JP2008065340A - Microscopic observation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a distinct image from a living body showing dynamic behavior, especially, moving in a short cycle. <P>SOLUTION: The microscopic observation device 1 for observing a biological sample S showing the dynamic behavior is equipped with: a first imaging apparatus 8 picking up the image of the biological sample S; a second imaging apparatus 9 picking up the image of the biological sample S at higher speed than the first imaging apparatus 8; and a controller 18 processing the image picked up by the second imaging apparatus 9 and arithmetically calculating a command signal for correcting the deviation of the image picked up by the first imaging apparatus 8. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microscope observation apparatus.

In recent years, in biological research, it has become possible to visualize ion concentration, membrane potential, etc. with a fluorescent probe using an optical microscope. So-called in-vivo observation for observing organs and the like has been performed. In in-vivo observation, the observation target has movements such as pulsation and respiratory movement, so that the observation position shifts and the focus is likely to be blurred.
As a method for eliminating such observation position deviation and out-of-focus blur, an apparatus is known in which an entire microscope including an imaging unit is made to follow the movement of an observation target (see, for example, Patent Document 1).

JP-A-7-222754

However, the apparatus of Patent Document 1 has a problem that it cannot be operated at high speed.
The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a microscope image capturing apparatus that can obtain a clear image from a living body that exhibits dynamic behavior and moves in a particularly short cycle.

In order to achieve the above object, the present invention provides the following means.
The present invention is a microscope observation apparatus for observing a biological sample exhibiting dynamic behavior, the first imaging apparatus for capturing an image of the biological sample, and the biological sample at a higher speed than the first imaging apparatus. And a command signal for correcting an image shift of the image picked up by the first image pickup device by processing the image picked up by the second image pickup device. There is provided a microscope observation device including a control device for calculation.

In the above invention, the image shift in the image picked up by the first image pickup device may be generated by the dynamic behavior of the biological sample.
In the above invention, the driving means for driving an objective lens for forming an image of the biological sample in a direction for correcting the image shift based on the command signal calculated by the control device is further provided. You may have.
Moreover, in the said invention, the light which injected from the said biological sample into the objective lens for forming the image of the said biological sample is branched toward the said 1st imaging device and the said 2nd imaging device. You may further have a beam splitter.

  According to the present invention, there is an effect that a clear image can be obtained from a living body that exhibits dynamic behavior and moves in a short cycle. Further, there is an effect that images can be obtained continuously and continuously from a living body moving with a large amplitude without losing the field of view.

Hereinafter, a microscope observation apparatus 1 according to a first embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 1, the microscope observation apparatus 1 according to the present embodiment guides the excitation light E from the stage 2 on which the sample S is mounted, the excitation light source 3 that emits the excitation light E, and the excitation light source 3. The illumination lens (illumination optical system) 4 and the excitation light E guided by the illumination lens 4 are guided to the sample S, while the objective lens 5 that collimates the fluorescence F generated in the sample S is collimated by the objective lens 5 A dichroic mirror 6 for branching the fluorescent light F from the excitation light E, a beam splitter 7 for further branching the branched fluorescent light F, and an imaging device (light detection unit) such as two CCDs for detecting the branched fluorescent light F 8 and 9. Reference numeral 20 in the figure denotes an imaging lens.

  The excitation light source 3, the illumination lens 4, the dichroic mirror 6, the beam splitter 7, and the two imaging devices 8 and 9 are provided in the microscope body 10. On the other hand, the objective lens 5 is separated from the microscope body 10 and supported by the objective lens driving device 11.

  The objective lens driving device 11 includes an arm 12 that arranges the objective lens 5 between the stage 2 and the microscope body 10, and a first drive mechanism 13 that moves the objective lens 5 held by the arm 12 in the vertical direction. And second and third drive mechanisms 14, 15 for moving the objective lens 5 in two horizontal directions, and swinging the objective lens 5 about an axis along two directions orthogonal to the optical axis of the objective lens 5. The fourth and fifth drive mechanisms 16 and 17 are provided. The first to third drive mechanisms 13 to 15 include, for example, motors 13a to 15a and a linear movement mechanism such as a ball screw (not shown) connected to the motors 13a to 15a. The fourth and fifth drive mechanisms 16 and 17 include, for example, motors 16a and 17a and speed reduction mechanisms 16b and 17b connected to the motors 16a and 17a. As the speed reduction mechanisms 16b and 17b, for example, as shown in FIG. 2, feed screws 16c and 17c and nuts 16d and 17d provided on the fixed side and operations of the nuts 16d and 17d provided on the moving side are transmitted. The thing provided with the nail | claw parts 16e and 17e, or the thing provided with the worm gears 16f and 17f as FIG. 3 shows are mentioned.

  The image pickup device 9 processes the image acquired by the image pickup device 9 to calculate the displacement amount and direction of the sample S, and also sends the sample to the drive mechanisms 13 to 17 of the objective lens drive device 11. A control device 18 that outputs a drive command for driving the objective lens 5 in a direction to correct the deviation of the optical axis of the objective lens 5 due to the displacement of S is connected.

In the microscope observation apparatus 1 according to the present embodiment configured as described above, as shown in FIG. 4, the objective lens 5 is moved in the Z-axis direction along the optical axis, the optical axis by the operation of the objective lens driving device 11. Can be moved in the X-axis and Y-axis directions orthogonal to each other, and in the A and B directions around the X-axis and Y-axis.
More specifically, as shown in FIGS. 5A and 5B, the objective lens 5 can be translated while being maintained in the X-axis, Y-axis, and Z-axis directions. It has become. Further, in the A and B directions around the X and Y axes, the objective lens 5 can be swung around its principal point H as shown in FIG.

  When the objective lens 5 is swung around its principal point H, as shown in FIGS. 6A and 6B, the observation surface of the sample S and the imaging surfaces of the imaging devices 8 and 9 are always provided. Arranged in a conjugate positional relationship. Therefore, even if the objective lens 5 is swung around the principal point H according to the inclination of the sample S, the conjugate positional relationship between the observation surface of the sample S and the imaging surfaces of the imaging devices 8 and 9 is not lost. The focus position can be prevented from being shifted, and the occurrence of one-sided blur can also be prevented.

In this case, when the objective lens 5 is swung around the principal point H, as shown in FIG. 6B, the fluorescence F emitted from the center position O ₀ of the observation range of the sample S is changed to the objective lens 5. And enters the center position I ₀ of the visual field range of the imaging devices 8 and 9 through a path indicated by a solid line in the figure. Therefore, in the imaging devices 8 and 9, the center position O ₀ of the observation range of the sample S to be observed is always maintained at the center position I ₀ of the visual field range, and the problem of image displacement does not occur. There is an advantage. In the figure, symbol O ₁ is an object point.

The operation of the microscope observation apparatus 1 according to the present embodiment configured as described above will be described below.
According to the microscope observation apparatus 1 according to the present embodiment, the excitation light E emitted from the excitation light source 3 is emitted from the microscope body 10 via the illumination lens 4 and the dichroic mirror 6 and is incident on the objective lens 5. The excitation light E incident on the objective lens 5 is applied to the sample S placed on the stage 2 through the objective lens 5. When the sample S is irradiated with the excitation light E, the fluorescent substance in the sample S is excited and fluorescence F is emitted.

  The fluorescence F generated in the sample S is collimated by the objective lens 5 and enters the microscope body 10 as substantially parallel light. The fluorescence F incident on the microscope body 10 is branched from the excitation light E by the dichroic mirror 6, further branched by the beam splitter 7, and detected by separate imaging devices 8 and 9.

  Image information acquired by being imaged by one imaging device 9 is sent to the control device 18, and is subjected to image processing by the control device 18, so that it is sent to the drive mechanisms 13 to 17 of the objective lens drive device 11. Command signals to be operated are calculated, and the drive mechanisms 13 to 17 are driven based on the command signals.

  When the sample S is displaced in the horizontal direction, the direction and amount of displacement are detected by image processing, and the control device is configured to translate the objective lens 5 in the horizontal direction by the same amount of displacement in the same direction. A command signal is sent from 18 to the second and third drive mechanisms 14 and 15, respectively. Thereby, since the objective lens 5 is translated so as to follow the displacement of the sample S, an image with less blur can be acquired.

  When the sample S is displaced in the optical axis direction, the amount of displacement is detected by image processing, and the control device 18 moves the objective lens 5 in the optical axis direction by the same amount of displacement. A command signal is sent to the first drive mechanism 13. As a result, the objective lens 5 is translated in the optical axis direction so as to follow the displacement of the sample S, so that a clear and focused image can be acquired.

  Further, when the sample S is tilted around the horizontal axis, the direction and the swing angle are detected by image processing, and the objective lens 5 is swung in the same direction by the same swing angle. A command signal is sent from the control device 18 to the fourth and fifth drive mechanisms 16 and 17. As a result, the objective lens 5 is swung so as to follow the inclination of the sample S, so that a clear image with less one-side blur can be acquired.

In this case, according to the microscope observation apparatus 1 according to the present embodiment, the objective lens 5 is swung around the principal point H, so that the images captured by the image capturing apparatuses 8 and 9 are misaligned by the swing. It does not occur, and it is not necessary to provide a special device for correcting image misalignment. Therefore, there exists an advantage that an apparatus can be comprised simply.
Thus, according to the microscope observation apparatus 1 according to the present embodiment, the microscope body 10 having a relatively large weight is fixed, and the relatively lightweight objective lens 5 is translated and swung, whereby the displacement of the sample S is changed. This makes it possible to compensate for blurring, defocusing, and out-of-view, and to obtain a clear image with little blurring. In addition, interruption of image acquisition due to out-of-view is eliminated, enabling continuous and continuous observation.

In the microscope observation apparatus 1 according to the present embodiment, the mechanism provided with the feed screws 16c and 17c or the worm gears 16f and 17f is exemplified as the mechanism for swinging the objective lens 5 of the objective lens driving device 11. Instead, as shown in FIG. 7, a multi-degree-of-freedom spherical ultrasonic motor 23 in which a plurality of ultrasonic motors 22 are arranged in contact with the periphery of a sphere 21 attached to the arm 12 may be used.
When the multi-degree-of-freedom spherical ultrasonic motor 23 is used, as shown in FIG. 8, the sphere 21 is formed hollow, the objective lens 5 is disposed therein, and the sample S is accommodated in the sphere 21 for observation. It is good also as performing.

  As a mechanism for swinging the objective lens 5, biaxial trapezoidal link mechanisms 24 and 25 may be employed as shown in FIG. By swinging the trapezoidal link mechanisms 24 and 25 using an actuator (not shown) provided in each of the trapezoidal link mechanisms 24 and 25, the objective lens 5 fixed to the tips of the trapezoidal link mechanisms 24 and 25 has two axes orthogonal to each other ( (X axis, Y axis).

  Further, as shown in FIG. 10, the objective lens driving device 11 includes a bobbin member 26 in which two hook-like portions 26a and 26b are connected by a cylindrical portion 26c in an external case 27 by an elastic member 28 such as a coil spring. As shown in FIG. 11, two ring-shaped coils 29a and 29b are arranged in the flanges 26a and 26b, and magnets 30a and 30b sandwiching the coils 29a and 29b are provided. It is good also as employ | adopting the electromagnetic linear motor 31 arrange | positioned in the position which opposes the diameter direction of the bowl-shaped parts 26a and 26b. The translational movement along the optical axis direction of the objective lens 5 is performed by the Z-axis stage 13 provided separately from the electromagnetic linear motor 31.

  FIG. 12 shows the energized state of the coil 29a (inner side) and the coil 29b (outer side) when the objective lens 5 is translated in the X-axis and Y-axis directions and when it swings in the A and B directions. For example, in order to translate the objective lens 5 in the X-axis direction, currents are supplied to the outer coils 29b of the upper and lower bowl-shaped portions 26a and 26b in the same direction, so that the Lorentz force between the magnet 30b and the coil 29b is increased. Utilizing this, it is possible to translate the objective lens 5 by simultaneously applying a force to the upper and lower flange portions 26a, 26b of the bobbin member 26 in the same direction in the X-axis direction. By switching the direction of the current flowing through the coil 29b, the translational movement direction of the objective lens 5 along the X-axis direction can be switched. In the case of translational movement in the Y-axis direction, current is supplied to the inner coils 29a of the upper and lower bowl-shaped portions 26a and 26b in the same manner.

  Further, in order to swing the objective lens 5 in the A direction around the X axis, the upper and lower hooks of the bobbin member 26 are made to flow by flowing currents in the opposite directions to the inner coils 29a of the upper and lower hooks 26a and 26b. The objective lens 5 can be swung in the A direction by generating a rotational force by simultaneously applying a force to the portions 26a and 26b in the opposite direction to the Y-axis direction. Similarly, in order to swing in the B direction around the Y axis, a reverse current is passed through the outer coils 29b of the upper and lower bowl-shaped portions 26a, 26b. Thereby, the single electromagnetic linear motor 31 can perform the X-axis and Y-axis translational movements of the objective lens 5 and the swinging movements in the A and B directions, and there is an advantage that the apparatus can be configured compactly.

  In the present embodiment, the objective lens 5 is separated from the microscope body 10 and controlled independently. However, as shown in FIG. 13, the objective lens 5 may be attached to the microscope body 10 by a parallel link type stage 32. .

The parallel link type stage 32 is a combination of six linear actuators 33, which translates the objective lens 5 in the X-axis, Y-axis, and Z-axis directions, and in the A and B directions around the X-axis and Y-axis. Can be swung. Examples of the linear actuator 33 include those using a piezoelectric element and those using a voice coil motor. Further, a linear actuator 33 of a method (for example, Japanese Patent Application Laid-Open No. 11-90867) that repeats rapid expansion and contraction of a piezoelectric element can be employed.
Further, the objective lens 5 may be swung along the spherical guide rail 34 as shown in FIG. 14 by using a method of repeating rapid expansion and contraction of the piezoelectric element. In the figure, reference numeral 35 denotes a piezoelectric element, reference numeral 36 denotes a weight as an inertial element, and reference numeral 37 denotes a slider that frictionally contacts the guide rail 34.

  In addition, as a method for detecting the amount of displacement in the optical axis direction, the tilt direction, and the swing angle according to the present embodiment, the method based on image processing has been described, but instead, a surface conjugate with the pupil of the imaging optical system is used. A so-called pupil division method may be used in which a plurality of photodetectors are arranged and in-focus information is obtained from output signals thereof. Further, a position detector by optical triangulation method may be arranged to acquire the surface position information of the sample.

Next, a microscope observation apparatus 40 according to the second embodiment of the present invention will be described below with reference to FIGS. 15 and 16.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation apparatus 1 according to the first embodiment described above, and the description thereof is omitted.

Microscope examination apparatus 40 according to this embodiment, as shown in FIG. 15 (a), in terms of swinging the objective lens 5 about its object point O _1, a microscope observation system according to a first embodiment 1 and different.
Also in this case, as shown in FIG. 15B, the focal plane of the sample S and the imaging planes of the imaging devices 8 and 9 are in a conjugate positional relationship. It is possible to obtain a clear image that is totally focused without blurring.

However, in the case of the present embodiment, when the objective lens 5 is swung, the center position O ₀ on the focal plane deviates from the center position I _{0 on the} imaging plane. A deviation correction mechanism 41 is provided. As shown in FIG. 16, the image shift correction mechanism 41 includes an imaging lens 42 and a pupil relay lens 43 disposed between the objective lens 5 and the imaging devices 8 and 9, and a rear of the pupil relay lens 43. It is conceivable to arrange a swingable correction mirror 44 disposed in the vicinity of the side focal position. Correction mirror 44 utilizes what can swing around an axis perpendicular to the optical axis, the inclination direction of the objective lens 5 by swinging the following angle A _CM in the opposite direction, to correct the image shift be able to.
A _CM = f _TL / 2f _PL × A _SP (1)
Here, f _TL is the focal length of the imaging lens 42, f _PL is the focal length of the pupil relay lens 43, and _ASP is the swing angle of the objective lens 5.

  The drive of the correction mirror 44 may be performed using an actuator (not shown) by the operation of the control device 18, or may have two rotation shafts and can freely swing three-dimensionally. Further, as shown in FIGS. 17 and 18, a mechanical transmission mechanism 45 may be used to interlock with the swing of the objective lens 5.

  17 and FIG. 18 will be described in more detail. One end of each of the links 47 and 48 having cylindrical end portions 47a and 48a is inserted into the elongated holes 46a and 46b provided in the arm 12 holding the objective lens 5. Deploy. Rotating shafts 51a and 51b of bevel gears 50a and 50b are connected to the other ends of the links 47 and 48 via universal joints 49, respectively. In the figure, reference numerals 52a and 52b are long holes that limit the operating directions of the links 47 and 48.

  With this configuration, when the arm 12 is translated in the horizontal direction, the displacement of the cylindrical end portions 47a and 48a in the long holes 46a and 46b is allowed, and the arm 12 is translated in the vertical direction. When moving, the universal joint 49 is bent while allowing displacement of the columnar ends 47a and 48a in the long holes 46a and 46b. As a result, no rotational force is transmitted to the rotating shafts of the bevel gears 50a and 50b by the translational movement in the X, Y, and Z directions.

Correction mirrors 44a and 44b are connected to the other bevel gears 53a and 53b that mesh with the bevel gears 50a and 50b via shafts 54a and 54b and gear trains 55a and 55b, respectively.
Thereby, when the arm 12 rotates around the X axis, the link 47 extending in the X axis direction is rotated around the axis, so that the rotational force is transmitted to the rotating shaft 51a of the bevel gear 50a, and the bevel gear 53a, shaft 54a and The first correction mirror 44a is swung through the gear train 55a. At this time, no rotational force is generated on the other link 48, and the second correction mirror 44b is kept stationary.

  Conversely, when the arm 12 rotates about the Y axis, the link 48 extending in the Y axis direction is rotated about the axis, so that the rotational force is transmitted to the rotating shaft 51b of the bevel gear 50b, and the bevel gear 53b, shaft 54b, and The second correction mirror 44b is swung through the gear train 55b. At this time, no rotational force is generated in the other link 47, and the first correction mirror 44a is kept stationary.

The relationship between the tilt angle of the arm 12 and the swing angle of the correction mirrors 44a and 44b is obtained by adjusting the number of teeth of the bevel gears 50a, 50b, 53a and 53b and the gear trains 55a and 55b. It is set to be a relationship.
By swinging the two correction mirrors 44a and 44b, the swing of the objective lens 5 around the X axis and the Y axis can be corrected.

Thus, according to the microscope apparatus 40 according to this embodiment, swinging the objective lens 5 to the object point O ₁ around, although it is necessary to image shift correction mechanism 41 and 45, the objective lens 5 Since the sample S can be observed with the object point O ₁ , that is, the center position of the visual field range on the object side of the objective lens 5 always matched with the center position O ₀ of the observation range, the objective lens 5 can be observed. There is an advantage that a clear image can be obtained without deteriorating aberration even when the objective lens 5 is swung, without impairing the optical performance.

Note that the microscope observation apparatus 40 according to the present embodiment has been described with respect to the case where the imaging apparatus 8 or 9 such as a CCD is provided, but instead of this, such as a galvanometer mirror that scans the excitation light E two-dimensionally. It is good also as applying to what has a light scanning part and has a photodetector like a photomultiplier tube as a light detection part. In this case, in order to correct an image shift by swinging the objective lens 5 around the object point O ₁ is the center position of the swing range of the galvanometer mirror may be set to be offset.

As the image acquisition timing, the objective lens 5 and the correction mirrors 44, 44a and 44b are oscillated by a high-speed actuator, and the objective lens 5 and the correction mirrors 44, 44a and 44b are placed at desired positions. Performing in a stationary state is preferable in that blurring can be reduced.
Further, since the objective lens 5 is driven by detecting the fluctuation of the sample S, the light detection unit 9 for detecting the fluctuation of the sample S is faster than the light detection unit 8 for image acquisition. It is preferable to do.

  In the above-described embodiment, two types of light having different wavelengths are emitted from the excitation light source 3. For example, long wavelength light is used for detecting displacement of the sample S, and fluorescence observation is performed using the short wavelength excitation light E. It is good also as performing. In this case, the beam splitter 7 may be a dichroic mirror.

  When the fluctuation of the sample S is periodic, for example, the objective lens 5 is continuously and periodically moved in the X, Y, Z, A, and B directions by a signal from a frequency oscillator such as an oscillator. It is also possible to adjust the driving frequency and amplitude in each vibration direction while watching the monitor.

It is a typical whole block diagram which shows the microscope observation apparatus which concerns on the 1st Embodiment of this invention. It is a schematic diagram which shows an example of the drive mechanism which rocks an objective lens among the objective lens drive mechanisms of the microscope observation apparatus of FIG. It is a schematic diagram which shows the other example of the drive mechanism of FIG. It is a perspective view which shows the relationship between the objective lens and coordinate axis in description of the microscope observation apparatus of FIG. It is a schematic diagram which shows the relationship between the operation direction of the sample in the microscope observation apparatus of FIG. 1, and the operation | movement form of an objective lens. FIG. 2 is a schematic diagram illustrating an optical path of an objective lens that swings around a principal point in the microscope observation apparatus of FIG. 1. It is a perspective view which shows the 1st modification of the objective lens drive mechanism in the microscope observation apparatus of FIG. It is a longitudinal cross-sectional view which shows the 2nd modification of the objective lens drive mechanism in the microscope observation apparatus of FIG. It is a perspective view which shows the 3rd modification of the objective lens drive mechanism in the microscope observation apparatus of FIG. It is a longitudinal cross-sectional view which shows the 4th modification of the objective lens drive mechanism in the microscope observation apparatus of FIG. It is a perspective view explaining the electromagnetic linear motor which comprises the objective-lens drive mechanism of FIG. It is a table | surface which shows the relationship between the operation direction of the electromagnetic linear motor of FIG. 11, and the energization state of each coil. It is a perspective view which shows the 5th modification of the objective lens drive mechanism in the microscope observation apparatus of FIG. It is a longitudinal cross-sectional view which shows the 6th modification of the objective lens drive mechanism in the microscope observation apparatus of FIG. It is a schematic diagram which shows the optical path of the objective lens which rocks | fluctuates centering on an object point in the microscope observation apparatus which concerns on the 2nd Embodiment of this invention. FIG. 16 is a schematic diagram illustrating correction of image misalignment in the microscope observation apparatus in FIG. 15. FIG. 16 is a perspective view illustrating an image shift correction mechanism that mechanically corrects image shift in the microscope observation apparatus of FIG. 15. FIG. 18 is a perspective view illustrating in detail a part of the image misalignment correction mechanism in FIG. 17.

Explanation of symbols

S sample (biological sample)
E Excitation light (light)
F fluorescence (return light)
H principal point O ₁ object point 1,40 Microscope observation device 3 Excitation light source (light source)
4 Imaging lens 5 Objective lens 8 Imaging device (light detection unit, first imaging device)
9 Imaging device (light detection unit, second imaging device)
DESCRIPTION OF SYMBOLS 10 Microscope main body 11 Objective lens drive device 18 Control apparatus 41, 45 Image shift correction mechanism (correction optical system)
44 Correction mirror (correction optical system)

Claims (4)

A microscope observation apparatus for observing a biological sample exhibiting dynamic behavior,
A first imaging device for capturing an image of the biological sample;
A second imaging device that captures an image of the biological sample at a higher speed than the first imaging device;
A microscope observation apparatus comprising: a control device that processes an image picked up by the second image pickup device and calculates a command signal for correcting an image shift of the image picked up by the first image pickup device.   The microscope observation apparatus according to claim 1, wherein an image shift in an image captured by the first imaging apparatus is generated by a dynamic behavior of the biological sample.   2. The driving device according to claim 1, further comprising a driving unit configured to drive an objective lens for forming an image of the biological sample in a direction for correcting the image shift based on the command signal calculated by the control device. Microscope observation device.   2. A beam splitter for branching light incident from the biological sample onto an objective lens for forming an image of the biological sample toward the first imaging device and the second imaging device. The microscope observation apparatus according to 1.

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