JP5261071B2 - Observation apparatus and observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an observation apparatus and an observation method which can obtain detailed information of an object such as a cell and display it effectively. <P>SOLUTION: The observation apparatus 1 for observing the surface or interior of an observation object 9 includes: light sources 11, 12; lenses 21 to 25; an aperture 31; an optical multiplexer 41; an optical demultiplexer 42; a half mirror 43; a photographing section 51; an analysis section 52; a display section 53; a light receiving section 61; a displacement detection section 62; a piezo actuator 71; a drive section 72; a mirror 73; a stage 81; a drive section 82; and a control section 90. The analysis section 52 determines the intensity component and phase component of second reflected light generated on the surface or in the interior of the observation object 9 on the basis of an interference light image obtained by setting an optical path length difference to be each target value successively by the phase shift method and photographing by the photographing section 51. The display section 53 displays the image of the second reflected light in the HSV color space with the phase component reflected in hue and with the intensity component reflected in saturation or brightness. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method and an apparatus for observing an observation object.

As an apparatus for observing or measuring an object using an interference optical system, an apparatus disclosed in Patent Document 1 is known. The apparatus disclosed in this document uses the fact that the amplitude peak of the interference fringe due to the reflected light from the object and the reflected light from the mirror depends on the optical path length difference between the two reflected lights. Observe or measure the object based on
Japanese Patent Laid-Open No. 9-2108016

  However, the apparatus disclosed in Patent Document 1 cannot obtain detailed information on an object such as a cell, and cannot display information effectively. The present invention has been made to solve the above-described problems, and an object of the present invention is to provide an observation apparatus and an observation method that can obtain detailed information of an object such as a cell and display it effectively. To do.

  The observation apparatus according to the present invention includes: (1) a light source that outputs light; and (2) the light output from the light source is bifurcated and output as first branched light and second branched light. The first reflected light generated by being reflected by the mirror is input, and the second reflected light generated by reflecting the second branched light on the surface or inside of the observation object is input, and the first reflected light and the second reflected light are input. An interference optical system that interferes with the reflected light and outputs the interference light; (3) an imaging optical system that forms the interference light output from the interference optical system; and (4) an image formed by the imaging optical system. An imaging unit that captures the interference light image, and (5) the optical path length from the light source to the imaging unit through the reference position of the observation object, and the optical path length from the light source to the imaging unit through the mirror Optical path length difference adjusting means for adjusting the optical path length difference, and (6) optical path length difference adjusting means so that the optical path length difference sequentially reaches each target value. And a control unit for controlling the optical path length difference adjusting operation according to.

  The observation apparatus according to the present invention further includes (7) an observation based on an interference light image picked up by the image pickup unit in which the optical path length difference is sequentially set to each target value by the optical path length difference adjusting means by the phase shift method. (8) The phase component of the second reflected light obtained by the analysis unit is reflected in the hue, and the analysis unit obtains the intensity component and phase component of the second reflected light generated on or inside the object. A display unit that displays an image of the second reflected light in the HSV color space by reflecting the obtained intensity component of the second reflected light in saturation or brightness.

  An observation method according to the present invention uses a light source, an interference optical system, an imaging optical system, an imaging unit, an optical path length difference adjusting unit, and a control unit as described above, and an optical path length difference by an optical path length difference adjusting unit by a phase shift method. Is sequentially set to each target value, and based on the interference light image captured by the imaging unit, the intensity component and the phase component of the second reflected light generated on the surface or inside of the observation object are obtained, and the obtained first The second reflected light is displayed as an image in the HSV color space by reflecting the phase component of the two reflected light in the hue and the intensity component of the second reflected light in the saturation or brightness.

  In the observation apparatus according to the present invention, the analysis unit obtains the intensity component and phase component of the plurality of sets of second reflected light in time series, and the display unit obtains the intensity of the plurality of sets of second reflected light obtained by the analysis unit. It is preferable to display these second reflected lights in time series in the HSV color space based on the component and the phase component. In the observation method according to the present invention, the intensity component and phase component of a plurality of sets of second reflected light are obtained in time series, and the HSV color space is obtained based on the obtained intensity components and phase components of the plurality of sets of second reflected light. Therefore, it is preferable to display the images of these second reflected lights in time series.

  In the observation apparatus according to the present invention, the analysis unit obtains the intensity component and the phase component of the second reflected light for each of the plurality of slice planes of the observation object, and the display unit obtains a plurality of sets of second obtained by the analysis unit. It is preferable to display an image of the second reflected light in the HSV color space based on the intensity component and the phase component of the reflected light. The observation method according to the present invention obtains the intensity component and phase component of the second reflected light for each of the plurality of slice planes of the observation object, and based on the obtained intensity component and phase component of the plurality of sets of second reflected light. It is preferable to display an image of these second reflected lights in the HSV color space.

  The observation apparatus according to the present invention preferably further includes an optical path length difference detecting unit that detects the optical path length difference, and the optical path length difference adjusting unit adjusts the optical path length difference based on a detection result by the optical path length difference detecting unit. is there. In the observation method according to the present invention, it is preferable that an optical path length difference detecting unit that detects an optical path length difference is further used, and the optical path length difference adjusting unit is adjusted based on a detection result by the optical path length difference detecting unit. is there.

  In the observation apparatus according to the present invention or the observation method according to the present invention, (a) the optical path length difference adjusting means includes a first moving means for moving one of the observation object and the mirror, and the observation object. And a second moving means for moving the other second object of the mirrors, adjusting the optical path length difference by the moving operation by the first moving means or the second moving means, and (b) the first moving means, (C) the second moving means has a position accuracy higher than that of the second moving means and (c) the second moving means is between the interference optical system and the second object. The second object is moved while maintaining the optical system, and (d) the control unit sets the first moving means so that the movement amount by the first moving means is within a predetermined range within the operating range at each target value of the optical path length difference. The second movement is performed by continuously or intermittently performing the movement operation by the two movement means. It is preferable that the optical path length difference during their movement to the feedback control of the movement operation by the first moving means so that the target value by stages.

  In the observation apparatus according to the present invention or the observation method according to the present invention, it is preferable that the observation object includes cells.

  According to the present invention, detailed information on an object such as a cell can be obtained and displayed effectively.

  The best mode for carrying out the present invention will be described below in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

  FIG. 1 is a configuration diagram of an observation apparatus 1 according to the present embodiment. The observation apparatus 1 shown in this figure is for observing the surface or the inside of an observation object 9, and includes light sources 11 and 12, lenses 21 to 25, an aperture 31, an optical multiplexer 41, an optical demultiplexer 42, A half mirror 43, an imaging unit 51, an analysis unit 52, a display unit 53, a light receiving unit 61, a displacement detection unit 62, a piezo actuator 71, a drive unit 72, a mirror 73, a stage 81, a drive unit 82, and a control unit 90 are provided.

The light source 11 outputs light λ ₁ having a relatively short coherent length, and is, for example, a tungsten lamp that can output broadband light having a wavelength band of 600 nm to 900 nm. The coherence length of the light λ ₁ output from the light source 11 is preferably 5 μm or less. On the other hand, the light source 12 outputs light λ ₂ having a relatively long coherence length, and is a semiconductor laser light source that outputs laser light having a wavelength of 1.31 μm, for example. The optical multiplexer 41 reflects the light λ ₁ output from the light source 11 and reached through the lens 21 and the aperture 31, and transmits the light λ ₂ output from the light source 12 to combine these lights. Wave and output to the lens 22.

The half mirror 43 divides the light λ ₁ and λ ₂ that have been combined by the optical multiplexer 41 and arrived through the lens 22 into a first branched light and a second branched light, and the first branched light to the lens 23. The second branched light is output to the lens 24. The half mirror 43 inputs the first reflected light, which is generated by the first branched light being reflected by the mirror 73 through the lens 23, again through the lens 23, and the second branched light is input through the lens 24 to the observation object 9. Second reflected light generated by being reflected on the surface or inside of the lens is input again through the lens 24, and the first reflected light and the second reflected light are caused to interfere with each other, and the interference light is output to the lens 25. That is, the half mirror 43 is an element constituting an interference optical system.

The optical demultiplexer 42 receives the light output from the half mirror 43 and passed through the lens 25, reflects the light λ ₁ among them, outputs the light λ ₁ to the imaging unit 51, transmits the light λ ₂ , and outputs it to the light receiving unit 61. To do. The lenses 23 to 25 are elements constituting an imaging optical system that forms an image on the imaging surface of the imaging unit 51 of the interference light λ ₁ output from the half mirror 43 and demultiplexed by the optical demultiplexer 42. The imaging unit 51 captures the image of the formed interference light λ ₁ and is, for example, a CCD camera. The light receiving unit 61 is adapted to detect the intensity of the light lambda ₂ which is output demultiplexed by the optical demultiplexer 42 from the half mirror 43, for example a photodiode.

  Here, the length of the optical path from the half mirror 43 reflected by the mirror 73 to the half mirror 43 again and the time from the half mirror 43 reflected by the reference position of the observation object 9 to the half mirror 43 again. The optical path length difference from the optical path length is ΔL. Note that the reference position of the observation object 9 may be the highest position of the observation object 9 (the position closest to the lens 24), or a stage on which the observation object 9 is placed, or an intermediate point between them. Or other positions.

As described above, since the coherent length of the light λ ₂ output from the light source 12 and reaching the light receiving unit 61 is relatively long, the intensity of the light λ ₂ reaching the light receiving unit 51 as shown in FIG. Changes periodically in a relatively wide optical path length difference ΔL. On the other hand, since the coherent length of the light λ ₁ that is output from the light source 11 and reaches the imaging unit 51 is relatively short, the intensity of the light λ ₁ that reaches the imaging unit 61 as shown in FIG. Changes periodically within a relatively narrow optical path length difference ΔL, and the closer the optical path length difference ΔL is to 0, the greater the amplitude of interference.

Using this, the analysis unit 52 acquires an interference light image of the light λ ₁ captured by the imaging unit 51 when the optical path length difference is set to each of a plurality of target values. This interference light image is generated by the mirror 73 when the optical path length from the half mirror 43 to the mirror 73 and the optical path length from the half mirror 43 to a certain slice surface of the observation object 9 are substantially equal to each other. It is an image due to interference between the reflected light and the second reflected light generated on the slice surface (and a range of about the coherence length of the light λ ₁ centered on the slice surface). Further, the analysis unit 52 performs a predetermined analysis based on the acquired interference light image. The display unit 53 displays the result of analysis by the analysis unit 52 as an image. The analysis by the analysis unit 52 and the image display by the display unit 53 will be described later.

The observation object 9 is preferably a translucent cell 93 placed on a thin film 92 formed on the main surface of a substantially flat substrate 91, for example, as shown in FIG. The thin film 92 is suitably used as a reference surface in feedback control described later by reflecting the light λ ₂ with a high reflectance. A slice plane S is set as a cross section of the cell 93 as the observation object 9. The slice plane S is a plane perpendicular to the optical axis of the lens 24, and its position is variable with respect to the optical axis direction.

The displacement detector 62 obtains an optical path length difference (or a change amount of the optical path length difference relative to a certain reference value) from the change in the intensity of the light λ ₂ detected by the light receiver 61. That is, the light source 12, the light receiving unit 61, and the displacement detecting unit 62 are elements constituting optical path length difference detecting means for detecting the optical path length difference. Note that the optical path length difference can be detected more accurately by applying minute vibrations to the mirror 73 by the piezo actuator 71 and modulating the optical signal with a certain optical path length difference as a center.

  The piezo actuator 71, the drive unit 72, the stage 81, and the drive unit 82 are elements that constitute optical path length difference adjusting means for adjusting the optical path length difference. The piezo actuator 71 is driven by the drive unit 72 to move the mirror 73 in a direction parallel to the optical axis of the optical system between the half mirror 43 and the mirror 73. At this time, the optical system between the half mirror 43 and the mirror 73 is maintained without moving the lens 23. The focus surface of the lens 23 coincides with the reflection surface of the mirror 73 with an accuracy of the depth of focus (for example, 0.5 μm).

  The stage 81 is driven by the drive unit 82 to move the observation object 9 in a direction parallel to the optical axis of the optical system between the half mirror 43 and the observation object 9. At this time, the optical system between the half mirror 43 and the observation object 9 is maintained without moving the lens 24. That is, the distance from the half mirror 43 to the focus surface on the observation object 9 side is maintained.

  The operating range of the piezo actuator 71 (first moving means) is narrower than the operating range of the stage 81 (second moving means). Further, the positional accuracy of the piezo actuator 71 is higher than the positional accuracy of the stage 81. As the drive unit 82 for moving the stage 81, for example, a long-distance moving piezo actuator or a rotation mechanism using a stepping motor can be used.

  Based on the detection result of the optical path length difference by the displacement detection unit 62, the control unit 90 uses the drive units 72 and 82 so that the optical path length difference sequentially becomes a plurality of target values, and the optical path by the piezo actuator 71 and the stage 81. Controls the length difference adjustment operation. In particular, the controller 90 causes the stage 81 to move continuously or intermittently so that the amount of movement by the piezo actuator 71 falls within a predetermined range within the operating range at each of the plurality of target values. The control unit 90 also feeds back the movement operation by the piezo actuator 71 so that the optical path length difference becomes each target value based on the detection result of the optical path length difference by the displacement detection unit 62 even during the movement operation by the stage 81. Control.

FIG. 4 is a diagram for explaining an optical path length difference adjusting operation by the piezo actuator 71 and the stage 81. In this figure, an optical system between the half mirror 43 and the mirror 73 is shown, an optical system between the half mirror 43 and the observation object 9 is shown, and a piezo actuator 71 for adjusting the optical path length difference. And stage 81 is shown. Here, the distance between the half mirror 43 and the lens 23 and x _1, the distance between the lens 23 and the mirror 73 and x _2. Further, the distance between the half mirror 43 and the lens 24 and y _1, the distance between the observation object 9 and the lens 24 and y _2. When the observation object 9 has the configuration shown in FIG. 3, y ₂ is an interval from the lens 24 to a certain slice surface of the observation object 9. The interval x ₂ is adjusted by a moving operation by the piezo actuator 71. The interval y ₂ is adjusted by the moving operation by the stage 81. The optical path length difference ΔL can be adjusted by changing the interval (x ₁ + x ₂ ) or the interval (y ₁ + y ₂ ) by the piezo actuator 71 or the stage 81.

Assuming that adjusting only distance y ₂ by the moving operation by the stage 81, since the operating range of the stage 81 is relatively wide, it is possible to adjust the optical path length difference in a wide dynamic range. However, in this case, since the position accuracy of the stage 81 is relatively low, the optical path length difference cannot be adjusted with high accuracy, and therefore the shape or the like of the observation object 9 cannot be measured with high accuracy.

On the other hand, if, in the case of adjusting only the interval x ₂ by the moving operation by the piezoelectric actuator 71, since it is relatively high positional accuracy of the piezoelectric actuator 71, it is possible to adjust the optical path length difference with high accuracy. However, in this case, since the operation range of the piezo actuator 71 is relatively narrow, the optical path length difference cannot be adjusted with a wide dynamic range. Therefore, the shape or the like of the observation object 9 can be measured with a wide dynamic range. I can't.

Moreover, if, when the perform the moving operation even over a wide range even within the operating range of the piezoelectric actuator 71, there is a case the difference between the focal distance and the distance x ₂ of the lens 23 becomes large, in which case The imaging surface of the interference light by the imaging optical system and the imaging surface of the imaging unit 51 are greatly displaced from each other, the interference light image captured by the imaging unit 51 becomes unclear, the shape of the observation object 9, etc. Cannot be measured with high accuracy.

  Therefore, in the present embodiment, in order to measure the surface or the inside of the observation object with high accuracy and a wide dynamic range, the control unit 90 has a movement amount by the piezo actuator 71 within the operation range at each of the plurality of target values. The moving operation by the stage 81 is performed continuously or intermittently so as to be within a predetermined range. The control unit 90 also feeds back the movement operation by the piezo actuator 71 so that the optical path length difference becomes each target value based on the detection result of the optical path length difference by the displacement detection unit 62 even during the movement operation by the stage 81. In the following, two preferred operation modes of the piezo actuator 71 and the stage 81 will be described.

FIG. 5 is a flowchart illustrating a first operation mode of the observation apparatus 1 according to the present embodiment. FIG. 6 is a diagram showing temporal changes of the interval x ₂ , the interval y _2, and the optical path length difference {(y ₁ + y ₂ ) − (x ₁ + x ₂ )} in the first operation mode. In the first operation mode, the control unit 90 causes the stage 81 to continuously perform the moving operation via the drive unit 82.

First, in step S <b> 11, the control unit 90 starts a moving operation by the stage 81 via the driving unit 82. The optical path length difference from one target value and to shift at regular time intervals Δt to the next target value, the amount of change in distance y ₂ at the time of the transition when the [Delta] y, the moving speed of the stage 81 is "[Delta] y / Δt ”. Thus, distance y ₂ between the observation object 9 and lens 24 will change substantially linearly with time. However, since it is a relatively low positional accuracy of the stage 81, a relatively large time variation interval y _2.

Therefore, in step S12, the control unit 90 feedback-controls the moving operation by the piezo actuator 71 via the drive unit 72 so that the optical path length difference becomes the target value. At this time, the interval x ₂ is adjusted by the piezo actuator 71, and the optical path length difference {(y ₁ + y ₂ ) − (x ₁ + x ₂ )} is set with high accuracy.

  In step S13, the control unit 90 determines whether or not a certain time Δt has elapsed since the optical path length difference was set to a certain target value. When the certain time Δt has elapsed, the process proceeds to the next step S14. In step S14, the control unit 90 determines whether or not there is a next target value. If there is a next target value, the process proceeds to the next step S15, and if there is no next target value, the process in step S18. Proceed to

In step S15, the control unit 90, before the optical path length difference shifts to the next target value, it is determined whether the amount of movement x ₂ by the piezoelectric actuator 71 at the target value after the shifting is out of a predetermined range . Then, the control unit 90, if the amount of movement x ₂ is determined to be outside the predetermined range the process proceeds to the step S17 through step S16, also, determines the amount of movement x ₂ is to be within a predetermined range In this case, the process immediately proceeds to step S17. In step S16, the control unit 90, the amount of movement x ₂ by the piezoelectric actuator 71 to adjust the speed of the moving operation by the stage 81 so as to fall within a predetermined range after the transition to the next target value.

  In step S <b> 17, the control unit 90 sets the optical path length difference to the next target value, and moves the piezo actuator 71 stepwise by Δx via the drive unit 72. Thereafter, returning to the process of step S12, the control unit 90 feedback-controls the movement operation by the piezo actuator 71 via the drive unit 72 so that the optical path length difference becomes a new target value. In step S <b> 18, the control unit 90 ends the moving operation by the stage 81 via the driving unit 82.

  As described above, in the first operation mode, the control unit 90 continuously performs the movement operation by the stage 81, and when the optical path length difference is shifted from a certain target value to the next target value, by the piezo actuator 71. The moving operation is performed stepwise, and during the period in which the optical path length difference is set to a certain target value, the moving operation by the piezo actuator 71 is feedback-controlled so that the optical path length difference becomes the target value. As described above, the control unit 90 controls the moving operation of the piezo actuator 71 and the stage 81, thereby making use of both the wide dynamic range of the moving operation of the stage 81 and the high positional accuracy of the moving operation of the piezo actuator 71. Thus, the surface shape and the like of the observation object 9 can be measured with high accuracy and a wide dynamic range.

  Further, in the first operation mode, before the optical path length difference is shifted from a certain target value to the next target value, the control unit 90 causes the movement amount by the piezo actuator 71 to be within a predetermined range at the target value after the shift. When it is off, the speed of the moving operation by the stage 81 is adjusted so that the moving amount falls within the predetermined range (steps S15 and S16). As described above, the control unit 90 adjusts the moving speed of the stage 81, so that even if the moving speed of the stage 81 and the accuracy of the target value change time interval Δt are insufficient, the moving amount by the piezo actuator 71 is increased. Can be maintained within a predetermined range, so that it is possible to maintain high-precision measurement of the surface shape and the like of the observation object 9. When the number of target values of the optical path length difference is N, the moving distance when the stage 81 is moved at a constant speed “Δy / Δt” within the time of N · Δt is sufficiently accurate (for example, an error). Is less than ± 1 μm), Steps S15 and S16 are not necessary, and it is only necessary to proceed to Step S17 immediately after Step S14.

FIG. 7 is a flowchart for explaining a second operation mode of the observation apparatus 1 according to the present embodiment. FIG. 8 is a diagram illustrating temporal changes of the interval x ₂ , the interval y _2, and the optical path length difference {(y ₁ + y ₂ ) − (x ₁ + x ₂ )} in the second operation mode. In the second operation mode, the control unit 90 causes the stage 81 to intermittently perform the moving operation via the drive unit 82.

In step S21, the control unit 90 feedback-controls the moving operation by the piezo actuator 71 via the drive unit 72 so that the optical path length difference becomes a target value. At this time, the stage 81 is not moved, since it is a relatively low positional accuracy of the stage 81, a relatively large time variation interval y _2. However, the distance x ₂ is adjusted by the piezo actuator 71, and the optical path length difference {(y ₁ + y ₂ ) − (x ₁ + x ₂ )} is set with high accuracy.

  In step S22, the control unit 90 determines whether or not a certain time Δt has elapsed since the optical path length difference was set to a certain target value. When the certain time Δt has elapsed, the process proceeds to the next step S23. In step S23, the control unit 90 determines whether or not there is a next target value. If there is a next target value, the process proceeds to the next step S24, and ends if there is no next target value.

In step S24, the control unit 90, before the optical path length difference shifts to the next target value, it is determined whether the amount of movement x ₂ by the piezoelectric actuator 71 at the target value after the shifting is out of a predetermined range . Then, the control unit 90, if the amount of movement x ₂ is determined to be outside the predetermined range the process proceeds to the step S26 through the step S25, also, determines the amount of movement x ₂ is to be within a predetermined range In this case, the process immediately proceeds to step S26.

At step S25, the control unit 90 stops after the amount of movement x ₂ by the piezoelectric actuator 71 after moving to the next target value moves the stage 81 to be within a predetermined range, also the stage 81 is moved During the period, the movement operation by the piezo actuator 71 is controlled so that the optical path length difference at that time becomes each target value. Note that when the movement of the stage 81 at this time, when the amount of movement x ₂ by the piezoelectric actuator 71 exceeds the upper limit of the predetermined range in the next target value, the amount of movement x ₂ by the piezoelectric actuator 71 is the lower limit of the predetermined range Try to be close. Conversely, if the amount of movement x ₂ by the piezoelectric actuator 71 exceeds the lower limit of the predetermined range in the next target value, the amount of movement x ₂ by the piezoelectric actuator 71 is set to be near the upper limit of the predetermined range.

  In step S <b> 26, the control unit 90 sets the optical path length difference to the next target value, and moves the piezo actuator 71 stepwise by Δx via the drive unit 72. Thereafter, returning to the process of step S21, the control unit 90 feedback-controls the moving operation by the piezo actuator 71 via the drive unit 72 so that the optical path length difference becomes a new target value.

  As described above, in the second operation mode, the control unit 90 feedback-controls the moving operation by the piezo actuator 71 so that the optical path length difference becomes each target value. As described above, the control unit 90 controls the moving operation of the piezo actuator 71 and the stage 81, thereby making use of both the wide dynamic range of the moving operation of the stage 81 and the high positional accuracy of the moving operation of the piezo actuator 71. Thus, the surface shape and the like of the observation object 9 can be measured with high accuracy and a wide dynamic range. If the position accuracy of the stage 81 is poor, the second operation mode is more effective than the first operation mode.

  Next, observation of the observation object 9 (particularly analysis by the analysis unit 52 and image display by the display unit 53) in the observation apparatus 1 according to the present embodiment and the observation method according to the present embodiment will be described in more detail.

The second reflected light E (x, y) reflected on the surface or inside of the observation object 9 and reaching the imaging surface of the imaging unit 51 is an intensity component R (x, y) and a phase component φ (x, y). Is represented by the following formula (1). The interference image picked up by the image pickup unit 51 is an image of the observation object 9 in which the second reflected light is generated such that the optical path lengths of the first reflected light and the second reflected light coincide with each other in the degree of the coherence length of the light λ ₁ . It reflects information on the slice plane (and a range of about the coherence length of the light λ ₁ centered on the slice plane). Note that x and y represent coordinate values of two orthogonal axes on the slice plane.

The intensity component R (x, y) of the second reflected light E (x, y) represents the reflectance of the light λ _{1 on} the slice surface, that is, information on the presence or absence of a reflector. The phase component φ (x, y) of the second reflected light E (x, y) represents information on the position of the reflector in the z direction (direction parallel to the optical axis) on the slice surface. For example, when a reflector (for example, a cell membrane, a nucleolus membrane, a lysosomal membrane, etc.) existing on the slice surface of the observation object 9 moves by Δz in the z direction, it reaches the imaging surface of the imaging unit 51. The phase component φ (x, y) of the second reflected light E (x, y) changes by 4πn ₁ Δz / λ ₁ . Here, n ₁ is the refractive index of the observation object 9. That is, the change amount of the phase component φ (x, y) represents the change amount of the position of the reflector in the z direction on the slice plane.

Such an intensity component R (x, y) and phase component φ (x, y) of the second reflected light E (x, y) are obtained by the phase shift method. The phase shift method, the piezoelectric actuator 71 is driven shifted optical path length difference ΔL is by lambda _1/4 under the control of the control unit 90 and drive unit 72 sequentially by the imaging unit 51 four interference light image I _{1 (x} , y), I ₂ (x, y), I ₃ (x, y), and I ₄ (x, y). Here, λ ₁ is the center wavelength of the low-coherent light output from the light source 11. From these four interference light images, the sine component A (x, y) and cosine component B (x, y) of the interference light image are obtained according to the following equation (2).

  Then, from the sine component A (x, y) and the cosine component B (x, y) of these interference light images, the intensity component R (x, y) of the second reflected light E (x, y) according to the following equation (3): y) and a phase component φ (x, y) are obtained. The sine component A (x, y) and the cosine component B (x, y) are the intensity component R (x, y) and phase component φ (x, y) of the second reflected light E (x, y). And expressed as the following equation (4). Further, the complex amplitude C (x, y) of the interference light image is expressed by the following equation (5) or (6). The analysis unit 52 obtains the complex amplitude C (x, y) of such an interference light image.

FIG. 9 is a diagram illustrating a sine component A (x, y) of the interference light image. FIG. 10 is a diagram illustrating the cosine component B (x, y) of the interference light image. FIG. 11 is a diagram illustrating the intensity component R (x, y) of the interference light image. FIG. 12 is a diagram showing the phase component φ (x, y) of the interference light image. These are all four interference light images I ₁ (x, y), I ₂ (x, y), I ₃ (x, y) obtained by the phase shift method for the HeLa cell as the observation object 9. , I ₄ (x, y). From the intensity component R (x, y) of the interference light image shown in FIG. 11, it can be seen that HeLa cells have various reflectors in the vicinity of the cell membrane and in the cells. The relationship between these reflectors and the organs of HeLa cells is considered as shown in FIG.

  There are various improved algorithms for the phase shift method, but any algorithm may be used. For example, according to an algorithm known as the Schwider-Hariharan Algorithm, based on five interference light images, the sine component A (x, y) and cosine component B (x, y) of the interference light image according to the following equation (7) Further, the intensity component R (x, y) and the phase component φ (x, y) of the second reflected light E (x, y) may be obtained from these.

In this embodiment, an arbitrary phase shift algorithm can be used. However, compared with the basic algorithm of the phase shift method according to the above equation (2), the improved algorithm of the phase shift method according to the above equation (7) has advantages such as being robust against the error of the phase shift amount. It is suitably used when the coherence length of the light λ ₁ is relatively short as in the embodiment.

FIG. 14 is a diagram illustrating a temporal change in the amount of phase shift when a plurality of sets of sine components A (x, y) and cosine components B (x, y) are acquired in time series. As shown in this figure, the piezoelectric actuator 71 is driven optical path length difference ΔL is shifted by lambda _1/4 every predetermined time by the control by the control unit 90 and drive unit 72, the phase shift amount is zero and 3λ _Increase / decrease is repeated between 1/4. Thereby, interference optical images I ₁ , I ₂ , I ₃ , I ₄ , I ₃ , I ₂ , I ₁ , I ₂ , I ₃ , I ₄ ,. Then, the first A ₁ , B ₁ , R ₁ and φ ₁ are obtained by using the first interference optical image I ₁ to the fourth interference optical image I ₄ . Also, the second A ₂ , B ₂ , R ₂ and φ ₂ are obtained by using the fourth interference optical image I ₄ to the seventh interference optical image I ₁ . Further, the seventh interference optical image I ₁ to the tenth interference optical image I ₄ are used to obtain the third A ₃ , B ₃ , R ₃ and φ ₃ .

In this way, the sine component A _n and cosine component B _n and intensity components R _n and the phase component phi _n of the second reflected light interference optical image is obtained sequentially at predetermined time intervals. Hereinafter, these are the sine component A (x, y, n), the cosine component B (x, y, n), the intensity component R (x, y, n), and the phase component φ (x, y, n). It may be written. Here, n in the notation of each component indicates that the component is obtained n-th and corresponds to a time variable.

  As described above, in the phase component φ (x, y, n), the optical path length from the half mirror 43 to the mirror 73 and the optical path length from the half mirror 43 to a certain slice surface of the observation object 9 are substantially equal to each other. Sometimes, it represents information on the position of the reflector in the z direction (direction parallel to the optical axis) on the slice surface. The change amount of the phase component φ (x, y, n) represents the change amount of the position of the reflector in the z direction on the slice surface. Therefore, as shown in FIG. 15, when the analysis unit 52 obtains a plurality of phase components φ (x, y, n) at regular time intervals, these are displayed in time series by the display unit 53 (moving images). Display), it is possible to observe the change in the position of the reflector in the z direction on the slice surface of the observation object 9.

Only the phase component φ (x, y, n) of the second reflected light is image-displayed by the display unit 53 in gray scale or pseudo color, so that the position of the reflector in the slice plane of the observation object 9 in the z direction. The state of change can be observed. However, in this case, the phase component φ (x, y, n) is displayed in gray scale or pseudo color regardless of the magnitude of the intensity component R (x, y, n). A position where (x, y, n) is small (that is, a position where the reflectance of the light λ ₁ is small) becomes noise. Therefore, for a person who views the image displayed on the display unit 53, the position where the amount of change in the phase component φ (x, y, n) is large (that is, the position of the reflector in the slice direction of the observation object 9 in the z direction). It is difficult to confirm the position where the amount of change is large.

  Therefore, the intensity component R (x, y, n) and the phase component φ (x, y, n) of the second reflected light obtained by the analyzing unit 52 are displayed on the display unit 53 as follows. . That is, the phase component φ (x, y, n) of the second reflected light obtained by the analyzing unit 52 is reflected on the hue H by the display unit 53, and the intensity component of the second reflected light obtained by the analyzing unit 52. R (x, y, n) is reflected in the saturation S or the brightness V, and the second reflected light is displayed as an image in the HSV color space.

  The hue H in the HSV color space is a parameter indicating the color type in the range of 0 to 360. Therefore, for example, if the phase component φ (x, y, n) can take a value in the range of −π to + π, the hue H is an expression of “360 × {φ + π (x, y, n)} / 2π”. It is represented by

  The saturation S in the HSV color space is a parameter indicating the vividness of the color in the range of 0-1. The brightness V in the HSV color space is a parameter indicating the brightness of the color in the range of 0 to 1. Therefore, for example, the intensity component R (x, y, n) normalized so that the maximum value is 1 is used, and the saturation S or brightness V is the normalized intensity component R (x, y, n). y, n) or represented by the expression “{1 + R (x, y, n)} / 2”.

  The correspondence between the two is set so that the saturation S or the lightness V becomes larger as the intensity component R (x, y, n) is larger. When the intensity component R (x, y, n) is represented by only one parameter of saturation S and brightness V, the other parameter is set to a fixed value (for example, value 1).

As described above, in the display unit 53, the phase component φ (x, y, n) is represented by the hue H, the intensity component R (x, y, n) is represented by the saturation S or the lightness V, and the HSV color. When the second reflected light is displayed as an image in space, the phase component φ (x, y, n) and the intensity component R (x, y, n) are represented in a common image. Thus, the intensity component R (x, y, n) is large position (i.e., the reflectance is large the position of the light lambda ₁₎ In so saturation S or lightness V is large, the display of the hue H becomes strong, it Therefore, noise is not a concern for humans who view the image displayed on the display unit 53. On the other hand, at a position where the intensity component R (x, y, n) is small (that is, a position where the reflectance of the light λ ₁ is small), the saturation S or the lightness V is small, and therefore the hue H display is weakened. The position where the amount of change in the phase component φ (x, y, n) is large for a person viewing the image displayed on the display unit 53 (that is, the position change amount in the z direction of the reflector on the slice surface of the observation object 9). It is easy to check the position where the

  A specific example of image display by the display unit 53 is as follows. When the intensity component R (x, y, n) is reflected only in the lightness V, the HSV color space is represented by the following (8) by the phase component φ (x, y, n) and the intensity component R (x, y, n). Expressed by an expression. When the intensity component R (x, y, n) is reflected only in the saturation S, the HSV color space is represented by the following (9) by the phase component φ (x, y, n) and the intensity component R (x, y, n). ) Expression. When the intensity component R (x, y, n) is reflected in both the saturation S and the brightness V, the HSV color space has a phase component φ (x, y, n) and an intensity component R (x, y, n). n) is expressed by the following equation (10).

  As a comparative example, when the intensity component R (x, y, n) is not reflected in either the saturation S or the brightness V, and both the saturation S and the brightness V are fixed values 1, the HSV color space Is expressed by the following equation (11) only with the phase component φ (x, y, n) out of the phase component φ (x, y, n) and the intensity component R (x, y, n).

  FIG. 16 is a diagram illustrating an example of image display by the display unit 53 when the intensity component R (x, y, n) is reflected only in the brightness V (in the case of the expression (8)). FIG. 17 is a diagram illustrating an example of image display by the display unit 53 when the intensity component R (x, y, n) is reflected only in the saturation S (in the case of the expression (9)). FIG. 18 is a diagram illustrating an example of image display by the display unit 53 when the intensity component R (x, y, n) is reflected in both the saturation S and the brightness V (in the case of the expression (10)). . FIG. 19 shows an example of image display by the display unit 53 in the case of the comparative example (in the case of equation (11)) in which the intensity component R (x, y, n) is not reflected in either the saturation S or the brightness V. FIG. In these figures, what is originally a pseudo color display is displayed in gray scale.

  FIG. 20 is a diagram showing the image (original pseudo color display image) shown in FIG. 18 in an R, G, B component. FIG. 21 is a diagram showing the image (original pseudo color display) shown in FIG. 19 in an R, G, B component. 20A and 21B, (a) displays the R component image in gray scale, (b) displays the G component image in gray scale, and (c) displays the B component image in gray scale. Display in scale.

In the image display of the comparative example (FIGS. 19 and 21), the position where the intensity component R (x, y, n) is small (that is, the position where the reflectance of the light λ ₁ is small) is also the phase component φ (x, y, n). ) Is displayed in pseudo color, so that the noise increases and a position where the amount of change in the phase component φ (x, y, n) is large for a human viewing the image displayed on the display unit 53 (that is, an observation object) 9), it is difficult to confirm the position of the reflector in which the position change amount in the z direction of the reflector is large.

On the other hand, in the image display according to the present embodiment (FIGS. 16 to 18 and 20), at a position where the intensity component R (x, y, n) is large (that is, a position where the reflectance of the light λ ₁ is large). Since the saturation S and the brightness V are large, the display of the hue H becomes strong, and noise is not anxious for a person who views the image displayed on the display unit 53. On the other hand, at the position where the intensity component R (x, y, n) is small (that is, the position where the reflectance of the light λ ₁ is small), the saturation S or the lightness V is small, and therefore the display of the hue H becomes weak and the display unit For a human viewing the image displayed in 53, a position where the change amount of the phase component φ (x, y, n) is large (that is, a position where the position change amount of the reflector in the z direction on the slice surface of the observation object 9 is large). ) Is easy to check.

  As described above, according to the observation device 1 according to the present embodiment or the observation method according to the present embodiment, detailed information of the observation object 9 such as a cell is obtained and effectively displayed, so that confirmation is easy. . This enables imaging of intracellular activity and is expected to be applied to cell research, drug discovery and regenerative medicine.

Further, the amount of change per phase of the phase component φ (x, y, n) of the second reflected light is obtained by the analysis unit 52 in the same manner as described above for each of the plurality of slice planes of the observation object 9. As a result, it is preferable that a change in the position of the reflector in the z direction is obtained for each position (x, y). In this case, not only the piezo actuator 71 but also the stage 81 is used, and the optical path length difference {(y ₁ + y ₂ ) − (x ₁ + x ₂ )} is adjusted stepwise as shown in FIG. 6 or FIG. In addition, in each step, a plurality of sets of intensity components R (x, y, n) and phase components φ (x, y, n) are set such that the phase shift amount changes with time as shown in FIG. ) Can be acquired. In this way, the tomographic observation of the observation object 9 becomes possible.

It is a lineblock diagram of observation device 1 concerning this embodiment. It is a figure which shows the relationship between the intensity | strength of the light which reaches | attains the imaging part 51 or the light-receiving part 51, and an optical path length difference. It is a figure which shows the structural example of the observation target object. FIG. 6 is a diagram for explaining an optical path length difference adjusting operation by a piezo actuator 71 and a stage 81. It is a flowchart explaining the 1st operation | movement aspect of the observation apparatus 1 which concerns on this embodiment. Is a diagram illustrating a _{- {(x 1 + x 2} ) (y 1 + y 2)} each time variation interval x _2, distance y ₂ and the optical path length difference in the first operation mode of the observation apparatus 1 according to this embodiment . It is a flowchart explaining the 2nd operation | movement aspect of the observation apparatus 1 which concerns on this embodiment. Is a diagram illustrating a _{- {(x 1 + x 2} ) (y 1 + y 2)} each time variation interval x _2, distance y ₂ and the optical path length difference in the second operation mode of the observation apparatus 1 according to this embodiment . It is a figure which shows the sine component A (x, y) of an interference light image. It is a figure which shows the cosine component B (x, y) of an interference light image. It is a figure which shows the intensity component R (x, y) of an interference light image. It is a figure which shows the phase component (phi) (x, y) of an interference light image. It is a figure which shows the relationship between the organ of a HeLa cell, and a reflector. It is a figure which shows the mode of the time change of the phase shift amount in the case of acquiring several sets of sine component A (x, y) and cosine component B (x, y) in a time series. It is a figure which shows the mode of acquisition of several phase component (phi) (x, y, n). It is a figure which shows the example of the image display by the display part 53 in case the intensity | strength component R (x, y, n) is reflected only in the lightness V (in the case of (8) Formula). It is a figure which shows the example of the image display by the display part 53 in case the intensity | strength component R (x, y, n) is reflected only in the saturation S (in the case of (9) Formula). It is a figure which shows the example of the image display by the display part 53 in case the intensity | strength component R (x, y, n) is reflected in both the saturation S and the lightness V (in the case of (10) Formula). It is a figure which shows the example of the image display by the display part 53 in the case of the comparative example (in the case of Formula (11)) in which intensity | strength component R (x, y, n) is not reflected in either saturation S or the brightness V. It is a figure which decomposes | disassembles and shows the image (original pseudo color display image) shown in FIG. 18 into each component of R, G, and B. It is a figure which decomposes | disassembles and shows the image (original pseudo color display image) shown in FIG. 19 into each component of R, G, and B.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Observation apparatus, 9 ... Observation object, 11, 12 ... Light source, 21-25 ... Lens, 31 ... Aperture, 41 ... Optical multiplexer, 42 ... Optical demultiplexer, 43 ... Half mirror, 51 ... Imaging part, 52 ... Analyzing unit, 53 ... Display unit, 61 ... Light receiving unit, 62 ... Displacement detecting unit, 71 ... Piezo actuator, 72 ... Drive unit, 73 ... Mirror, 81 ... Stage, 82 ... Drive unit, 90 ... Control unit.

Claims (11)

A light source that outputs light;
The light output from the light source is bifurcated and output as first branched light and second branched light. The first reflected light generated by reflecting the first branched light by a mirror is input, and the second branched light is input. An interference optical system that inputs second reflected light that is generated when light is reflected from or on the surface of the observation object, interferes with the first reflected light and the second reflected light, and outputs the interference light;
An imaging optical system for imaging the interference light output from the interference optical system;
An imaging unit that captures an interference light image formed by the imaging optical system;
An optical path length difference between an optical path length from the light source through the reference position of the observation object to the imaging unit and an optical path length from the light source through the mirror to the imaging unit is adjusted. Optical path length difference adjusting means;
A control unit for controlling the optical path length difference adjusting operation by the optical path length difference adjusting means so that the optical path length difference is sequentially set to each target value;
The optical path length difference is sequentially set to each target value by the optical path length difference adjusting means by the phase shift method, and is generated on the surface or inside of the observation object based on the interference light image captured by the imaging unit. An analysis unit for obtaining an intensity component and a phase component of the second reflected light;
In the HSV color space, the phase component of the second reflected light obtained by the analyzing unit is reflected in hue, and the intensity component of the second reflected light obtained by the analyzing unit is reflected in saturation or brightness. A display unit for displaying an image of the second reflected light;
An observation apparatus comprising: The analysis unit obtains a plurality of sets of intensity components and phase components of the second reflected light in time series,
The display unit displays the second reflected light in time series in the HSV color space based on the intensity component and phase component of the plurality of sets of the second reflected light obtained by the analysis unit.
The observation apparatus according to claim 1. The analysis unit determines an intensity component and a phase component of the second reflected light for each of a plurality of slice planes of the observation object;
The display unit displays an image of the second reflected light in the HSV color space based on the intensity component and phase component of the plurality of sets of the second reflected light obtained by the analyzing unit.
The observation apparatus according to claim 1. Further comprising optical path length difference detecting means for detecting the optical path length difference,
The optical path length difference adjusting means adjusts the optical path length difference based on a detection result by the optical path length difference detecting means;
The observation apparatus according to claim 1. The optical path length difference adjusting means moves a first moving means for moving one of the observation object and the mirror, and a second second object of the observation object and the mirror. A second moving means, and adjusting the optical path length difference by a moving operation by the first moving means or the second moving means,
The first moving means has an operating range narrower than the operating range of the second moving means and a positional accuracy higher than the positional accuracy of the second moving means;
The second moving means moves the second object while maintaining the optical system between the interference optical system and the second object;
The controller continuously or intermittently moves the second moving means so that the moving amount by the first moving means falls within a predetermined range within the operating range at each target value of the optical path length difference. Feedback control of the movement operation by the first movement means so that the optical path length difference becomes each target value even during the movement operation by the second movement means,
The observation apparatus according to claim 1. A light source that outputs light;
The light output from the light source is bifurcated and output as first branched light and second branched light. The first reflected light generated by reflecting the first branched light by a mirror is input, and the second branched light is input. An interference optical system that inputs second reflected light that is generated when light is reflected from or on the surface of the observation object, interferes with the first reflected light and the second reflected light, and outputs the interference light;
An imaging optical system for imaging the interference light output from the interference optical system;
An imaging unit that captures an interference light image formed by the imaging optical system;
An optical path length difference between an optical path length from the light source through the reference position of the observation object to the imaging unit and an optical path length from the light source through the mirror to the imaging unit is adjusted. Optical path length difference adjusting means;
A control unit for controlling the optical path length difference adjusting operation by the optical path length difference adjusting means so that the optical path length difference is sequentially set to each target value;
Use
The optical path length difference is sequentially set to each target value by the optical path length difference adjusting means by the phase shift method, and is generated on the surface or inside of the observation object based on the interference light image captured by the imaging unit. Obtaining an intensity component and a phase component of the second reflected light;
Reflecting the obtained phase component of the second reflected light in hue, reflecting the intensity component of the second reflected light in saturation or brightness, and displaying the second reflected light in an HSV color space.
An observation method characterized by that. Obtaining a plurality of sets of intensity components and phase components of the second reflected light in time series;
Based on the obtained intensity components and phase components of the plurality of sets of the second reflected light, the second reflected light is displayed in time series in the HSV color space.
The observation method according to claim 6. Obtaining an intensity component and a phase component of the second reflected light for each of a plurality of slice planes of the observation object;
Based on the obtained intensity component and phase component of the plurality of sets of the second reflected light, the second reflected light is image-displayed in the HSV color space.
The observation method according to claim 6. Further using the optical path length difference detecting means for detecting the optical path length difference,
Adjusting the optical path length difference by the optical path length difference adjusting means based on the detection result by the optical path length difference detecting means;
The observation method according to claim 6. The optical path length difference adjusting means moves a first moving means for moving one of the observation object and the mirror, and a second second object of the observation object and the mirror. A second moving means, and adjusting the optical path length difference by a moving operation by the first moving means or the second moving means,
The first moving means has an operating range narrower than the operating range of the second moving means and a positional accuracy higher than the positional accuracy of the second moving means;
The second moving means moves the second object while maintaining the optical system between the interference optical system and the second object;
The controller continuously or intermittently moves the second moving means so that the moving amount by the first moving means falls within a predetermined range within the operating range at each target value of the optical path length difference. Feedback control of the movement operation by the first movement means so that the optical path length difference becomes each target value even during the movement operation by the second movement means,
The observation method according to claim 6. The observation method according to claim 6, wherein the observation object includes a cell.