JP2019109181A - Phase image acquisition device and phase image acquisition method - Google Patents

Abstract

To provide a device and a method capable of acquiring a phase image repeatedly at high speed.SOLUTION: A phase image acquisition device 1A comprises an interference image acquisition unit 2 and a calculation unit 3. In a first phase image acquisition step, the interference image acquisition unit 2 acquires an interference image in each state in which difference in optical path length of a two-beam interferometer is stabilized in each of a plurality of set values different from one another, and the calculation unit 3 obtains a first phase image on the basis of a plurality of interference images acquired by the interference image acquisition unit 2. In a second phase image acquisition step, the interference image acquisition unit 2 acquires an interference image in each state in which difference in optical path length of the two-beam interferometer is stabilized in a fixed set value so that the amount of interference light received by each pixel of an imaging device in a region of interest corresponds to a phase value one to one, and the calculation unit 3 obtains a second phase image on the basis of the interference image acquired by the interference image acquisition unit 2 and the first phase image.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an apparatus and method for acquiring a phase image of an object.

  Patent Documents 1 and 2 disclose inventions of apparatuses and methods capable of acquiring a phase image of an object using a phase shift method. The phase image acquisition device described in these documents comprises: a light source; a two-beam interferometer which splits the light output from the light source into two light beams and then combines the two light beams to output interference light; And an image pickup device for receiving the interference light output from the two-beam interferometer. This phase image acquisition device stabilizes the optical path length difference between two light beams in the two light beam interferometer with each of a plurality of different set values, acquires an interference image of the object in each state with an imager, and A phase image is determined based on the plurality of acquired interference images.

International Publication No. 2016/121250 JP 2008-281484 A

D. Pogany, et al, "Single-Shot Thermal Energy Mapping with Semiconductor Devices with the Nanosecond Resolution Using Holographic Interferometry," IEEE ELECTRONIC DEVICE LETTERS, VOL. 23, NO. 10, OCTOBER 2002.

  Conventional phase image acquisition devices including those described in Patent Documents 1 and 2 obtain one phase image based on a plurality of interference images by the phase shift method, and each interference image is acquired. In this case, it is necessary to stabilize the optical path length difference of the two-beam interferometer with a predetermined set value. In order to stabilize the optical path length difference sequentially with each of a plurality of set values, the optical path length difference is set by feedback control using a piezo element after changing the optical path length difference from a certain set value to the next set value. It takes time to stabilize at the value. Therefore, in the conventional phase image acquisition apparatus, it takes time to acquire one phase image, and the frame rate of the phase image that can be repeatedly acquired is limited to about 50 fps.

  Non-Patent Document 1 describes a technique intended to obtain a phase image of an object at high speed. The technique described in this document acquires an interference image consisting of two-dimensional interference fringes with high spatial frequency by an off-axis interference optical system, and performs spatial Fourier transform on the interference image. Determine the phase image. However, the technique described in Non-Patent Document 1 has a low spatial resolution of the obtained phase image, and in order to enhance the spatial resolution of the phase image, it is necessary to use an imager with a large number of pixels. Therefore, it takes time to output interference image data from the imaging device, and it is difficult to repeatedly acquire a phase image at high speed.

  The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide an apparatus and method capable of acquiring a phase image repeatedly at high speed.

  The phase image acquisition apparatus according to the present invention includes: (1) a light source, a two-beam interferometer that splits the light output from the light source into two beams, combines the two beams, and outputs interference light; And an imaging device for receiving the interference light output from the measuring device, wherein the difference in optical path length between the two light beams is variable in the two-beam interferometer, and the interference image of the object is imaged in a state where the difference in optical path length is stabilized. And an operation unit for obtaining a phase image of the object based on the interference image acquired by the interference image acquisition unit. Then, in the phase image acquisition device of the present invention, (a) in the first phase image acquisition step, the interference image acquisition unit stabilizes the optical path length difference of the two-beam interferometer with each of a plurality of different setting values. An interference image is obtained in each state, and the operation unit obtains a first phase image based on the plurality of interference images acquired by the interference image acquisition unit. (B) In the second phase image acquisition step, the interference image acquisition unit Is a state in which the optical path length difference of the two-beam interferometer is stabilized at a constant set value so that the light quantity and the phase value of the interference light received by each pixel of the imager in the region of interest become one to one correspondence. An image is acquired, and the calculation unit obtains a second phase image based on the interference image and the first phase image acquired by the interference image acquisition unit.

  In the phase image acquisition device according to one aspect of the present invention, in the second phase image acquisition step, the interference image acquisition unit determines that the light amount of interference light received by each pixel of the imaging device in the region of interest is less than the saturation light amount of the pixel In the range, the output light intensity of the light source or the exposure time of the imager is increased as compared to that in the first phase image acquisition step.

  The phase image acquisition apparatus according to another aspect of the present invention further includes a power supply unit for supplying a temporally intensity-modulated injection current to the object, and in the second phase image acquisition step, the interference image acquisition unit An interference image is acquired in synchronization with the intensity modulation.

  The phase image acquiring apparatus according to still another aspect of the present invention further includes an infrared light source for irradiating a temporally intensity-modulated infrared light to the object, and in the second phase image acquiring step, the interference image acquiring unit The interference image is acquired in synchronization with the intensity modulation of infrared light.

  In the phase image acquisition apparatus according to still another aspect of the present invention, the interference image acquisition unit further includes a spatial light phase modulator provided on the optical path of any one of two light fluxes in the two light flux interferometer. In the second phase image acquisition step, the range in which the light amount and the phase value of the interference light received by each pixel of the image pickup device are in a one-to-one correspondence relationship is expanded by spatial phase modulation by a spatial light phase modulator. .

  In the phase image acquiring apparatus according to still another aspect of the present invention, the interference image acquiring unit outputs temporally wavelength-modulated laser light which is multiplexed with the output light of the light source and input to the two-beam interferometer. The optical system further includes a laser light source, and a light detector that receives interference light of laser light output from the two-beam interferometer and outputs a detection signal, and detects an optical path length difference of the two-beam interferometer based on the detection signal. And stabilize the optical path length difference based on the detection result.

  In a phase image acquisition device according to still another aspect of the present invention, a light source outputs incoherent light.

  The phase image acquisition method according to the present invention comprises: (1) a light source, a two-beam interferometer that splits the light output from the light source into two light beams, combines the two light beams, and outputs interference light; And an imaging device for receiving the interference light output from the measuring device, wherein the difference in optical path length between the two light beams is variable in the two-beam interferometer, and the interference image of the object is imaged in a state where the difference in optical path length is stabilized. And an arithmetic unit for obtaining a phase image of the object based on the interference image acquired by the interference image acquisition unit. Then, according to the phase image acquisition method of the present invention, (a) the interference image acquisition unit stabilizes the optical path length difference of the two-beam interferometer with each of a plurality of different set values and acquires interference images in each state. A first phase image acquisition step of obtaining a first phase image based on the plurality of interference images acquired by the interference image acquisition unit by the operation unit; (b) each of the imaging devices in the region of interest by the interference image acquisition unit An interference image is acquired as a state in which the optical path length difference of the two-beam interferometer is stabilized at a fixed set value so that the light amount of the interference light received by the pixel and the phase value become one to one correspondence. And a second phase image acquisition step of obtaining a second phase image based on the interference image acquired by the interference image acquisition unit and the first phase image.

  In the phase image acquisition method according to one aspect of the present invention, in the second phase image acquisition step, the interference image acquisition unit determines that the light amount of interference light received by each pixel of the imaging device in the region of interest is less than the saturation light amount of the pixel In the range, the output light intensity of the light source or the exposure time of the imager is increased as compared to that in the first phase image acquisition step.

  In the phase image acquisition method according to another aspect of the present invention, the object is an electric circuit, and in the second phase image acquisition step, an injection current temporally intensity-modulated by the power supply unit is applied to the electric circuit to acquire an interference image. An interference image is acquired by the unit in synchronization with the intensity modulation of the injection current.

  In the phase image acquisition method according to still another aspect of the present invention, the object is a cell, and in the second phase image acquisition step, the cell is irradiated with infrared light temporally intensity-modulated by an infrared light source, and interference is caused. The image acquisition unit acquires an interference image in synchronization with the intensity modulation of infrared light.

  In a phase image acquisition method according to still another aspect of the present invention, in the second phase image acquisition step, a spatial light phase modulator provided on the optical path of any one of two luminous fluxes of two luminous fluxes in a two luminous flux interferometer By spatial phase modulation, the range in which the light amount of the interference light received by each pixel of the imaging device and the phase value have a one-to-one correspondence relationship is expanded.

  In the phase image acquiring method according to still another aspect of the present invention, the interference image acquiring unit causes the laser light source to output temporally wavelength-modulated laser light, and combines the laser light with the output light of the light source. The interference light of the laser beam output from the two-beam interferometer is received by the light detector and output as a detection signal, and the optical path length difference of the two-beam interferometer is calculated based on the detection signal. It detects and stabilizes the optical path length difference based on the detection result.

  In still another aspect of the phased image acquisition method of the present invention, the light source outputs incoherent light.

  According to the present invention, a phase image can be acquired repeatedly at high speed.

FIG. 1 is a diagram showing a configuration of a phase image acquisition device 1A of the first embodiment. FIG. 2 is a diagram showing the configuration of a sample. FIG. 3 is a timing chart for explaining the operation of the phase image acquisition apparatus 1A of the first embodiment and a phase image acquisition method using the same. FIG. 4 is a timing chart for explaining the operation of the phase image acquisition device 1A of the first embodiment and a phase image acquisition method using the same. FIG. 5 is a diagram showing four interference images I1 to I4 acquired by the interference image acquisition unit 2 in the first phase image acquisition step. FIG. 6 is a diagram showing the first phase image Φ obtained by the calculation unit 3 in the first phase image acquisition step. FIG. 7 is a view showing an interference image acquired by increasing the integrated light quantity of light incident on one pixel of the imaging device 18. FIG. 8 is a diagram showing three interference images acquired by the interference image acquisition unit 2 in the second phase image acquisition step. FIG. 9 is a diagram showing the relationship between the phase value of the phase image of FIG. 6 and the luminance value of the interference image of FIG. FIG. 10 is a diagram showing the relationship between the phase value of the phase image of FIG. 6 and the luminance value of the interference image of FIG. FIG. 11 is a diagram showing three phase images obtained by the calculation unit 3 in the second phase image obtaining step. FIG. 12 is an enlarged view of a part of the phase image of FIG. 11 (t = 0.000 seconds). FIG. 13 is a view for explaining the undesirable condition in the second phase image acquisition step. FIG. 14 is a diagram for explaining the undesirable condition in the second phase image acquisition step. FIG. 15 is a diagram for describing interference image acquisition preferable conditions in the second phase image acquisition step. FIG. 16 is a diagram for describing a method of setting or designating a region of interest. FIG. 17 is a diagram for describing a method of setting or designating a region of interest. FIG. 18 is a diagram for describing a method of setting or designating a region of interest. FIG. 19 is a flowchart for explaining the operation of the phase image acquisition device 1A of the first embodiment and the phase image acquisition method of the first embodiment. FIG. 20 is a diagram showing the configuration of a phase image acquisition device 1B of the second embodiment. FIG. 21 is a timing chart for explaining the operation of the phase image acquisition device 1B of the second embodiment and the phase image acquisition method using the same. FIG. 22 is a diagram showing the configuration of a phase image acquisition device 1C of the third embodiment. FIG. 23 is a diagram showing an example of the configuration of a sample. FIG. 24 is a timing chart for explaining the operation of the phase image acquisition device 1C of the third embodiment and a phase image acquisition method using the same. FIG. 25 is a diagram showing the configuration of a phase image acquisition device 1D according to the fourth embodiment. FIG. 26 is a diagram showing the configuration of a phase image acquisition device 1E of the fifth embodiment. FIG. 27 is a diagram showing the configuration of the reference mirror 15E.

  Hereinafter, with reference to the accompanying drawings, modes for carrying out the present invention will be described in detail. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description. The present invention is not limited to these exemplifications, is shown by the claims, and is intended to include all modifications within the scope and meaning equivalent to the claims.

First Embodiment
FIG. 1 is a diagram showing a configuration of a phase image acquisition device 1A of the first embodiment. The phase image acquisition device 1A includes the interference image acquisition unit 2 and the calculation unit 3. The interference image acquisition unit 2 includes a light source 11, a beam splitter 12, an objective lens 13, an objective lens 14, a reference mirror 15, a tube lens 16, a beam splitter 17, an imaging device 18, a piezo element 21, a photodetector 22, and a phase control circuit. Including 23.

  The interference image acquisition unit 2 includes a Michelson interferometer as a two-beam interferometer, and acquires an interference image of an object. The calculation unit 3 obtains a phase image of the object based on the interference image acquired by the interference image acquisition unit 2.

  The subject is not limited to a specific cell or biological sample. For example, target cells include cultured cells, immortalized cells, primary cultured cells, cancer cells, adipocytes, liver cells, cardiomyocytes, nerve cells, glial cells, somatic stem cells, embryonic stem cells, pluripotent stem cells, iPS Cells, and cell clusters (colony or spheroid) produced from the cells, and the like can be mentioned. The object is not limited to a living body, and examples include industrial samples such as metal surface, semiconductor surface, glass surface, inside of semiconductor element, resin material surface, liquid crystal, polymer compound and the like.

  In the following description of the present embodiment, it is assumed that the object is a cell 73 in the culture solution 72 contained in the container 70, as shown in FIG. Inside the bottom of the container 70 a reflection enhancing coating 71 is provided.

  The light source 11 outputs light. Preferably, the light source 11 outputs incoherent light. The light source 11 is, for example, a lamp based light source such as a halogen lamp, a light emitting diode (LED) light source, a superluminescent diode (SLD) light source, an amplified spontaneous emission (ASE) light source, or the like.

  The beam splitter 12 is optically coupled to the light source 11 to constitute a Michelson interferometer which is a two-beam interferometer. The beam splitter 12 may be, for example, a half mirror in which the ratio of transmittance to reflectance is 1: 1. The beam splitter 12 splits the light output from the light source 11 into two light fluxes to be a first split light and a second split light. The beam splitter 12 outputs the first branched light to the objective lens 13 and outputs the second branched light to the objective lens 14.

  Further, the beam splitter 12 receives the first branched light reflected by the reflection enhancing coating 71 and passed through the objective lens 13, and receives the second branched light reflected by the reference mirror 15 and passed through the objective lens 14. Then, the beam splitter 12 combines the input first branched light and the input second branched light, and outputs interference light to the tube lens 16.

  The objective lens 13 is optically coupled to the beam splitter 12, and focuses the first branched light output from the beam splitter 12 on the cells 73 in the container 70. The objective lens 13 also receives the first branched light reflected by the reflection enhancing coating 71 and outputs the first branched light to the beam splitter 12.

  The objective lens 14 is optically coupled to the beam splitter 12 and condenses the second branched light output from the beam splitter 12 on the reflection surface of the reference mirror 15. The objective lens 14 also receives the second branched light reflected by the reflection surface of the reference mirror 15 and outputs the second branched light to the beam splitter 12.

  The tube lens 16 is optically coupled to the beam splitter 12 forming the interference optical system, and images the interference light output from the beam splitter 12 on the imaging surface of the imaging device 18 through the beam splitter 17. The beam splitter 17 splits the light arriving from the tube lens 16 into two, outputs one of the split lights to the imager 18, and outputs the other split light to the photodetector 22. The beam splitter 17 may be, for example, a half mirror.

  The imager 18 is optically coupled to the beam splitter 17 and receives the interference light arriving from the beam splitter 17 to acquire an interference image. The imager 18 is an image sensor such as, for example, a CCD area image sensor and a CMOS area image sensor.

  The piezo element 21 moves the reflective surface in a direction perpendicular to the reflective surface of the reference mirror 15. The piezoelectric element 21 can adjust the optical path length difference (that is, the phase difference) between the two light beams in the two light beam interferometer by the movement of the reflection surface. The piezo element 21 can determine the position of the reflection surface of the reference mirror 15 with a resolution less than the wavelength. In the two-beam interferometer, the optical path length difference between the two beams is variable.

  Assuming that the optical distance from the beam splitter 12 to the reflection enhancing coating 71 is L1 and the optical distance from the beam splitter 12 to the reflecting surface of the reference mirror 15 is L2, the optical path length between the two light beams in the two light beam interferometer The difference is 2 (L1-L2). If this optical path length difference is equal to or less than the coherent length of the output light of the light source 11, the imager 18 can acquire a clear interference image. Assuming that the central wavelength of the output light of the light source 11 is λ0, the phase difference Δφ between the two light beams in the two light beam interferometer is represented by the following equation.

  The light detector 22 is optically coupled to the beam splitter 17, receives the interference light arriving from the beam splitter 17, and outputs a detection signal. The photodetector 22 is, for example, a photodiode, an avalanche photodiode, or a photomultiplier, and may be a line sensor (linear sensor), a CCD area image sensor, a CMOS area image sensor, or the like.

  The phase control circuit 23 is electrically connected to the light detector 22, and receives a detection signal output from the light detector 22. The phase control circuit 23 is electrically connected to the piezo element 21 and controls the adjustment operation of the optical path length difference by the piezo element 21. The phase control circuit 23 detects an optical path length difference between the two light beams in the two light beam interferometer based on the input detection signal. Then, the phase control circuit 23 controls the adjustment operation of the optical path length difference by the piezo element 21 by feedback control based on the detection result. As a result, the optical path length difference between the two light beams in the two-beam interferometer can be stabilized at the set value (locked state).

  The interference image acquisition unit 2 can capture and acquire an interference image of the object (cell 73) with the image pickup device 18 in the locked state. The calculation unit 3 obtains a phase image of the object based on the interference image acquired by the imaging device 18 of the interference image acquisition unit 2. Arithmetic unit 3 may be a computer such as a personal computer and a tablet terminal. In addition, the calculation unit 3 includes an input unit (keyboard, mouse, tablet terminal, etc.) for receiving an input from the operator, and a display unit (display, tablet terminal, etc.) for displaying an interference image, a phase image, etc. It is also good. Further, it is preferable that the calculation unit 3 has a function of displaying an image or the like on the screen and receiving specification of the area by the operator on the screen.

  The light output from the light source 11 is split into two light beams by the beam splitter 12 to be the first split light and the second split light, and the beam splitter 12 outputs the first split light and the second split light. The first branched light output from the beam splitter 12 is focused on the cells 73 in the container 70 by the objective lens 13, and is reflected by a reflection enhancing coating 71 provided on the inside of the bottom of the container 70. The first branched light reflected by the reflection enhancing coating 71 is input to the beam splitter 12 through the objective lens 13. The second branched light output from the beam splitter 12 is focused by the objective lens 14 on the reflection surface of the reference mirror 15, and is reflected by the reflection surface. The second branched light reflected by the reflection surface of the reference mirror 15 is input to the beam splitter 12 through the objective lens 14.

  The first split light input from the objective lens 13 to the beam splitter 12 and the second split light input from the objective lens 14 to the beam splitter 12 are multiplexed by the beam splitter 12 and interference light from the beam splitter 12 Is output. The interference light passes through the tube lens 16 and is branched into two by the beam splitter 17 and is received by the imaging device 18 and the light detector 22 respectively. A detection signal is output from the light detector 22 that has received the interference light, and the phase control circuit 23 detects the difference in optical path length between the two light beams in the two light beam interferometer based on the detection signal. Then, by feedback control on the piezo element 21 by the phase control circuit 23, the optical path length difference between the two light beams in the two light beam interferometer is stabilized at the set value (locked state). An interference image is acquired by the imaging device 18 that receives the interference light in the locked state, and the interference image is output to the calculation unit 3. Then, the operation unit 3 obtains a phase image of the object (cell 73) based on the interference image.

  FIGS. 3 and 4 are timing charts for explaining the operation of the phase image acquisition apparatus 1A of the first embodiment and the phase image acquisition method using the same. These figures show an example of the time change of the output light intensity of the light source 11, the exposure period of the imaging device 18, and the time change of the phase difference of the interference light (the phase difference between the two light beams in the two light beam interferometer). There is. The operation of the phase image acquisition apparatus 1A and the phase image acquisition method are divided into a first phase image acquisition step, a condition search step, and a second phase image acquisition step.

  In the first phase image acquisition step, the phase image acquisition device 1A obtains one phase image from the plurality of interference images by the phase shift method. That is, the interference image acquisition unit 2 acquires the interference image in each state in a state in which the optical path length difference of the two-beam interferometer is stabilized with each of a plurality of different setting values. The calculation unit 3 obtains a first phase image based on the plurality of interference images acquired by the interference image acquisition unit 2.

  For example, the interference image acquisition unit 2 stabilizes the phase difference of the interference light at a certain initial phase by feedback control using the piezo element 21, the light detector 22, and the phase control circuit 23, and stabilizes the phase difference. The interference image I1 is acquired by the imaging device 18 in the state as described above. Next, the interference image acquisition unit 2 stabilizes the phase difference of the interference light at “initial phase + π / 2” using the piezo element 21, the light detector 22, and the phase control circuit 23, and stabilizes the phase difference. The interference image I2 is acquired by the imaging device 18 in the state as described above. Similarly, the interference image acquisition unit 2 acquires the interference image I3 with the imaging device 18 in a state in which the phase difference of the interference light is stabilized at “initial phase + π”, and the phase difference of the interference light is “initial phase + 3π The interference image I4 is acquired by the imaging device 18 in a state of being stabilized at 1/2.

  The calculation unit 3 calculates the first phase image Φ by performing the following calculation using the four interference images I1 to I4. Note that I1 to I4 and Φ are functions of the pixel position (x, y), and the calculation of the following equation is performed for each pixel. arg is an operator for acquiring complex number transformations. i is an imaginary unit.

  In the second phase image acquisition step, the phase image acquisition device 1A obtains one phase image from one interference image without using the phase shift method. If an interference image is repeatedly acquired N times, a phase image can be repeatedly acquired N times. The interference image acquisition unit 2 sets the optical path length difference of the two-beam interferometer to a fixed set value so that the light amount and the phase value of the interference light received by each pixel of the imaging device 18 in the region of interest become one to one correspondence. An interference image is repeatedly acquired as a stabilized state. The calculation unit 3 obtains a second phase image based on each interference image acquired by the interference image acquisition unit 2 and the first phase image Φ.

  In the second phase image acquisition step, the interference image acquisition unit 2 secures a one-to-one correspondence between the light amount of the interference light received by each pixel of the imaging device 18 in the region of interest and the phase value. In the first phase image acquisition step, the output light intensity of the light source 11 or the exposure time of the image pickup device 18 is within a range where the light amount of interference light received by each pixel of the image pickup device 18 in the region is less than the saturation light amount of the pixel. It is preferable to make it relatively large. Hereinafter, a suitable condition in the case of interference image acquisition by the interference image acquisition unit 2 in the second phase image acquisition step is referred to as “interference image acquisition suitable condition”.

  FIG. 3 shows a case where the output light intensity of the light source 11 is increased in the second phase image acquisition step as compared to the first phase image acquisition step. FIG. 4 shows the case where the exposure time of the imaging device 18 is made longer in the second phase image acquisition step as compared to the first phase image acquisition step. In the condition search step, interference light image acquisition suitable conditions are searched by changing the output light intensity of the light source 11 or the exposure time of the image pickup device 18. Then, in the second phase image acquisition step, the interference image acquisition unit 2 acquires an interference image under the interference image acquisition suitable condition.

  Below, an embodiment is described in more detail, showing the composition and the example of an image of a concrete example. An LED having a central wavelength of 633 nm was used as the light source 11. The focal length of each of the objective lens 13 and the objective lens 14 was 4.5 mm. The focal length of the tube lens 16 was 125 mm. The pixel size of the imaging device 18 was 4.8 μm × 4.8 μm. The number of pixels of the imaging field of view was 400 pixels × 300 pixels. The size of the imaging surface corresponding to the imaging field of view was 1.92 mm × 1.44 mm. The field size at the sample surface was 69.12 μm × 51.84 μm. Hamster ovary-derived cultured cells CHO were used as cells 73 of the sample. In the first phase image acquisition step, the time required for acquiring each interference image by the interference image acquisition unit 2 was 67 msec, and the exposure time of the imaging device 18 was 300 μsec.

  FIG. 5 is a diagram showing four interference images I1 to I4 acquired by the interference image acquisition unit 2 in the first phase image acquisition step. In these interference images, the light amount of the interference light received by each pixel of the imaging device 18 is equal to or less than the saturation light amount of the pixel. FIG. 6 is a diagram showing the first phase image Φ obtained by the calculation unit 3 in the first phase image acquisition step. The first phase image Φ is after phase unwrapping.

Assuming that the offset phase amount in each pixel is φ _offset and the phase difference is Δφ in the interference image, the luminance value I of each pixel is expressed by the following equation. Each parameter in this equation is also a function of pixel position (x, y), and the relationship of this equation is established for each pixel. In the actual interference image, since there is a light component not contributing to the interference, the DC component DC on the right side of the following equation does not generally have the value 0. When the phase difference Δφ changes in the range of 0 to 2π, the luminance value I of the pixel changes sinusoidally. That is, in the region of interest, the intensity I of the interference light can be reduced by appropriately setting the phase difference Δφ.

  As compared with the first phase image acquisition step, in the second phase image acquisition step, the integrated light amount of light incident on one pixel of the imaging device 18 is increased to acquire an interference image. When increasing the integrated light quantity of light, the light quantity of the light source 11 may be increased (FIG. 3) or the exposure time per one exposure of the image pickup device 18 may be increased (FIG. 4). In this embodiment, the exposure time of the imaging device 18 at the time of acquiring the interference image in the first phase image acquisition step is 300 μsec, while the imaging at the time of acquiring the interference image in the second phase image acquisition step The exposure time of the device 18 is 900 μsec.

  FIG. 7 is a view showing an interference image acquired by increasing the integrated light quantity of light incident on one pixel of the imaging device 18. At the time of acquisition of the interference image of FIG. 7, the exposure time of the imaging device 18 is increased by three times as compared to the time of acquisition of the interference image of FIG. An amount of photoelectrons is generated, and the pixel of the interference image is saturated. However, in the region of interest A within the frame in FIG. 7, a relatively dark image is obtained because the phase difference Δφ is relatively small. In the condition search step, such a condition is searched in the region of interest to set an interference image acquisition suitable condition in the second phase image acquisition step.

  In the second phase image acquisition step, N interference images are repeatedly acquired by using N interference images without using the phase shift method under the interference image acquisition suitable conditions searched in the condition search step. Get it repeatedly. In the present embodiment, the interference image acquisition unit 2 sets the exposure time of the imaging device 18 for acquiring each interference image to 900 μsec, and acquires 3000 interference images for 3 seconds. The frame rate was 1000 fps.

  FIG. 8 is a diagram showing three interference images acquired by the interference image acquisition unit 2 in the second phase image acquisition step. This figure shows interference at the start of acquisition (t = 0.000 seconds), 1 second after acquisition start (t = 1.000 seconds) and 2 seconds after acquisition start (t = 2.000 seconds). The image is shown. In 3000 interference images acquired at a frame rate of 1000 fps, small granular objects with a diameter of about 1 μm are often seen in the region of interest A in FIG. 7, and they move at high speed randomly in the field of view with time. I was able to see it.

  In order to observe high-speed movement of such granular objects (phase objects), it is desirable that the S / N of the interference image be high. However, in the off-axis method, the amount of light can not be made high because of the relationship with the amount of saturated light of the imaging device. On the other hand, in the phase image acquiring apparatus and the phase image acquiring method of the present embodiment, the interference condition of the region of interest is set as a condition that darkens the image as much as possible, so that the imager 18 can emit The S / N of the interference image can be increased while avoiding saturation of the charge amount.

  FIG. 9 is a diagram showing the relationship between the phase value of the phase image of FIG. 6 and the luminance value of the interference image of FIG. In FIG. 9, the horizontal axis represents the luminance value of the interference image, and the vertical axis represents the phase value of the phase image, which is plotted for each pixel. The phase image of FIG. 6 and the interference image of FIG. 7 have the same field of view. The curve in FIG. 9 is a fitting curve represented by the following equation. When a1 to a3 were determined by the least squares method, they were a1 = 240.5, a2 = 89.6, and a3 = 1.594.

  Referring to FIG. 9, even using the fitting curve, the phase value can not be uniquely determined from the luminance value. For example, the phase value of a pixel whose luminance value is 200 is either 0.44 radian or 2.65 radian as estimated by the fitting curve, but can not be determined. This uncertainty is resolved by limiting the area of interest. When the region of interest A is defined in the interference image as shown in FIG. 7, the relationship between the luminance value and the phase value for the pixels in the region of interest A is shown in FIG. 10 as a one-to-one correspondence. Become. FIG. 10 is a diagram showing the relationship between the phase value of the phase image of FIG. 6 and the luminance value of the interference image of FIG.

  Therefore, by limiting to the region of interest A, the phase value can be determined from the luminance value of each pixel by the following equation, and the phase image can be determined. Here, the arccos function is an inverse function of the cos function. However, as the phase value obtained by this equation, among the two values obtained by the calculation of arc cos, the value of the range of the limited phase value in FIG. 10 is adopted.

  FIG. 11 is a diagram showing three phase images obtained by the calculation unit 3 in the second phase image obtaining step. This figure shows interest at the start of acquisition (t = 0.000 seconds), 1 second after acquisition start (t = 1.000 seconds) and 2 seconds after acquisition start (t = 2.000 seconds). The phase image in the region A is shown. The second phase image shown in FIG. 11 is obtained based on the interference image of FIG. 8 and the first phase image of FIG.

  In this embodiment, in this way, in the second phase image acquisition step, the interference image is assumed to be in a state in which the phase difference Δφ between the two light beams in the two light beam interferometer is stabilized by the interference image acquisition unit 2 at a constant set value. While repeatedly acquiring at high speed, the phase image can be repeatedly acquired at high speed by the calculation unit 3. In addition, by appropriately setting interference image acquisition suitable conditions at the time of interference image acquisition by the interference image acquisition unit 2 in the second phase image acquisition step, it is possible to obtain a phase image having a high S / N. For example, in FIG. 12 showing an enlarged part of the phase image of FIG. 11 (t = 0.000 seconds), a fine granular form having an optical thickness of 8.4 nm and a diameter of 1.2 μm at a location indicated by an arrow. The phase object of was observed. It was possible to observe the movement of the granules around the cell membrane as a moving image.

  Next, interference image acquisition suitable conditions in the second phase image acquisition step will be described using FIGS. 13 to 15. FIG. 13 and FIG. 14 are diagrams for explaining the unfavorable conditions in the second phase image acquisition step. FIG. 15 is a diagram for describing interference image acquisition preferable conditions in the second phase image acquisition step. In these figures, the horizontal axis represents the luminance value of the interference image, and the vertical axis represents the phase value of the phase image, which is plotted for each pixel.

  The condition shown in FIG. 13 is not preferable because there are pixels in the region of interest that would exceed the saturation charge amount of the imaging device 18. Under such conditions, since the pixels of the image pickup device 18 are saturated even if the local phase change of the object is captured, the phase change can not be originally detected. Therefore, it is understood that it is better to shoot under the condition that the light quantity of the interference image in the region of interest is as low as possible and the image becomes dark.

  The condition shown in FIG. 14 is not preferable because the light amount of the interference image in the region of interest is the lowest. This is because, under this condition, two estimated phase values appear with respect to the luminance value of the interference image, so that the phase value can not be estimated from the interference image. Also, with regard to the increase and decrease of the luminance in the interference image, it is not known whether the increase or decrease is positively correlated with the phase change or negatively correlated.

  The interference image acquisition preferred conditions shown in FIG. 15 do not have the problems of the conditions of FIGS. 13 and 14 described above, that is, the interference image acquisition preferred conditions are between the intensity and the phase value of the interference image of the region of interest. The interference image of the region of interest is set to have a low light intensity as much as possible within a range where one-to-one correspondence is possible. Under this condition, one-to-one correspondence based on the fitting curve is possible between the luminance of the interference image and the phase value measured in advance, and moreover, it is ideal that no pixel has reached the saturation charge amount. Conditions are realized.

Next, a method of setting or designating a region of interest will be described with reference to FIGS. In the region-of-interest designation method shown in FIG. 16, the operator designates the region of interest A using, for example, a mouse on the screen on which the phase image is displayed (FIG. 16 (a)). Then, the calculation unit 3 obtains an offset phase amount φ _offset that can satisfy the interference image acquisition suitable condition in the region of interest A (FIG. 16 (b)). In the second phase image acquisition step, the interference image acquisition unit 2 locks the optical path length difference of the two-beam interferometer with this offset phase amount φ _offset .

In the region-of-interest designation method shown in FIG. 17, the operator designates the region of interest A using, for example, a mouse on the screen on which the phase image is displayed (FIG. 17A). Then, the calculation unit 3 finds a region of interest B that can satisfy the interference image acquisition suitable condition in the region of interest A, displays the region of interest B, and at the region of interest B, an interference image acquisition suitable condition An offset phase amount φ _offset that can be satisfied is determined (FIG. 17 (b)). When the arithmetic unit 3 can satisfy the interference image acquisition suitable condition in the entire region of interest A designated by the operator, an offset phase amount capable of satisfying the interference image acquisition suitable condition in the entire region of interest A What is necessary is just to obtain φ _offset . In the second phase image acquisition step, the interference image acquisition unit 2 locks the optical path length difference of the two-beam interferometer with this offset phase amount φ _offset .

In the region-of-interest designation method shown in FIG. 18, the computing unit 3 determines and displays a range in which the interference image acquisition suitable condition can be satisfied for each of a plurality of values of the offset phase amount φ _offset (FIG. 18A to (C)). The operator selects and designates one of them. In the second phase image acquisition step, the interference image acquisition unit 2 sets the optical path length difference of the two-beam interferometer with the offset phase amount φ _offset specified by the operator. Lock it.

  Next, the operation of the phase image acquisition device 1A of this embodiment and the procedure of the phase image acquisition method of this embodiment will be described using FIG. FIG. 19 is a flowchart for explaining the operation of the phase image acquisition device 1A of the first embodiment and the phase image acquisition method of the first embodiment.

  In step S1, optical adjustment is performed on the two-beam interferometer so that the interference image can be acquired by the interference image acquisition unit 2. In step S2 (first phase image acquisition step), a plurality of interference images are acquired by the interference image acquisition unit 2 by the phase shift method under the condition that each pixel of the imaging device 18 is not saturated, and calculation is performed based on the plurality of interference images The part 3 calculates a first phase image.

In step S3 (condition search step), either or both of the increase in the output light quantity of the light source 11 and the increase in the exposure time of the image pickup device 18 are performed, and then the offset phase amount φ _offset is shifted to make a plurality of interference images To search for interference image acquisition suitable conditions. In addition, since interference image acquisition suitable conditions can also be obtained by calculation based on the plurality of interference images and the first phase image acquired in step S2, the offset phase amount φ _offset is shifted here to make a plurality of interference images It is not necessary to search for interference image acquisition suitable conditions by acquiring.

In step S4 (second phase image acquisition step), the interference image acquisition unit 2 locks the offset phase amount φ _offset to the predetermined value obtained in step S3 and captures a plurality of interference images in time series, and the operation unit 3 A plurality of second phase images are calculated in time series on the basis of the correspondence between the luminance value and the phase value of the interference image.

When the region of interest is designated by the method of designating the region of interest shown in FIG. 16, the calculation of the offset phase amount φ _offset by the calculation unit 3 is performed before step S4. The designation of the region of interest A by the operator may be any time before this.

When the region of interest is specified by the region of interest specifying method shown in FIG. 17, the setting of the region of interest B by the operation unit 3 and the calculation of the offset phase amount φ _offset following this are performed in steps S2 and S4. Takes place during The designation of the region of interest A by the operator may be any time before this.

When the region of interest is designated by the method of designating the region of interest shown in FIG. 18, the display of the range satisfying the interference image acquisition preferable condition for each of the plurality of offset phase amounts φ _offset by the operation unit 3 and the subsequent operation The selection by the person is performed between step S2 and step S4.

  When the region of interest is designated by the method of designating the region of interest shown in FIGS. 16 to 18, an instruction from the operator is required. Here, the phase image acquisition device 1A is in an idle state while waiting for an instruction by the operator. It is not preferable that this instruction waiting period takes a long time between step S2 and step S4. The reason is that if the phase distribution of the entire sample changes significantly during the instruction standby period after acquiring the first phase image and determining the correspondence between the luminance and the phase value, the correspondence relationship must not be remeasured again. It is because it becomes impossible.

  Next, conditions that the optical system of the phase image acquisition device 1A desirably satisfies will be described. In the case of incoherent light, the light path difference in the two-beam interferometer is as small as possible, and the chromatic dispersion between the two light paths in the two-beam interferometer is as small as possible. Less is desirable. By using incoherent light, it is desirable that speckle noise be as small as possible in the interference image. Further, it is desirable that the curvature of the phase distribution (in particular, the curvature of a spherical surface) be as small as possible in the phase image by using the objective lens 13 and the objective lens 14 with the same or substantially the same optical conditions.

Second Embodiment
FIG. 20 is a diagram showing the configuration of a phase image acquisition device 1B of the second embodiment. The phase image acquisition device 1B of the second embodiment includes the power supply unit 31 and the timing control circuit 32 in addition to the interference image acquisition unit 2 and the calculation unit 3 provided in the phase image acquisition device 1A of the first embodiment shown in FIG. Further comprising

  The phase image acquisition device 1B observes the generation and diffusion of heat in the electric circuit 81 as an object at high speed. When a voltage is applied to an integrated circuit, power consumption in the circuit generates heat and a refractive index change occurs around the heat generation site. The change in refractive index due to this heat generation changes the phase of light transmitted through the integrated circuit. It is desirable to perform imaging at high speed because the influence of this heat generation spreads spatially due to diffusion in a short time.

  Therefore, in the phase image acquisition apparatus 1B of this embodiment, the power supply unit 31 which supplies the injection circuit whose intensity is temporally modulated to the electric circuit 81, the timing of voltage application from the power supply unit 31 to the electric circuit 81, and the interference image acquisition unit And a timing control circuit 32 for controlling the timing of interference image acquisition according to the second embodiment. In the second phase image acquisition step, the power supply unit 31 applies the temporally intensity-modulated injection current to the electric circuit 81, and the interference image acquisition unit 2 performs intensity modulation of the injection current under control of the timing control circuit 32. Synchronize and acquire an interference image.

  FIG. 21 is a timing chart for explaining the operation of the phase image acquisition device 1B of the second embodiment and the phase image acquisition method using the same. This figure shows the time change of the output light intensity of the light source 11, the period of voltage application from the power supply unit 31 to the electric circuit 81, the exposure period of the imaging device 18, and the phase difference of the interference light (two light in the two-beam interferometer The example of the time change of the phase difference between bundles is shown. The operation of the phase image acquisition apparatus 1B and the phase image acquisition method are divided into a first phase image acquisition step, a condition search step, and a second phase image acquisition step, as in the first embodiment.

  The voltage applied from the power supply unit 31 to the electric circuit 81 is, for example, one that is periodically intensity-modulated in a rectangular wave shape. Since the phase change due to heat generation accompanying voltage application in the electric circuit 81 is a local minute amount, voltage application does not have to be performed in the first phase image acquisition step and the condition search step. In the second phase image acquisition step, the interference image acquisition unit 2 acquires an interference image in synchronization with the intensity modulation of the injection current in the power supply unit 31 under the control of the timing control circuit 32.

  As an operation example of this timing control, the interference image acquisition unit 2 acquires one or more interference images during a period in which a voltage is applied from the power supply unit 31 to the electric circuit 81, and a voltage from the power supply unit 31 to the electric circuit 81 Acquire one or more interference images during a period in which no And the calculating part 3 calculates | requires a phase image based on each interference image. In the case of acquiring a plurality of phase images in each of the voltage application period and the non-application period, it is possible to observe a state in which a portion where a phase change occurs after the voltage application start spatially spreads along with heat diffusion. Further, by obtaining a difference image representing the difference between the phase images of the voltage application period and the non-application period, it is possible to clearly recognize a portion where the temperature has risen by the voltage application.

Third Embodiment
FIG. 22 is a diagram showing the configuration of a phase image acquisition device 1C of the third embodiment. The phase image acquisition device 1C of the third embodiment includes an infrared light source 41 and a timing control circuit in addition to the interference image acquisition unit 2 and the calculation unit 3 included in the phase image acquisition device 1A of the first embodiment shown in FIG. And 42.

  The phase image acquisition device 1C observes at high speed the phenomenon of the refractive index change that occurs when the cell 73 as the object irradiated with infrared light absorbs heat. When the cell is irradiated with infrared light of a wavelength included in the absorption wavelength range of a substance contained in the cell (eg, fat etc.), the local site of the cell generates heat due to local light absorption, and the local refractive index change Occurs. Due to the change in refractive index due to this heat generation, the phase of light transmitted through cells changes. It is desirable to perform imaging at high speed because the influence of this heat generation spreads spatially due to diffusion in a short time.

  Therefore, the phase image acquisition device 1C according to the present embodiment includes an infrared light source 41 for irradiating the cells 73 with infrared light whose intensity is temporally modulated, timing of infrared light irradiation from the infrared light sources 41 to the cells 73, and And a timing control circuit 42 that controls the timing of interference image acquisition by the interference image acquisition unit 2. In the second phase image acquisition step, the infrared light source 41 irradiates the cells 73 with temporally intensity-modulated infrared light, and the interference image acquisition unit 2 generates infrared light under the control of the timing control circuit 42. An interference image is acquired in synchronization with the intensity modulation.

  FIG. 23 is a diagram showing an example of the configuration of a sample. The cells 73 as an object are in the culture fluid 72 contained in the container 70. The bottom of the container 70 is an infrared light transmission window 74 for transmitting infrared light output from the infrared light source 41 disposed below.

  FIG. 24 is a timing chart for explaining the operation of the phase image acquisition device 1C of the third embodiment and a phase image acquisition method using the same. This figure shows the time change of the output light intensity of the light source 11, the period of infrared light irradiation from the infrared light source 41 to the cell 73, the exposure period of the imaging device 18, and the phase difference of the interference light (in two-beam interferometer The example of the time change of the phase difference between two light beams is shown. The operation of the phase image acquisition device 1C and the phase image acquisition method are divided into a first phase image acquisition step, a condition search step, and a second phase image acquisition step, as in the first embodiment.

  The infrared light emitted from the infrared light source 41 to the cell 73 is, for example, periodically modulated in intensity in a rectangular wave shape. Since the phase change due to heat generation caused by the infrared light irradiation in the cells 73 is a local minute amount, infrared irradiation may not be performed in the first phase image acquisition step and the condition search step. In the second phase image acquisition step, the interference image acquisition unit 2 acquires an interference image in synchronization with the intensity modulation of infrared light irradiation in the infrared light source 41 under the control of the timing control circuit 42.

  As an operation example of this timing control circuit, the interference image acquisition unit 2 acquires one or more interference images in a period in which the infrared light source 41 irradiates the cell 73 with infrared light, and At 73, one or more interference images are acquired during a period when infrared light is not irradiated. And the calculating part 3 calculates | requires a phase image based on each interference image. When a plurality of phase images are acquired in each of the infrared light irradiation period and the non-irradiation period, it is possible to observe that the site where the phase change occurs after the start of the infrared light irradiation spatially spreads along with the thermal diffusion. It is possible. Further, by obtaining a difference image representing the difference between the phase image of each of the infrared light irradiation period and the non-irradiation period, it is possible to clearly recognize the portion whose temperature has risen by the infrared light irradiation.

  The method of the present embodiment can be used as an index for appropriately selecting the wavelength of infrared light when it is desired to selectively stimulate a specific cell by infrared light irradiation. For example, in the observation field of view, cells to which stimulation is to be applied and cells to which stimulation is not to be cocultured. And the phase change accompanying infrared light irradiation is imaged by the method of this embodiment. In this phase image, if a relatively large phase change occurs in one cell and a relatively small phase change occurs in the other cell, the infrared light wavelength selectively causes thermal stimulation to the cell. Suggest that you can add In this way, critical wavelength bands in cell stimulation can be searched.

Fourth Embodiment
FIG. 25 is a diagram showing the configuration of a phase image acquisition device 1D according to the fourth embodiment. The phase image acquisition device 1D includes an interference image acquisition unit 2D and an arithmetic unit 3. Compared with the configuration of the phase image acquisition device 1A of the first embodiment shown in FIG. 1, the phase image acquisition device 1D of the fourth embodiment shown in FIG. It differs in the point provided with part 2D. The interference image acquisition unit 2D in the fourth embodiment further includes a spatial light phase modulator 24 and a phase modulation control circuit 25 in addition to the configuration of the interference image acquisition unit 2 in the first embodiment.

  The spatial light phase modulator 24 is provided on the light path of one of the two light fluxes in the two light flux interferometer. In FIG. 25, the spatial light phase modulator 24 is provided on the optical path between the beam splitter 12 and the objective lens 14 and is shown as a reflective type. In the second phase image acquisition step, the spatial light phase modulator 24 spatially modulates the phase of the light when reflecting the light, so that the light amount and the phase value of the interference light received by each pixel of the image pickup device 18 Can expand the range in which there is a one-to-one correspondence. The phase modulation control circuit 25 controls spatial phase modulation by the spatial light phase modulator 24 in response to an instruction from the arithmetic unit 3.

  As described above, in order to obtain the phase image based on the interference image in the second phase image acquisition step, there is a one-to-one correspondence between the light amount of the interference light received by each pixel of the imaging device 18 in the region of interest and the phase value. It is necessary for correspondence to exist. Although it is also necessary that the light quantity of the interference light received by each pixel of the image pickup device 18 in the region of interest is equal to or less than the saturated light quantity of the pixel, this condition is the light quantity of the interference light of each pixel in the region of interest It is satisfied if there is a one-to-one correspondence between the and the phase value (see FIG. 15).

  In the second phase image acquisition step, in order to acquire a phase image of a wide area, it is necessary to expand the range in which the light amount of the interference light received by each pixel of the imaging device 18 has a one-to-one correspondence. There is. However, for example, when the object is a cell, there is usually a phase difference of half the wavelength of light or more between the central portion and the peripheral portion of the cell. Therefore, no matter how the overall phase difference between the two light beams in the two-beam interferometer is optimized, one part of the cell satisfies the interference image acquisition preferable condition, while the other part And the phase value do not have a one-to-one correspondence, or the light quantity of the interference light exceeds the saturated light quantity of the pixel. That is, it is difficult to secure a one-to-one correspondence between the light amount of the interference light and the phase value with the entire cell as the region of interest.

Therefore, in the second phase image acquisition step, each pixel of the image pickup device 18 receives light by spatially phase modulating the light reflected by the spatial light phase modulator 24 in the second phase image acquisition step. The range in which the light amount of the interference light and the phase value have a one-to-one correspondence relationship is expanded, and the range in which the interference image acquisition preferable condition is satisfied is expanded. Note that spatial phase modulation by the spatial light phase modulator 24 means that the offset phase amount φ _offset in the above equation (3) is adjusted for each pixel.

  The arithmetic unit 3 writes the phase modulation pattern in the spatial light phase modulator 24 via the phase modulation control circuit 25 so as to cancel the phase distribution of the object obtained in the first phase image acquisition step, and the interference light Make the phase flat in a wide area. By performing such an operation in advance, it is possible to satisfy the interference image acquisition suitable condition in a wide region of interest in the visual field.

  Although the spatial light phase modulator 24 is provided on the optical path between the beam splitter 12 and the objective lens 14 in FIG. 25, it is provided on the optical path between the beam splitter 12 and the objective lens 13. May be Although the spatial light phase modulator 24 is shown as a reflective type in FIG. 25, it may be a transmissive type. Instead of the reference mirror 15, a reflective spatial light phase modulator may be provided.

Fifth Embodiment
In the first to fourth embodiments described so far, one light source 11 is used to obtain an interference image and to detect and control the phase difference of the two-beam interferometer. In this configuration, as described in Patent Document 1, phase displacement is detected by applying high-speed modulation (for example, 20 kHz) to the position of the reference mirror 15, and the frequency slower than this high-speed modulation is cut off frequency With the feedback control system, the stabilization of the phase difference between the two beams in the two-beam interferometer in the later frequency band will be performed.

  In this case, the frequency of the high-speed mechanical modulation that can be applied to the reference mirror 15 is at most about 20 kHz to 30 kHz, so the band of feedback control is limited to 2 kHz to 3 kHz. Therefore, in the first to fourth embodiments, the frame rate of the phase image that can be repeatedly acquired in the second phase image acquisition step is limited to the band of this feedback control. The configuration of the fifth embodiment can exceed this limit by using a laser light source for phase detection.

  FIG. 26 is a diagram showing the configuration of a phase image acquisition device 1E of the fifth embodiment. The phase image acquisition device 1E includes an interference image acquisition unit 2E and an arithmetic unit 3. Compared to the configuration of the phase image acquisition device 1A of the first embodiment shown in FIG. 1, the phase image acquisition device 1E of the fifth embodiment shown in FIG. It differs in the point provided with the part 2E. The interference image acquisition unit 2E in the fifth embodiment is different from the interference image acquisition unit 2 in the first embodiment in that it further includes the laser light source 26, the dichroic mirror 27, and the current modulation unit 28, and reference is made to the reference mirror 15. The difference is that a mirror 15E is included, and a dichroic mirror 17E is included instead of the beam splitter 17. FIG. 27 is a diagram showing the configuration of the reference mirror 15E. The reference mirror 15E has two reflecting surfaces 151 and 152 parallel to each other.

  The laser light source 26 outputs the wavelength-modulated laser light to the dichroic mirror 27. The laser light wavelength is different from the wavelength of the light output from the light source 11. The current modulation unit 28 temporally modulates the wavelength of the laser light output from the laser light source 26 by temporally modulating the driving current to be supplied to the laser light source 26.

  The dichroic mirror 27 is optically coupled to both the light source 11 and the laser light source 26, combines the laser light output from the laser light source 26 and the output light from the light source 11 and outputs the light to the beam splitter 12 coaxially. The laser light output from the laser light source 26 is multiplexed coaxially with the output light of the light source 11 and input to the two-beam interferometer, and after being branched into two light beams in the same manner as the output light of the light source 11, The two light beams are combined by the beam splitter 12 and output to the dichroic mirror 17E. However, in the reference mirror 15E, the output light of the light source 11 is reflected by the reflecting surface 151, and the laser light output from the laser light source 26 is transmitted through the reflecting surface 151 and reflected by the reflecting surface 152.

  The dichroic mirror 17E transmits the interference light of the output light of the light source 11 out of the interference light arriving from the beam splitter 12, and outputs the interference light to the imaging device 18, reflects the interference light of the laser light, and outputs the light to the light detector 22. Do. The light detector 22 receives the interference light of the laser light that has arrived from the dichroic mirror 17E and outputs a detection signal. The phase control circuit 23 detects the optical path length difference between the two light beams in the two-beam interferometer based on this detection signal, and controls the operation of the piezo element 21 by feedback control based on the detection result to calculate the optical path length difference. Stabilize.

  Since the reflecting surface 151 for reflecting the output light of the light source 11 and the reflecting surface 152 for reflecting the laser light are separately provided in the reference mirror 15E, the two light fluxes of the output light of the light source 11 for acquiring an interference image are obtained. When the optical path length difference between the two light beams in the interferometer is equal to or less than the coherent length, the optical path lengths of the two light beams in the two light beam interferometer are unbalanced for laser light. If the wavelength of the laser light is sinusoidally modulated at the frequency f in such a state, the phase of the interference light of the laser light received by the light detector 22 is also temporally modulated accordingly. The detection signal output from the light detector 22 that receives the interference light of the laser light includes a component of the frequency f and a component of the frequency 2f. The phase control circuit 23 can detect and stabilize the optical path length difference between the two light beams in the two light beam interferometer based on such a detection signal.

  In the invention described in Patent Document 2, the oscillation wavelength of laser light is modulated at a frequency of 30 kHz, and the phase difference is detected based on the detection signal output from the light detector that has received the interference light of the laser light. The phase difference is stabilized by controlling the position of the reference mirror by feedback control using a slower frequency as the cutoff frequency.

  In this embodiment, for example, the frequency of wavelength modulation of laser light may be several hundreds kHz, and about 30 kHz which is the mechanical limit of the piezo element can be set as the cutoff frequency in feedback control. The frame rate of the phase image that can be repeatedly acquired in the acquisition step can be set to a high speed of 10000 fps or more.

  1A to 1E phase image acquisition device 2, 2D, 2E interference image acquisition unit 3, operation unit 11, light source 12, beam splitter 13, 14 objective lens 15, 15E reference mirror, 16 Tube lens, 17: beam splitter, 17 E: dichroic mirror, 18: image pickup device, 21: piezo element, 22: photodetector, 23: phase control circuit, 24: spatial light phase modulator, 25: phase modulation control circuit, Reference Signs List 26 laser light source 27 dichroic mirror 28 current modulation unit 31 power supply unit 32 timing control circuit 41 infrared light source 42 timing control circuit 70 container 70, reflection enhancement coating 72 ... culture solution, 73 ... cell, 74 ... infrared light transmission window, 81 ... electric circuit.

Claims (14)

A light source, a two-beam interferometer that splits the light output from the light source into two light beams and then combines the two light beams to output interference light; and receives the interference light output from the two-beam interferometer And an imaging device, wherein the optical path length difference between the two luminous fluxes is variable in the two-beam interferometer, and an interference image of the object is captured and acquired by the imaging device in a state where the optical path length difference is stabilized. An interference image acquisition unit,
An operation unit for obtaining a phase image of the object based on the interference image acquired by the interference image acquisition unit;
Equipped with
In the first phase image acquisition step, the interference image acquisition unit stabilizes the optical path length difference of the two-beam interferometer with a plurality of setting values different from one another and acquires an interference image in each state, and the arithmetic unit Determining a first phase image based on the plurality of interference images acquired by the interference image acquisition unit;
In the second phase image acquisition step, the interference image acquisition unit is configured such that the light amount of the interference light received by each pixel of the image pickup device in the region of interest and the phase value have a one-to-one correspondence. The interference image is acquired as a state in which the optical path length difference is stabilized at a constant set value, and the operation unit acquires the second phase image based on the interference image acquired by the interference image acquisition unit and the first phase image. Ask,
Phase image acquisition device. In the second phase image acquisition step, the interference image acquisition unit outputs the output light of the light source in a range where the light amount of the interference light received by each pixel of the image pickup device in the region of interest is equal to or less than the saturation light amount of the pixel. Increasing the intensity or the exposure time of the imager as compared to the first phase image acquisition step;
The phase image acquisition device according to claim 1. The power supply unit further includes: a power supply unit for supplying a temporally intensity-modulated injection current to the object;
In the second phase image acquisition step, the interference image acquisition unit acquires an interference image in synchronization with intensity modulation of the injection current.
The phase image acquisition device according to claim 1. It further comprises an infrared light source for irradiating the object with infrared light whose intensity is temporally modulated.
In the second phase image acquisition step, the interference image acquisition unit acquires an interference image in synchronization with the intensity modulation of the infrared light.
The phase image acquisition device according to claim 1. The interference image acquisition unit further includes a spatial light phase modulator provided on the light path of any one of two light fluxes of the two light flux interferometer in the two light flux interferometer;
In the second phase image acquiring step, a range in which the light amount of the interference light received by each pixel of the image pickup device and the phase value have a one-to-one correspondence by spatial phase modulation by the spatial light phase modulator Expanding,
The phase image acquisition device according to any one of claims 1 to 4. The interference image acquisition unit is a laser light source that outputs a temporally wavelength-modulated laser beam that is multiplexed with the output light of the light source and input to the two-beam interferometer, and is output from the two-beam interferometer And a photodetector for receiving the interference light of the laser beam and outputting a detection signal, and detecting an optical path length difference of the two-beam interferometer based on the detection signal, and based on the detection result Stabilize the optical path length difference,
The phase image acquisition device according to any one of claims 1 to 5. The light source outputs incoherent light,
The phase image acquisition device according to any one of claims 1 to 6. A light source, a two-beam interferometer that splits the light output from the light source into two light beams and then combines the two light beams to output interference light; and receives the interference light output from the two-beam interferometer And an imaging device, wherein the optical path length difference between the two luminous fluxes is variable in the two-beam interferometer, and an interference image of the object is captured and acquired by the imaging device in a state where the optical path length difference is stabilized. An interference image acquisition unit,
An operation unit for obtaining a phase image of the object based on the interference image acquired by the interference image acquisition unit;
Using
The interference image acquisition unit stabilizes the optical path length difference of the two-beam interferometer with each of a plurality of different setting values, acquires an interference image in each state, and the operation unit acquires the interference image acquisition unit A first phase image acquisition step of determining a first phase image based on the plurality of acquired interference images;
The light path length difference of the two-beam interferometer is set to a constant value so that the light quantity and the phase value of the interference light received by each pixel of the image pickup device in the region of interest become one-to-one correspondence by the interference image acquisition unit. Acquiring an interference image as a stabilized state, and calculating a second phase image based on the interference image acquired by the interference image acquisition unit and the first phase image by the operation unit; ,
A phase image acquisition method comprising: In the second phase image acquisition step, the interference image acquisition unit outputs the output light of the light source in a range where the light amount of interference light received by each pixel of the image pickup device in the region of interest is equal to or less than the saturation light amount of the pixel. Increasing the intensity or the exposure time of the imager as compared to the first phase image acquisition step;
The phase image acquisition method according to claim 8. The object is an electrical circuit,
In the second phase image acquisition step, an injection current temporally intensity-modulated by a power supply unit is applied to the electric circuit, and an interference image is acquired by the interference image acquisition unit in synchronization with the intensity modulation of the injection current.
The phase image acquisition method according to claim 8 or 9. The object is a cell,
In the second phase image acquisition step, the cells are irradiated with infrared light temporally intensity-modulated by an infrared light source, and the interference image acquisition unit acquires interference images in synchronization with the intensity modulation of the infrared light. Do,
The phase image acquisition method according to claim 8 or 9. In the second phase image acquisition step, each pixel of the image pickup device is subjected to spatial phase modulation by a spatial light phase modulator provided on the optical path of any one of two luminous fluxes in the two luminous flux interferometer. Expand the range in which the light amount of the interference light received by the light and the phase value have a one-to-one correspondence,
The phase image acquisition method according to any one of claims 8 to 11. The interference image acquisition unit causes the laser light source to output temporally wavelength-modulated laser light, combines the laser light with the output light of the light source, and causes the two-beam interferometer to input the two-beam interference. The interference light of the laser beam output from the meter is received by the light detector to output a detection signal, and the difference in optical path length of the two-beam interferometer is detected based on the detection signal, and the detection result is Stabilize the optical path length difference,
The phase image acquisition method according to any one of claims 8 to 12. The light source outputs incoherent light,
The phase image acquisition method according to any one of claims 8 to 13.