 in the vertical direction. The measuring head 6 has an optical axis arranged vertically downward with an interval above the stage 24.

In the living body observation apparatus 20 according to the present embodiment, the living body observing apparatus according to the first embodiment is not provided with a focus adjusting means on the measurement head 6 side and the focus adjusting means is configured by the lifting mechanism 26 of the stage 24. This is different from the observation apparatus 1.
The control device 7 receives information from the distance sensor 16 provided in the measuring head 6 and outputs a vertical movement command to the elevating mechanism 26 so that the output fluctuation from the distance sensor 16 becomes zero. It is supposed to be.

  According to the biological observation apparatus 20 according to the present embodiment configured as described above, the sample A in the operating state regardless of the dynamic behavior of the sample A, like the biological observation apparatus 1 according to the first embodiment. A clear image with reduced blurring can be obtained. In addition to this, unlike the first embodiment in which the focal position C is adjusted on the measurement head 6 side, the stage 24 cancels the dynamic behavior at the focal position C according to the dynamic behavior of the sample A. Is moved up and down, so that there is an advantage that the imaging optical system 19 which dislikes vibrations can be fixed.

Next, a living body observation apparatus 30 according to a third embodiment of the present invention will be described below with reference to FIG.
The biological observation apparatus 30 according to the present embodiment is different from the biological observation apparatuses 1 and 20 according to the first and second embodiments in the control device 7.

  As shown in FIG. 5, the control device 7 of the living body observation apparatus 30 according to the present embodiment sequentially receives position information from the distance sensor 16 and calculates a variation distance ΔLn of the surface of the sample A with respect to a predetermined reference distance L. The dynamic distance of the sample A is received by receiving the variable distance calculator 31, the variable distance ΔLn calculated by the variable distance calculator 31, and the time information tn generated from the clock 32 and storing them in association with each other. A history storage means 33 for storing a history of behavior, a fluctuation distance estimation unit 34 for calculating an estimated value ΔLn + 1 of a fluctuation distance in the next step based on the history stored in the history storage means 33, and the actual fluctuation The switching means 35 for selecting either the distance ΔLn or the estimated value ΔLn + 1 of the fluctuation distance, and either the fluctuation distance ΔLn or the fluctuation distance estimation value ΔLn + 1 Based on and a movement command calculator 36 which calculates a focus position moving command.

  According to the living body observation apparatus 30 according to the present embodiment configured as described above, when the position information from the distance sensor 16 is input to the control apparatus 7, the variable distance calculation unit 31 performs the sample A based on the position information. A surface fluctuation distance ΔLn is calculated. Until the history of the dynamic behavior of the sample A is created, the fluctuation distance ΔLn calculated by the fluctuation distance calculation unit 31 remains as it is, and the basis for calculating the focal position movement command to the focus adjusting means such as the linear actuator 17 Then, the movement command calculated based on ΔLn is output from the focal position movement command calculation unit 36. In this case, the fluctuation distance ΔLn calculated by the fluctuation distance calculation unit 31 is input to the history storage means 33 together with the time tn when the fluctuation distance ΔLn occurs, and is stored as a dynamic behavior history as shown in FIG. The

  For example, a dynamic behavior that occurs at a substantially constant cycle, such as a heartbeat, does not change abruptly, and the next behavior can be predicted by considering a certain history. The fluctuation distance estimation unit 34 calculates and outputs the next fluctuation distance estimated value ΔLn + 1 based on the history stored in the history storage unit 33. The estimation may be performed based on, for example, the average of the fluctuation distance for the past several cycles, the increase / decrease of the frequency, or the like.

  Then, after a predetermined time has elapsed, or when necessary, the switching means 35 is operated to select the fluctuation distance estimated value ΔLn + 1, so that the fluctuation distance estimated value ΔLn + 1 becomes a movement command to the focus adjusting means. It will be the basis of calculation. That is, according to the living body observation apparatus 30 according to the present embodiment, the next dynamic behavior is estimated in advance based on the history of the dynamic behavior of the sample A. It is possible to prevent the focal position adjustment operation from deviating from the dynamic behavior, and to follow the dynamic position of the sample A more accurately.

It is a whole lineblock diagram showing the living body observation device concerning a 1st embodiment of the present invention. It is a figure explaining the focus position adjustment of the biological observation apparatus of FIG. It is a whole block diagram which shows the modification of the biological observation apparatus of FIG. It is a whole block diagram which shows the other modification of the biological observation apparatus of FIG. It is a whole block diagram which shows the biological observation apparatus which concerns on the 2nd Embodiment of this invention. It is a block diagram which shows the control apparatus of the biological observation apparatus which concerns on the 3rd Embodiment of this invention. It is a graph which shows the log | history of the dynamic behavior of the biological body produced in the control apparatus of FIG.

Explanation of symbols

A Sample (living body)
C Focus position 1, 20, 30 Living body observation device 3 Photodetector (imaging means)
7 Control device 16 Distance sensor (behavior detection means)
17 Linear actuator (focus adjustment means)
19 imaging optical system 24 stage 33 history storage means 34 behavior estimation means

Claims (7)

An imaging means for imaging an observation target part of a living body exhibiting dynamic behavior;
An imaging optical system disposed between the imaging means and the observation target site;
Focus adjusting means for adjusting the focus position of the imaging optical system;
Behavior detecting means for detecting the dynamic behavior of the living body;
A living body observation apparatus comprising: a control device that controls the focus adjustment means so that the focus position coincides with an observation target site based on the dynamic behavior of the living body detected by the behavior detection means.   The living body observation apparatus according to claim 1, wherein the behavior detecting unit is a sensor that detects a surface position of the living body.   The living body observation apparatus according to claim 1, wherein the focus adjustment unit includes a variable focus lens that changes a focal length based on a control signal from the control apparatus.   The living body observation apparatus according to claim 1, wherein the focus adjustment unit includes a linear actuator that moves a focus position of the imaging optical system based on a control signal from the control apparatus. Equipped with a stage for placing a living body,
The living body observation apparatus according to claim 1, wherein the focus adjustment unit includes a linear actuator that displaces the stage based on a control signal from the control apparatus.   6. The control unit according to claim 2, wherein the control unit controls the focus adjustment unit to maintain the focus of the imaging optical system at a position in a depth direction that is shifted by a predetermined distance from the surface position of the living body detected by the sensor. A biological observation apparatus according to claim 1.   The control means stores a history storage means for storing a history of the dynamic behavior of the living body detected by the behavior detection means, and a behavior for estimating the dynamic behavior of the living body based on the history stored in the history storage means The living body observation apparatus according to any one of claims 1 to 6, further comprising an estimation unit, wherein the focus adjustment unit is controlled based on the estimated dynamic behavior.

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