JP2008298860A - Living body observation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a clear observation image of a living body which vibrates periodically without blurring. <P>SOLUTION: The living body observation device 1 is equipped with a storage part 4 for storing the reference waveform of the vibration of the living body P which vibrates periodically, an observation optical system 3 for obtaining the observation image of the living body P, a drive part 5 for moving an observation range by the observation optical system 3, and a control part 6 for controlling the drive part 5 such that the observation range by the observation optical system 3 moves in accordance with the vibration of the living body P based on the reference waveform stored in the storage part 4. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a living body observation apparatus for observing a living body alive (in vivo).

  Conventionally, when a living body is observed with a microscope while living, the observation image is blurred due to vibrations such as respiration and pulsation of the living body, and there is a disadvantage that observation with a clear observation image cannot be performed. In order to solve this, an observation apparatus having a stabilizer that presses and fixes a living body during observation has been devised (see, for example, Patent Document 1).

JP 2005-338631 A

  However, in this observation apparatus, since the living body is pressed in the optical axis direction, vibration along the optical axis direction of the living body can be suppressed, and the inconvenience of shifting the focus of the observation optical system can be avoided. It is difficult to solve the problem that the vibration in the intersecting direction cannot be suppressed with high accuracy and the observed image is blurred.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a living body observation apparatus that can clearly obtain an observation image of a living body that periodically vibrates without blurring.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a storage unit that stores a reference waveform of vibration of a living body that periodically vibrates, an observation optical system that acquires an observation image of the living body, a drive unit that moves an observation range of the observation optical system, and the storage And a control unit that controls the drive unit so that the observation range of the observation optical system moves in a form that matches the vibration of the living body based on a reference waveform stored in the unit.

  According to the present invention, based on the reference waveform, when the drive unit is driven by the operation of the control unit, the observation range by the observation optical system is moved in a form that matches the vibration of the living body. That is, since the living body repeats periodic vibration, it is possible to obtain an observation image of the living body as if it is stationary by moving the observation range according to the reference waveform stored in advance in the storage unit. It becomes possible. Thereby, a clear observation image without blurring can be acquired by the observation optical system. Further, by storing the reference waveform in the storage unit in advance, it is possible to shorten the time required for processing and to accurately follow the observation range with the vibration of the living body.

In the above invention, a waveform acquisition unit that acquires a reference waveform of biological vibration is provided, the storage unit stores the reference waveform acquired by the waveform acquisition unit for at least one cycle, and the control unit includes a storage unit The driving unit may be controlled by repeatedly using a reference waveform stored in the memory.
By doing in this way, the reference | standard waveform of a biological vibration is acquired by the action | operation of a waveform acquisition part, and the reference | standard waveform for at least 1 period is memorize | stored in a memory | storage part. And a control part can follow an observation range over a long time with respect to the biological body which vibrates periodically by controlling a drive part repeatedly using the memorized reference waveform. In addition, the storage capacity of the storage unit can be saved.

In the above invention, the waveform acquisition unit may generate a vibration waveform at each position where the observation range is moved in the vibration direction of the living body when the observation range by the observation optical system is narrower than the amplitude of vibration of the living body. A reference waveform for at least one cycle may be generated by acquiring and synthesizing the plurality of acquired vibration waveforms.
Depending on the magnification of the observation optical system, the observation range may be narrower than the amplitude of the vibration of the living body. Even if the vibration waveform of the living body in a single observation range is acquired, the observation range is synchronized with the vibration of the living body. Is difficult. According to the present invention, the vibration waveform is acquired at each position where the observation range is moved in the vibration direction of the living body, and these are combined to generate a reference waveform for at least one period, thereby including the entire vibration of the living body. The reference waveform can be acquired, and the observation range can be easily synchronized with the vibration of the living body, and a clear observation image can be acquired.

Moreover, in the said invention, the said waveform acquisition part is good also as acquiring a reference | standard waveform based on the motion of the feature point in the observation range of the biological body by the said observation optical system.
By doing in this way, the reference waveform which fully reflected the vibration form of the living body can be easily acquired. In this case, in the observation image in the observation range, a high-intensity region is extracted as a feature point, and a reference waveform can be acquired by detecting a temporal change in the position. When there are few high-luminance regions in the observation image of a living body, the fluorescent beads can be easily extracted as feature points by attaching a marker such as a fluorescent bead that generates fluorescence by excitation light to the surface of the living body. it can.

Moreover, in the said invention, the said waveform acquisition part is good also as acquiring the reference | standard waveform by acquiring the vibration waveform of a biological body for multiple periods, and averaging the acquired vibration waveforms of multiple periods.
By doing so, it is possible to relax the irregularity of the vibration of the living body and generate a reference waveform that is more easily matched with all vibration waveforms.

  According to the present invention, there is an effect that an observation image of a living body that periodically vibrates can be obtained clearly without blurring.

A living body observation apparatus 1 according to a first embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the living body observation apparatus 1 according to the present embodiment includes a stage 2 on which a sample P such as a mouse is placed, and an observation optical system 3 that acquires an observation image of the sample P on the stage 2. A storage unit 4 that stores a reference waveform of vibration of the sample P placed on the stage 2, and a drive unit 5 that drives the observation optical system 3 based on the reference waveform stored in the storage unit 4. And a control unit 6 for controlling the drive unit 5.

  The observation optical system 3 includes an objective lens 7 that collects light such as reflected light or fluorescence from the sample P, an imaging lens 8 that forms an image of the light collected by the objective lens 7, and the imaging A beam splitter 9 that branches the light collected by the lens 8, two cameras 10 and 11 that respectively capture the light branched by the beam splitter 9, and a display that displays an image captured by one camera 10 Part 12.

  The other camera 11 is a high-speed camera (hereinafter referred to as the high-speed camera 11) having a sufficiently higher frame rate than the one camera 10 and processes an image taken by the high-speed camera 11. It is connected to the image processing unit 13. The image processing unit 13 generates a reference waveform of the vibration of the sample P placed on the stage 2 based on the image taken by the high speed camera 11 and stores it in the storage unit 4.

  The drive unit 5 includes an actuator (not shown) such as an ultrasonic motor that moves the objective lens 7 provided in the observation optical system 3 in two directions (X and Y directions) orthogonal to the optical axis, And a position detector (not shown) such as an encoder for detecting the position. The control unit 6 drives the actuator using the reference waveform stored in the storage unit 4 as a target value signal, and feeds back the position of the objective lens 7 detected by the position detector so that the error becomes zero. It comes to control.

Here, a method for generating a reference waveform of the vibration of the sample P by the image processing unit 13 will be described.
In order to generate the reference waveform, first, the sample P is placed on the stage 2, and an image of the sample P is acquired by the high-speed camera 11 (for example, a frame rate of 1000 fps) as shown in the flowchart of FIG. (Step S1), the acquired image is stored in an image memory (not shown) (Step S2), a high luminance area in each image is extracted and labeled (Step S3), and the barycentric position of the labeled area is determined. The calculation is performed (step S4), and the center of gravity position is stored in a memory (not shown) (step S5). Then, Steps S1 to S5 are repeated until the number of images necessary for generating the reference waveform is acquired (Step S6).

  In general, since the high-speed camera 11 has a short exposure time and low sensitivity, the image becomes dark and it is difficult to clearly extract a high-luminance region. Therefore, photographing is performed by attaching a substance that emits light such as the fluorescent beads 14 to the surface of the sample P. As shown in FIG. 3, by using a spherical body having a size corresponding to a plurality of pixels 15 of the high-speed camera 11, as the fluorescent beads 14, the position of the center of gravity can be accurately calculated.

  The extraction of the high luminance region can be easily performed by binarizing each acquired image based on a predetermined threshold, for example. As a result, the region occupied by the fluorescent beads 14 emitting fluorescence is extracted, and the feature point of the sample P can be set by calculating the position of the center of gravity. A time-series movement trajectory of the feature points set for each image in this way can be generated as a reference waveform.

More specifically, as shown by a thick frame in FIG. 4A, the observation range R including the trajectory Q of the entire vibration range AB of the vibration of the sample P (when vibrating between the signs A and B). And a case where only a part of the entire vibration range AB is observed as the observation range R as shown by a thick frame in FIG.
Here, the sample P actually vibrates in the X, Y, and 2 directions. However, in order to simplify the explanation, the sample P is described as being vibrated only in the X direction and not in the Y direction. Even in the case of simultaneous vibration in the X, Y, and 2 directions, the reference waveform can be generated by the same procedure.
Moreover, about the direction (Z direction) along the optical axis of the objective lens 7, a vibration shall be suppressed by using a stabilizer, for example.

  First, as shown in FIG. 4, when the observation range R including the entire vibration range AB is observed with the low-magnification objective lens 7, a predetermined value between the minimum value A and the maximum value B of the amplitude ( For example, time series data is acquired and stored in a time range in which the intermediate value M is included three times or more. Next, as shown in FIG. 4B, time-series data from the time t1 at which the predetermined value M is first reached to the time t3 at which the predetermined value M is thirdly extracted. Thereby, since the vibration waveform for 1 period is cut out, this can be memorize | stored in the memory | storage part 4 as a reference | standard waveform.

  Next, as shown in FIG. 5, when only a part of the vibration range AB is observed with the high-magnification objective lens 7, the moving direction of the observation range R is changed as shown in the flowchart of FIG. 0 is input to the flag Flag_limit to be initialized (step S11), and the initial observation range R (for example, the observation range CD, hereinafter, also referred to as the observation range CD using the minimum value C and the maximum value D) is used. Move (step S12). In this observation range CD, as shown in FIG. 5B, time series data is acquired and stored in a time range in which the minimum value C is included three times or more (step S13). Next, time-series data from the time t1 at which the minimum value C is first reached to the time t3 at which the minimum value C is reached for the third time is cut out (step S14). As a result, the vibration waveform for one period of the observation range CD is cut out, and this is temporarily stored (step S15).

  Next, it is determined whether or not there is a state in which the vibration waveform for one cycle exceeds the maximum value D of the observation range CD (step S16). As indicated by the broken line, the objective lens is moved by one step in the X direction by the width dimension of the observation range R (step S17). Here, it is moved to the observation range DE. Thereafter, steps S13 to S17 are repeated.

  When the maximum value Pmax of the time-series data does not reach the maximum value G of the observation range FG as in the observation range FG shown in FIG. 7, that is, in step S16, the maximum of the observation range FG is obtained as a vibration waveform for one cycle. If it is determined that there is no state exceeding the value G, it is determined that the end of the vibration range AB has been reached, and it is determined whether or not the acquisition of the reference waveform has ended (step S18).

  Then, in order to reverse the movement direction of the observation range R, 1 is input to the flag Flag_limit (step S19), and the observation range HC is moved from the first observation range CD in the opposite direction by the width dimension of the observation range R. Move (step S20). Next, time series data is acquired and stored in a time range in which the maximum value C is included three or more times in the observation range HC (step S13). Next, time series data from the time t1 at which the maximum value C is first reached to the time t3 at which the maximum value C is reached for the third time is cut out (step S14).

  Then, a vibration waveform for one period is cut out and temporarily stored for the observation range HC (step S15). Next, it is determined whether or not there is a state where the vibration waveform for one cycle is below the minimum value H of the observation range HC (step S16). Is moved in the X direction by the width dimension of the observation range HC (step S17), and steps S13 to S17 are repeated thereafter.

  Thereafter, as shown in the observation range IJ of FIG. 8, when the minimum value Pmin of the time series data does not reach the minimum value J of the observation range IJ, that is, in step S16, the observation range IJ is converted into a vibration waveform for one cycle. If it is determined that there is no state below the minimum value J, it is determined that one end A of the vibration range AB has been reached, it is determined that acquisition of the reference waveform has ended (step S18), The process ends.

As a result, the vibration waveform over the entire vibration range AB of the vibration of the sample P is temporarily stored in a state divided into a plurality in the vibration direction. A vibration waveform over the range AB is generated.
Specifically, in all adjacent observation ranges R, the time axis is adjusted so that the time of the minimum value of one observation range R and the time of the maximum value of the other observation range R coincide.

  For example, in the observation range CD and the observation range DE cut out for one period, as shown in FIG. 9, a time TD1 at which the maximum value D is first at the beginning of the observation range CD, and a minimum value D at the beginning of the observation range DE. Since the time TD2 is shifted by ΔTD, the time series data of the region DE is shifted by ΔTD, and the vibration waveforms of the observation range CD and the observation range DE are synthesized. By performing this operation for all observation ranges R, as shown in FIG. 10, a vibration waveform over the entire vibration range AB is synthesized, and this is stored in the storage unit as a reference waveform.

  Since the reference waveform of the vibration of the sample P is generated and stored in the storage unit 4 in this way, the control unit 6 uses the reference waveform for one cycle of the vibration of the sample P stored in the storage unit 4 as the target value. It outputs as a signal and it controls to make the actuator of the drive part 5 track. The control unit 6 continuously outputs target value signals for a plurality of cycles at a timing when the peak value of the target value signal read from the storage unit 4 coincides with one end of the vibration of the sample P.

As a result, the objective lens 7 is moved in a direction and at a speed that matches the direction and speed of the vibration in synchronization with the vibration of the sample P, so that the observation image photographed by the camera 10 vibrates as if. It looks as if it is still. That is, according to the living body observation apparatus 1 according to the present embodiment, there is an advantage that an observation image of the vibrating sample P can be clearly obtained without blurring like a still image.
In addition, according to the living body observation apparatus 1 according to the present embodiment, since the reference waveform of the vibration of the sample P is stored in the storage unit 4 in advance, the processing for controlling the actuator is easy, and the vibration of the sample P Therefore, the objective lens 7 can be accurately followed without time delay.

  In the living body observation apparatus 1 according to the present embodiment, the driving unit 5 that drives the objective lens 7 includes an actuator such as an ultrasonic motor, but instead, a voice coil motor that can be linearly driven. A linear motor or a piezo motor may be employed. Further, linear drive may be realized by combining a ball screw with a rotary motor such as a DC motor.

  Further, a stepping motor may be employed. In this case, control may be performed in an open loop without feeding back a signal from a position detector such as an encoder.

  In this embodiment, the vibration waveform for one cycle is acquired and stored in the storage unit 4 as a reference waveform. Instead, after acquiring the vibration waveforms for a plurality of cycles, The average waveform may be stored as a reference waveform. By doing so, the irregularity of vibration of the sample P made of a living body can be relaxed, and the objective lens 7 can be made to follow the dynamic behavior of the sample P with higher accuracy.

  Further, in the present embodiment, the reference waveform is generated in the living body observation apparatus 1, but instead, the reference waveform generated by another apparatus is stored in the storage unit 4 and based on this. The objective lens 7 may be driven and controlled. Further, the stage 2 may be driven and controlled instead of the driving control of the objective lens 7. Further, the observation range R may be moved by driving and controlling other optical components incorporated in the observation optical system 3.

It is a whole lineblock diagram showing a living body observation device concerning one embodiment of the present invention. 2 is a flowchart for explaining acquisition of time-series data in a method for generating a reference waveform of sample vibration by the biological observation apparatus of FIG. In the flowchart of FIG. 2, it is a figure explaining the fluorescent bead used in order to clarify the vibration of a sample. In the living body observation apparatus of FIG. 1, when (a) the whole vibration range is included in an observation range, (a) The relationship between the locus | trajectory of a fluorescent bead and an observation range, (b) It is a figure which shows the time series data of a motion of a fluorescent bead. 1 shows (a) the relationship between the locus of the fluorescent beads and the observation range when the observation range includes only a part of the entire vibration range, and (b) the time series data of the movement of the fluorescent beads. FIG. 6 is a flowchart illustrating a method for generating a reference waveform of sample vibration in the case of FIG. It is a figure which shows the case where the observation range of FIG. 5 is arrange | positioned at the end of the locus | trajectory of a fluorescent bead. It is a figure which shows the case where the observation range of FIG. 5 is arrange | positioned at the end of the locus | trajectory of a fluorescent bead. It is a figure explaining the process which synthesize | combines the reference waveform of a vibration based on the some image acquired according to the flowchart of FIG. It is a figure which shows an example of the reference waveform for 1 period of the vibration synthesize | combined by the synthetic | combination process of FIG.

Explanation of symbols

P Sample (living body)
R observation range 1 living body observation apparatus 3 observation optical system 4 storage unit 5 drive unit 6 control unit 11 high-speed camera (waveform acquisition unit)

Claims (5)

A storage unit for storing a reference waveform of a vibration of a living body that vibrates periodically;
An observation optical system for acquiring an observation image of a living body;
A drive unit for moving an observation range by the observation optical system;
A living body observation apparatus comprising: a control unit that controls the driving unit so that an observation range by the observation optical system moves in a form that matches vibration of a living body based on a reference waveform stored in the storage unit. A waveform acquisition unit for acquiring a reference waveform of biological vibration is provided,
The storage unit stores at least one period of the reference waveform acquired by the waveform acquisition unit,
The living body observation apparatus according to claim 1, wherein the control unit controls the driving unit by repeatedly using a reference waveform stored in the storage unit.   The waveform acquisition unit acquires a vibration waveform at each position obtained by moving the observation range in the vibration direction of the living body when the observation range by the observation optical system is narrower than the amplitude of vibration of the living body, The living body observation apparatus according to claim 2, wherein a reference waveform for at least one cycle is generated by synthesizing the vibration waveforms.   The living body observation apparatus according to claim 2, wherein the waveform acquisition unit acquires a reference waveform based on a movement of a feature point in a living body observation range by the observation optical system.   The living body observation apparatus according to claim 2, wherein the waveform acquisition unit acquires a vibration waveform of a living body for a plurality of periods, and averages the acquired vibration waveforms of a plurality of periods to acquire a reference waveform.

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