JPWO2016163560A1 - Digital holographic microscope - Google Patents

Abstract

A fluorescence holographic microscope and a phase image detection digital holographic microscope are combined to provide a digital holographic microscope capable of simultaneously measuring a fluorescence image and a phase image in three dimensions. A first holographic optical system that obtains a phase three-dimensional image by interference light obtained by superimposing the object light passing through the sample stage 4 and the reference light not passing by using the first laser light 5; and a second laser A second holographic optical system that obtains a fluorescence three-dimensional image by fluorescence excitation light using the light 6, an imaging unit 3, and a bifocal Fullel lens (SPFL) 2 having polarization dependency are provided. The respective optical systems are arranged so that the object light and the fluorescent signal light are coaxially superimposed, and the bifocal lens 2 is disposed on the superimposed optical axis. The imaging means 3 acquires a phase three-dimensional image and a fluorescence three-dimensional image simultaneously as a hologram. The phase three-dimensional image and the fluorescence three-dimensional image are separated, and the object light and the fluorescence signal light are reconstructed from the respective interference intensity distributions.

Description

  The present invention relates to a digital holographic microscope capable of simultaneously measuring three-dimensional fluorescence and phase.

Dynamic three-dimensional imaging of living cells is an important measurement technique in the field of bioapplications. For example, a fluorescent labeling technique is known in which a fluorescent molecule is introduced into specific DNA in a cell nucleus using a fluorescence microscope, and changes in cells undergoing mitosis are measured. Visualization of the interaction between nuclei and proteins in cells stained with fluorescent molecules enables various observations. A confocal laser scanning microscope is known as one capable of measuring a three-dimensional fluorescence image with high resolution and high contrast. However, since the confocal laser scanning microscope requires scanning of the focal point for each point of the measurement object, it takes time to obtain a three-dimensional fluorescence image, and measurement of depth and depth of a dynamic object is fast. There is an essential limitation that simultaneous measurement of a plurality of cells at different positions is not suitable.
There is also a need for a technique for simultaneously observing not only the fluorescence image but also the structure of the cell nucleus and cell wall. Structural observation means include a phase contrast microscope and a differential interference microscope. The phase contrast image is effective for measuring the shape of a transparent object.

If the fluorescence image and the phase image can be observed at the same time, different information can be acquired at the same time, so that more detailed information can be provided in the bioreaction.
Recently, an optical microscope capable of simultaneously observing a fluorescence image and a phase difference image is commercially available. However, a filter needs to be replaced, and there is a restriction that a measurement range is limited to one point or two dimensions.

  On the other hand, a digital holographic microscope (DHM) is known as a tool that enables instantaneous three-dimensional measurement. In the digital holographic microscope, three-dimensional information is acquired as hologram information, and light wave information of the object with respect to the depth position is reconstructed by performing back propagation calculation of the light wave in the computer. The characteristics of the digital holographic microscope are that it does not require special fluorescent staining and enables three-dimensional observation of living cells (label-free), and has an auto-focusing function that can arbitrarily change the focal position by computer reconstruction. It is mentioned that quantitative phase measurement is possible. For this reason, even in a situation where cells move three-dimensionally, it is possible to obtain a reconstructed image in focus automatically by reconstructing calculation.

  When applying a digital holographic microscope to cell observation, it is necessary to acquire a hologram of fluorescence information. However, in order to realize holography technology using fluorescence, the problem is that the fluorescence is incoherent light that is very unlikely to interfere. There is. In recent years, methods for making holograms from incoherent light have been proposed one after another, and research toward a Fluorescence Digital Holographic Microscope (FDHM) has been activated all over the world. Most have many optical elements (for example, refer patent document 1).

  The hologram recording apparatus disclosed in Patent Document 1 solves the problem that light with low coherence (for example, natural light or fluorescence) cannot be observed, and uses a spatial light modulator (wavefront modulation element). Thus, the object light including the first component light and the second component light having different polarization directions are made to have different wavefront shapes of the first component light and the second component light, and the second component with respect to the first component light. In the phase shift amount of light, a distribution that periodically changes in space is generated, and the first component light and the second component light are caused to interfere with each other to form a hologram. As a result, a reproduced image of the subject can be obtained from a hologram recorded by one imaging using light having low coherence passing through a single optical path. However, this method has a problem that it is limited to observation of a three-dimensional fluorescence image with low coherence.

  In addition, a fluorescence digital holographic microscope (FDHM) is suitable for targeting measurement, but has a problem that it is ineffective for a substance that does not generate fluorescence. In addition, when performing fluorescence observation, there is also a problem that a measurement target is limited because harmful fluorescent molecules are used. Therefore, from the viewpoint of expanding the application of the digital holographic microscope (DHM), the appearance of a multimodal digital holographic microscope capable of observing various information such as phase images, fluorescent images, and polarized light is desired. Yes.

The present inventors have already prototyped a digital holographic microscope capable of two-dimensional fluorescence imaging and phase imaging and reconstructed it from a hologram image for the purpose of realizing a multimodal digital holographic microscope (Non-Patent Document). 1). The prototype microscope acquires a fluorescent image and a phase image sequentially, and does not measure the fluorescent image and the phase image at the same time.
Hereinafter, unless otherwise specified, in this specification, a fluorescence image and a fluorescence three-dimensional image, and a phase image and a phase three-dimensional image are used in the same meaning.

Japanese Patent Laying-Open No. 2015-1726

Osamu Matoba, “Prototype of hybrid digital holographic microscope capable of fluorescence and phase imaging”, Optics & Photonics Japan 2014, No.6, P15.

The prototype digital holographic microscope described above has the functions of both a fluorescence microscope and a phase image detection digital holographic microscope, but each image is acquired sequentially by switching the shutter. It is not a combination of an image detection digital holographic microscope and does not mean that a fluorescence image and a phase image can be measured three-dimensionally at the same time.
In view of such circumstances, an object of the present invention is to provide a digital holographic microscope in which a fluorescence digital holographic microscope and a phase image detection digital holographic microscope are combined, and a fluorescence image and a phase image can be simultaneously measured three-dimensionally. .

In order to achieve the above object, a digital holographic microscope of the present invention comprises the following 1) to 4).
1) First holographic optical system 2 that acquires a phase three-dimensional image by interference light obtained by superimposing object light that passes through the observation target sample and reference light that does not pass using the first laser light 2) Second Second holographic optical system for acquiring a three-dimensional fluorescence image of the sample to be observed by the fluorescence excitation light using laser light 3) Imaging means 4) Double focus lens having polarization dependence

  Then, the respective optical systems are arranged so that the object light and the fluorescent signal light are superimposed on the same axis, and the bifocal lens of 4) is arranged on the superimposed optical axis. An imaging means simultaneously acquires a phase three-dimensional image and a fluorescence three-dimensional image as a hologram. The phase three-dimensional image and the fluorescence three-dimensional image are separated from the hologram image acquired by the imaging means, and the object light and the fluorescence signal light are reconstructed from the respective interference intensity distributions.

According to the above configuration, the two optical systems of the first holographic optical system that acquires the phase three-dimensional image from the observation target sample and the second holographic optical system that acquires the fluorescent three-dimensional image are used as a common optical path. The two hologram images can be acquired by one imaging means by superimposing the object light and the fluorescent signal light on the same axis.
Here, the double-focus lens having polarization dependence is a lens having strong anisotropy in the polarization direction of the photoelectric field and having two focal lengths.

  As the bifocal lens having polarization dependency, a sub-wavelength periodic structured Fresnel lens (SPFL) having a sub-wavelength periodic structure is preferably used. In the case of SPFL, the subwavelength periodic structure, which is a diffraction grating structure having a shorter period than the wavelength of light, is diffracted by the polarization state of the light wave. In addition, the Fresnel lens is a lens in which a normal lens is folded back with the wavelength of light to reduce the thickness, and the cross section is a saw-like shape in which prisms are arranged. The bifocal Fresnel lens has a function of diffracting one beam light into two parts and focusing on different places.

Further, a liquid crystal spatial light modulator may be used as a double focus lens having polarization dependency. In the liquid crystal spatial light element, different actions can be generated with respect to the polarization state of the incident photoelectric field depending on the orientation of the liquid crystal molecules. For example, when acting as an ordinary ray with respect to linearly polarized light in a certain direction, it acts as an extraordinary ray with respect to linearly polarized light perpendicular thereto. Therefore, it is possible to realize a double-focus lens having polarization dependency that acts only on a specific linear polarization direction.
The fluorescent signal light in the digital holographic microscope of the present invention generates two diffracted lights by the double lens realized by the liquid crystal spatial light modulator. Both of these two diffracted lights have the same polarization, and self-interference occurs. In this respect, the function differs from the spatial light modulation element in Patent Document 1 described above that forms a hologram by causing the first component light and the second component light having different polarization directions to interfere with each other (in the case of the present invention, for the fluorescence hologram). The fluorescent signal light and the object light for phase hologram correspond to the first component light and the second component light of Patent Document 1.)

In the digital holographic microscope of the present invention, the double-focus lens having polarization dependency is designed so as to act only on the second holographic optical system and not on the first holographic optical system.
The bifocal lens is designed so that the polarization of the object light of the first holographic optical system that acquires the phase three-dimensional image is insensitive and does not cause lens action, and the fluorescence three-dimensional image is acquired. The second holographic optical system is designed to have a strong sensitivity to the polarization of fluorescence. The polarization of the fluorescence of the second holographic optical system will cause self-interference due to the bifocal lens having two focal lengths.

In the digital holographic microscope of the present invention, the fluorescence three-dimensional image is a concentric hologram, the phase three-dimensional image is an equiangular hologram, and separates the fluorescence three-dimensional image and the phase three-dimensional image on the spatial frequency plane.
Since the fluorescence polarization of the second holographic optical system self-interferences in the three-dimensional fluorescence image, the interference pattern is concentric. On the other hand, the phase three-dimensional image can be formed into an interference pattern having a carrier frequency by using equi-tilt interference. The concentric interference pattern and the equiangular interference pattern can be separated on the spatial frequency plane, and the three-dimensional fluorescence image and the three-dimensional phase image can be separated on the spatial frequency plane from the image captured by the imaging means. . By Fourier transforming the two hologram patterns, the components of the equi-tilt interference pattern can be extracted by a bandpass filter on the spatial frequency plane. By acquiring the reference light intensity distribution used for the equi-tilt interference pattern in advance, it is possible to extract only the concentric interference pattern.

In the digital holographic microscope of the present invention, the second holographic optical system may be capable of switching the excitation laser light source. The wavelength of the excitation laser light source is switched according to the fluorescence characteristics of the observation target sample. Thus, by switching the excitation laser light source according to the type of fluorescent staining reagent, it is possible to select a target cell and measure cell state changes such as division and shape change.
That is, using the digital holographic microscope of the present invention, a fluorescence three-dimensional image acquired using one excitation laser light source and a fluorescence three-dimensional image acquired using another excitation laser light source were simultaneously acquired. From the three-dimensional phase image, a cell observation method for observing cell behavior can be realized.

In the digital holographic microscope of the present invention, it is preferable that the first holographic optical system is a transmission type and the second holographic optical system is a reflection type (an epi-illumination type). In addition, both the first and second holographic optical systems may be turned upside down depending on the sample installation state.
In the second holographic optical system that acquires a three-dimensional fluorescence image of the sample to be observed by fluorescence excitation light, the fluorescence signal is very weak. Therefore, when the transmission type is used, it is very difficult to cut only strong fluorescence excitation light. This is because it becomes difficult.
When the second holographic optical system is made to be a transmission type instead of a reflection type (epi-illumination type), the fluorescence excitation light that excites the fluorescence signal is sufficiently cut, and the dichroic mirror that passes only the fluorescence signal or the specification of the fluorescence wavelength It is necessary to use a band-pass filter that passes the laser wavelength for phase measurement and phase measurement.

In the digital holographic microscope of the present invention, when observing cells, it is preferable to simultaneously acquire spatiotemporal information of cell nuclei from a three-dimensional fluorescence image and spatiotemporal structures of cell nuclei and cell walls from a three-dimensional phase image.
Multidimensional information obtained from a plurality of physical quantities has not been obtained by measurement in the conventional bioimaging field, and enables observation that has been impossible with the digital holographic microscope of the present invention. The digital holographic microscope of the present invention enables simultaneous and high-speed photographing of a three-dimensional fluorescence image and a three-dimensional phase image as a powerful measurement rule in the field of bioimaging.
That is, by using the digital holographic microscope of the present invention, it is possible to realize a cell observation method that simultaneously acquires spatiotemporal information of cell nuclei from a fluorescence three-dimensional image and spatiotemporal structures of cell nuclei and cell walls from a phase three-dimensional image.

  According to the digital holographic microscope of the present invention, the fluorescence digital holographic microscope and the phase image detection digital holographic microscope are combined, and there is an effect that the fluorescence three-dimensional image and the phase three-dimensional image can be measured simultaneously.

Configuration diagram of optical system of digital holographic microscope of embodiment 1 Configuration diagram of first holographic optical system for acquiring phase three-dimensional image Configuration diagram of second holographic optical system for obtaining fluorescent three-dimensional image Illustration of separation and reproduction principle of fluorescence hologram and phase hologram Example of acquired hologram image Fluorescence image and phase image separated from hologram image Illustration of bifocal Fresnel lens (SPFL) Configuration diagram of optical system of digital holographic microscope of embodiment 2 Example of measurement image of Greater Canada (1) Example of measurement image of Greater Canada (2) Configuration diagram of optical system of digital holographic microscope of embodiment 4 Phase image and fluorescence image of two-color fluorescent beads

  Hereinafter, an example of an embodiment of the present invention will be described in detail with reference to the drawings. The scope of the present invention is not limited to the following examples and illustrated examples, and many changes and modifications can be made.

  FIG. 1 shows a configuration of an optical system according to an embodiment of a digital holographic microscope. The digital holographic microscope shown in FIG. 1 uses the two optical systems of the transmission type digital holographic microscope shown in FIG. 2 and the reflection type fluorescence microscope shown in FIG. Two holograms of a phase three-dimensional image and a fluorescence three-dimensional image can be simultaneously acquired by one image pickup unit by superimposing them.

First, the fluorescent molecules of the observation target sample on the sample stage 4 are excited using the excitation laser light source 6 in the wavelength band of 355 nm to 550 nm. The excited fluorescent molecules emit a fluorescent signal having a long wavelength from an excitation laser light source having a wavelength band of 355 nm to 550 nm, and enter the objective lens 15 together with the excitation laser light reflected from the glass substrate surface of the sample stage 4. A dichroic mirror 16 is installed to sufficiently attenuate the excitation laser light and emphasize the fluorescent signal light to obtain a fluorescent three-dimensional image.
In the fluorescence three-dimensional image, the polarization component of fluorescence self-interfers with the bifocal Fresnel lens (SPFL) 2 that is a bifocal lens, so that the interference pattern is concentric. As shown in FIG. 7, the SPFL2 is a Fresnel lens having a sub-wavelength periodic structure having _two focal lengths (f ₁ , f ₂ ). Due to the diffraction grating structure (subwavelength periodic structure) having a period shorter than the wavelength of light, the light wave is diffracted by its polarization state. One diffracted wave is generated from one light beam by a Fresnel lens having two focal lengths. The two diffracted waves are in focus at different locations.

  At the same time, the measurement object of the sample to be observed on the sample stage 4 is illuminated using the He—Ne laser light source 5 having the wavelength 633 (nm). The He-Ne laser light is divided into an object light path that passes through the measurement object and an empty reference light path by the beam splitter 12 to constitute a Mach-Zehnder interferometer. Since the wavelength of the He—Ne laser beam that passes through the measurement object is longer than the wavelength of the excitation laser beam in the wavelength band of 355 nm to 550 nm, it is propagated without being affected by the dichroic mirror 16 and is again interfered with the reference beam by the beam splitter 18. can do. At this time, an off-axis hologram, that is, a hologram having an equitilt interference pattern is obtained by slightly setting an angle between the object beam and the reference beam. The amplitude distribution and the phase distribution of the object light are extracted from the acquired hologram of the equal inclination interference pattern using the Fourier transform method. In the off-axis method, the wavefront of the object light of the observation target sample is reproduced by propagating back to the original object position.

Then, two holograms of a phase three-dimensional image and a fluorescence three-dimensional image are simultaneously acquired by the image sensor 3 used as an imaging unit.
As described above, since the fluorescence polarization component self-interferences with the bifocal Fresnel lens (SPFL) 2 in the three-dimensional fluorescence image, the interference pattern is concentric. On the other hand, the phase three-dimensional image becomes an equitilt interference pattern. The concentric interference pattern and the equi-angle interference pattern can be separated on the spatial frequency plane, and the phase hologram of the phase three-dimensional image and the fluorescence hologram of the fluorescence three-dimensional image are spatially separated from the image captured by the image sensor 3. It can be separated in terms of frequency.
By providing the SPFL2, a half-wave plate 20 is provided so that the phase hologram light is not affected, and the polarization of the phase hologram light is adjusted to a polarization state that is not affected by the SPFL2. In addition, a half-wave plate 21 is provided to align the polarization direction of the fluorescence excitation light source with the polarization direction in which the SPFL 2 works.

  Here, the signal in which the phase hologram and the fluorescence hologram are superimposed can be expressed as the following Equation 1.

In the above equation 1, o _p represents the complex amplitude distribution of the object light wave for phase holograms, r _p represents the complex amplitude distribution of the reference light wave. Further, o _F is a complex amplitude distribution of the object light wave for a fluorescence hologram incident on the double focus lens. Here, f ₁ and f ₂ indicate two focal lengths of the bifocal lens. G is a function representing the phase function of the lens and the Fresnel diffraction propagation to the imaging surface.
If r _p is assumed to be a plane wave expressed by the following Expression 2, the phase hologram is the off-axis hologram. Therefore, the extraction of the phase hologram can be performed on the spatial frequency plane by Fourier transforming the above Equation 1.

  Formula 3 below is the result of Fourier transform of Formula 1 above. Here, A is the amplitude of the reference light for the phase hologram. FIG. 4A is an interference image in which two holograms of a phase hologram and a fluorescence hologram are superimposed, and illustrates the following Equation 3. The first, second, fifth, sixth, seventh and eighth terms on the right side of Equation 3 below are terms based on self-interference concentric holograms, which are signals appearing near the origin on the spatial frequency plane (see FIG. 4 (2)). ). Further, the third and fourth terms on the right side are terms based on an off-axis hologram, and are signals present at positions shifted from the origin on the spatial frequency plane (see FIG. 4 (2)). Therefore, by multiplying the window function, the two holograms of the fluorescence hologram that appears near the origin and the phase hologram that exists at a position shifted from the origin are separated on the spatial frequency plane shown in FIG. 4B (FIG. 4). (See (3)). Therefore, the third and fourth terms on the right side, which are terms based on the off-axis hologram, can be extracted as in the following equations 4 and 5.

Here, the amplitude A of the reference light phase hologram, since it is possible to previously measured, it is possible to find the object beam o _p and its complex conjugate distribution phase hologram from the equation 4 and equation 5. By substituting the above formulas 4 and 5 into the above formula 3, the following formula 6 is obtained as a fluorescence hologram.
Here, G ₁ and G ₂ in Equation 3 above represent Fourier transforms of g (O _F (x, y), f ₁ ) and g (O _F (x, y), f ₂ ), respectively. Yes.

  As described above, by performing Fourier transform on the two patterns of the phase hologram and the fluorescence hologram, it is possible to extract the component of the equiangular interference pattern of the signal existing at the position shifted from the origin on the spatial frequency plane by the band pass filter. it can. Also, by acquiring the intensity distribution of the reference light used for the equi-angle interference pattern in advance, it is possible to extract only the concentric interference pattern.

(Example of imaged hologram)
Here, an example of the imaged hologram is shown. The image sensor 3 used was Photometrics CoolSNAPKINO-SPK (4.54 μm pitch, 14 bits). The objective lens 15 used was an objective lens 40 times as large as MDPlan 40 (NA = 0.65) manufactured by Olympus. As an observation object, TetraSpeck (registered trademark) fluorescent beads of 4 μm in diameter from Invitrogen were used.

FIG. 5 shows an example of a hologram image in which a fluorescence image and a phase hologram are individually acquired by an image sensor at a fluorescence excitation center wavelength 532 (nm) and a fluorescence center wavelength 550 (nm).
FIG. 5 (1) is a hologram image of fluorescent beads (an enlarged view of the fluorescent bead portion is also appended), which is Fourier transformed to obtain the spatial frequency plane of FIG. 5 (2). A hologram existing at a position shifted from the origin of the spatial frequency plane is extracted, and the phase distribution of FIG. 5 (3) is acquired (an enlarged phase image is also appended).
FIG. 5 (4) shows a three-dimensional display of the reconstructed phase distribution. From FIG. 5 (4), the phase change amount of the fluorescent beads can be seen.

The fluorescent image of the fluorescent beads and the phase image obtained by the process of FIG. 5 are shown in FIGS. 6 (1) and (2), respectively. A phase 3D plot is shown in FIG. From FIG. 6 (1) and (2), the fluorescent bead image and the phase image of the fluorescent bead can be confirmed. FIG. 6 (3) shows that the shape of the fluorescent beads is close to a convex spherical shape. Using the constructed experimental system, it was confirmed that measurement of a 4 μm size fluorescence image, a 10 μm size cell shape phase image, and the like was possible.
The digital holographic microscope of the present invention can measure cell shape change and nucleus movement in real time. By further implementing an autofocus function for searching for an imaging position in the depth position direction, observation of a fluorescence three-dimensional image and a phase three-dimensional image may be realized in real time.

In the digital holographic microscope of Example 2 described above, the first holographic optical system that acquires a phase hologram is a transmission type, and the second holographic optical system that acquires a fluorescence hologram is a reflection type. As described in the above, the second holographic optical system for acquiring the fluorescence hologram may be a transmission type.
FIG. 8 shows a configuration diagram of an optical system of the digital holographic microscope of the second embodiment.

  Similar to the optical system of the digital holographic microscope of the first embodiment, the measurement object of the sample to be observed on the sample stage 4 is illuminated using the He—Ne laser light source 5 having a wavelength of 633 (nm). The He-Ne laser light is divided by the beam splitter 12 into an object light path that passes through the measurement object and a reference light path that has nothing, and constitutes a Mach-Zehnder interferometer. Since the wavelength of the He—Ne laser beam that passes through the measurement object is longer than the excitation laser light source in the wavelength band of 532 nm, which will be described later, it is propagated without being influenced by the dichroic mirror 33 and again interferes with the reference light by the beam splitter 18. Can do. At this time, an off-axis hologram (a hologram with an equal inclination interference pattern) is acquired by slightly setting an angle between the object beam and the reference beam.

  Further, the excitation laser light source 6 having a wavelength band of 532 nm is used to excite fluorescent molecules through the observation target sample placed on the surface of the sample stage 4 from the back surface of the glass substrate of the sample stage 4. The excited fluorescent molecule emits a fluorescent signal having a wavelength longer than 532 (nm) and enters the objective lens 15 together with the excitation laser light transmitted through the sample stage 4. Since the fluorescence signal by the fluorescence excitation light of the sample to be observed is weak, a dichroic mirror 33 that cuts the fluorescence excitation light sufficiently and allows only the fluorescence signal to pass when the transmission type is used as in this embodiment is installed. The excitation laser beam is sufficiently attenuated, and the fluorescence signal light is emphasized to obtain a fluorescence three-dimensional image. In the fluorescence three-dimensional image, the polarization component of the fluorescence self-interfers with the bifocal Fresnel lens (SPFL) 2 having the sub-wavelength periodic structure, so that the interference pattern is concentric.

  Then, the image sensor 3 simultaneously acquires two holograms of a phase three-dimensional image and a fluorescence three-dimensional image. The interference pattern of the fluorescence hologram is concentric, and the interference pattern of the phase hologram is an equitilt interference pattern. As in the first embodiment, the concentric interference pattern and the equiangular interference pattern can be separated on the spatial frequency plane, and the phase hologram of the three-dimensional phase image and the three-dimensional fluorescence are obtained from the image captured by the image sensor 3. The fluorescence hologram of the image can be separated on the spatial frequency plane. Further, by providing the SPFL 2, the half-wave plate 20 is provided so that the phase hologram light is not affected, and the polarization of the phase hologram light is adjusted to a polarization state that is not affected by the SPFL 2. In addition, a half-wave plate 21 is provided to align the polarization direction of the fluorescence excitation light source with the polarization direction in which the SPFL 2 works.

  Conventionally, microscopic observation in the field of bioimaging has a limitation that only cell movement in the vicinity of the focal plane can be observed. On the other hand, in digital holography, it is known that when a 20 × objective lens is used, a 1000 × depth range can be observed. Using the digital holographic microscope of Example 1 or 2, it is possible to measure the reaction in different cells with respect to a three-dimensional cell mass or depth, and to multitrack the three-dimensional position of a large number of cells.

A reconstructed image focused in three dimensions is reproduced in real time at 30 frames per second by determining whether the reconstructed image is different in time or not. An autofocus function is provided for tracking the position where the depth distribution is focused on the phase distribution information. It is also possible to construct a digital holographic microscope that can support a 10-digit time scale of 10 ⁻⁵ seconds to 10 ⁵ seconds as the time resolution. In addition, it is possible to simultaneously observe the effects of drugs on cancer cells having different depths, and a reconstructed image focused on all aspects can be provided.

FIGS. 9 and 10 show images of chloroplast protoplast flow observed in the digital holographic microscope of Example 1 in the canard moth. FIG. 9 (1) is a photograph taken by a quasi-canada model (a sectional view showing a two-layer structure is also appended), FIG. 9 (2) is a hologram, FIG. 9 (3) is a spatial frequency plane, and FIG. 9 (4) is a reproduced phase. The image is shown.
Chloroplasts emit autofluorescence without staining (see FIG. 10 (1)). Therefore, a fluorescence hologram and a phase hologram are acquired simultaneously, and the structure of the cell wall and the chloroplast are simultaneously measured by phase measurement (see FIG. 10 (2)). Since the chloroplast is moving three-dimensionally, the hologram images arranged in time series are reconstructed in real time.

  Further, as shown in FIG. 9 (1), the giant canadian has a two-layer structure of a front surface and a back surface. In the digital holography measurement by the digital holographic microscope of Example 1, the crosstalk noise is large because the coherence length is long when the structure exists in the depth. In digital holography, information with different depth positions can be removed or reduced by a filter, so that depth resolution can be improved by signal processing.

  FIG. 11 shows the configuration of an optical system according to another embodiment of the digital holographic microscope. Similar to the digital holographic microscope shown in FIG. 1, two optical systems of a transmission type digital holographic microscope and a reflection type fluorescent microscope are used as a common optical path, and object light and fluorescent signal light are coaxially superimposed to form one imaging means. Thus, two holograms of a phase three-dimensional image and a fluorescence three-dimensional image can be acquired simultaneously.

The difference from the digital holographic microscope shown in FIG. 1 is that two excitation laser light sources (41, 42) are provided. Specifically, a blue laser having a wavelength of 473 nm and a green laser having a wavelength of 532 nm are provided. The blue laser is mainly intended to excite fluorescence of 500 to 530 nm. On the other hand, the green laser mainly aims to excite fluorescence of 540 to 600 nm. It is assumed that the fluorescent marker varies depending on the cell to be observed. Therefore, the observation target sample can be irradiated with a laser having a wavelength excited by the fluorescent marker.
For example, in the case of the configuration shown in FIG. 11, the excitation laser light source (41, 42) that excites fluorescent molecules of the observation target sample on the sample stage 4 is switched by the two shutters (43, 44) and the beam splitter 46. . The shutter (43, 44) is switched by a shutter control signal (51, 52) output from the shutter control device 50. An ND (Neutral Density) filter 47 ahead of the beam splitter 46 is for reducing the amount of laser light without affecting the color.

One of the two excitation laser light sources (41, 42) is used to excite fluorescent molecules of the observation target sample on the sample stage 4. The excited fluorescent molecules emit a fluorescent signal having a long wavelength from the excitation laser light source, and enter the objective lens 15 together with the excitation laser light reflected by the glass substrate surface of the sample stage 4. Further, the shutter control device 50 switches the two shutters (43, 44), and excites the fluorescent molecules of the observation target sample on the sample stage 4 using the other excitation laser light source.
In each of the three-dimensional fluorescent images of the two excitation laser light sources (41, 42), the polarization component of the fluorescence self-interfers with the bifocal Fresnel lens (SPFL) 2 which is a bifocal lens, so the interference pattern is concentric. It becomes.

  Simultaneously with the acquisition of the three-dimensional fluorescence image, the measurement object of the observation target sample on the sample stage 4 is illuminated using the He—Ne laser light source 5 with a wavelength of 633 (nm) to create an off-axis hologram. The image sensor 3 simultaneously acquires two holograms of a phase three-dimensional image and a fluorescence three-dimensional image.

As a sample to be observed, a sample having a diameter of 2.5 to 4.5 μm and mixed with yellow and Nile red two-color fluorescent beads was used. FIG. 12 shows the result of obtaining the phase image and the fluorescence image of the two-color fluorescent beads using the digital holographic microscope having the configuration of the optical system in FIG. However, in this embodiment, the fluorescence is a two-dimensional image. FIG. 12 (1) shows a phase image of two-color fluorescent beads. FIGS. 12 (2) and 12 (3) show a fluorescent image of yellow fluorescent beads and a fluorescent image of Nile red fluorescent beads, respectively. The fluorescent image of the yellow fluorescent beads in FIG. 12 (2) is a fluorescent image when the fluorescent molecules of the sample to be observed on the sample stage 4 are excited using a blue laser light source. The fluorescent image of the Nile red fluorescent beads in FIG. 12 (3) is a fluorescent image when the fluorescent molecules of the observation target sample on the sample stage 4 are excited using a green laser light source. Six fluorescent beads can be confirmed from the phase image of FIG. Further, four fluorescent beads can be confirmed in the fluorescent image of FIG. 12 (2), and two fluorescent beads can be confirmed in the fluorescent image of FIG. 12 (3). From these facts, it was found that μ-order size fluorescent particles can be identified and confirmed for each color using the constructed experimental system.
FIG. 12 (4) shows one phase profile of these fluorescent beads. From this figure, the size of the fluorescent beads (about 5 μm) and the phase difference (about 2.6 rad) can be known.

In the digital holographic microscope of the present invention, it is possible to measure changes in cell shape and movement of cell nuclei in real time, and by switching the excitation laser light source according to the type of fluorescent staining reagent, the target cell You can select to measure the shape change and movement.
In this embodiment, two excitation laser light sources are used, but three or more laser light sources may be used. Further, the fluorescent image may be acquired by switching the shutter control signal output from the shutter control device at high speed.

  The present invention is useful for a microscope in the field of bioimaging.

1,30 Digital holographic microscope 2 Bifocal lens (SPFL)
3 Image sensor 4 Sample stage 5 He-Ne laser light source 6, 41, 42 Excitation laser light source 10, 11, 19, 32, 34, 35, 36 Lens 12, 18, 46 Beam splitter 13, 14, 31, 45 Reflection Mirror 15 Objective lens 16, 33 Dichroic mirror 17 Tube lens 20, 21 1/2 wavelength plate 43, 44 Shutter 47 ND filter 50 Shutter control device 51, 52 Shutter control signal

Claims (11)

A first holographic optical system that obtains a phase three-dimensional image by interference light obtained by superimposing the object light passing through the observation target sample and the reference light not passing using the first laser light;
A second holographic optical system that acquires a three-dimensional fluorescence image of the observation target sample by fluorescence excitation light using a second laser beam;
Imaging means;
A bifocal lens with two focal lengths having polarization dependence;
With
Each optical system is disposed so that the object light and the fluorescent signal light are coaxially superimposed, and the double focus lens is disposed on the superimposed optical axis,
The imaging means simultaneously acquires the phase three-dimensional image and the fluorescence three-dimensional image as holograms, separates the phase three-dimensional image and the fluorescence three-dimensional image, and extracts the object light and the fluorescence signal from respective interference intensity distributions. A digital holographic microscope characterized by reconstructing light.   The digital holographic microscope according to claim 1, wherein the bifocal lens is a bifocal Fresnel lens having a sub-wavelength periodic structure.   The digital holographic microscope according to claim 1, wherein the double focus lens includes a liquid crystal spatial light modulator.   The digital lens according to any one of claims 1 to 3, wherein the double focus lens acts only on the second holographic optical system and does not act on the first holographic optical system. Holographic microscope.   The digital holographic microscope according to claim 1, wherein the second holographic optical system self-interfers with the double focus lens.   The fluorescent three-dimensional image is a concentric hologram, the phase three-dimensional image is an isometric hologram, and the fluorescent three-dimensional image and the phase three-dimensional image are separated on a spatial frequency plane. The digital holographic microscope according to any one of 1 to 5.   The digital holographic microscope according to any one of claims 1 to 6, wherein the second holographic optical system can switch an excitation laser light source.   The digital holographic microscope according to claim 1, wherein the first holographic optical system is a transmissive type and the second holographic optical system is a reflective type.   9. The digital holographic microscope according to claim 1, wherein spatiotemporal information of a cell nucleus is obtained from the fluorescence three-dimensional image, and a spatiotemporal structure of a cell nucleus and a cell wall is obtained simultaneously from the phase three-dimensional image. . Using the digital holographic microscope according to any one of claims 1 to 8,
A cell observation method for simultaneously acquiring spatiotemporal information of a cell nucleus by a fluorescence three-dimensional image and a spatiotemporal structure of a cell nucleus and a cell wall by a phase three-dimensional image. Using the digital holographic microscope according to claim 7,
The behavior of the cell is observed from a three-dimensional fluorescence image acquired using one excitation laser light source, a three-dimensional fluorescence image acquired using another excitation laser light source, and a phase three-dimensional image acquired simultaneously. Cell observation method.