JP2022127657A - Digital Holographic Microscope Realizing Highly Efficient Measurement of Fluorescent Images - Google Patents

Abstract

A digital holographic microscope capable of obtaining a three-dimensional fluorescence distribution in a highly efficient and simple system by fluorescence holography is provided. Kind Code: A1 In a digital holographic microscope equipped with a fluorescence holographic optical system for acquiring a fluorescence image of a sample to be observed by fluorescence signal light, the fluorescence holographic optical system converts two polarized light beams whose polarization directions are orthogonal to each other. and an optical system for generating an off-axis hologram and acquiring a fluorescence image when recombining the light obtained by separating the fluorescence signal light into two. The separating means uses a polarizing prism using birefringence to separate the fluorescence signal light into mutually orthogonal linearly polarized light, and a Wollaston prism or the like is used. [Selection drawing] Fig. 1

Description

The present invention relates to a digital holographic microscope that efficiently acquires a three-dimensional fluorescence distribution by fluorescence holography.

Dynamic three-dimensional imaging of living cells is an important measurement technique in the bioapplication field. For example, a fluorescent labeling technique is known in which fluorescent molecules are introduced into specific DNA in cell nuclei using a fluorescence microscope to measure changes in cells undergoing mitosis. Various observations can be made by visualizing intracellular nuclei and protein interactions stained with fluorescent molecules. A confocal laser scanning microscope is known as a microscope capable of measuring fluorescence images with high resolution and high contrast. However, with a confocal laser scanning microscope, it is necessary to scan the focal point for each point of the object to be measured, so it takes time to obtain a fluorescent image. There is an essential limitation that simultaneous measurement of a plurality of different cells is unsuitable.
There is also a demand for techniques for simultaneously observing structures such as cell nuclei and cell walls in addition to fluorescence images. Means for structural observation include a phase-contrast microscope, a differential interference microscope, and the like. Phase-contrast images are effective for shape measurement of transparent objects. If the fluorescence image and the phase image can be observed simultaneously, different information can be obtained at the same time, so that it becomes possible to provide more detailed information on the biological reaction.

A digital holographic microscope is known as a tool that enables instantaneous three-dimensional measurement. In a digital holographic microscope, three-dimensional information is acquired as hologram information, and light wave information of an object with respect to depth positions is reconstructed by performing back propagation calculation of light waves in a computer. Features of digital holographic microscopes include the ability to observe living cells in three dimensions without the need for special fluorescent staining, an auto-focusing function that can arbitrarily change the focus position through computer reconfiguration, and quantitative phase measurement. is possible. Therefore, even in a situation where cells move three-dimensionally, a reconstructed image that is automatically in focus can be obtained by reconstruction calculation.

When a digital holographic microscope is applied to cell observation, it is necessary to obtain a hologram of fluorescence information. , methods of making holograms from incoherent light have been proposed one after another, and research toward fluorescence digital holographic microscopes is active.
Also, although the fluorescence digital holographic microscope is suitable for targeting measurement, it has the problem that it is powerless for substances that do not generate fluorescence. In addition, when performing fluorescence observation, there is a problem that measurement objects are restricted due to the use of harmful fluorescent molecules. Therefore, from the viewpoint of expanding the application of digital holographic microscopes, multimodal digital holographic microscopes that can observe various information such as phase images, fluorescence images, and polarization are required.

One of the inventors of the present invention has already proposed a digital holographic microscope that combines a fluorescence digital holographic microscope and a phase image detection digital holographic microscope and can simultaneously measure a fluorescence image and a phase image (Patent Documents 1 and 2). ).
However, since the digital holographic microscope utilizes the interference of two light waves, it is necessary to expand the light wave spatially, divide it into two light waves, and superimpose them again. You need to increase your energy. As a result, the light energy irradiated to the observed object increases, and when the observed object is a living cell, phototoxicity that damages the cell becomes a serious problem. When applying a digital holographic microscope to the field of bioimaging, it is necessary to further improve the light energy utilization efficiency (higher efficiency) in order to avoid this phototoxicity.

Re-table 2018/070451 Re-table 2016/163560

SUMMARY OF THE INVENTION In view of the above situation, an object of the present invention is to provide a digital holographic microscope capable of acquiring a three-dimensional fluorescence distribution by fluorescence holography with a highly efficient and simple system. In addition, the present invention provides a digital holographic microscope in which a fluorescence digital holographic microscope and a phase image detection digital holographic microscope are combined, a fluorescence image and a phase image can be measured simultaneously, and a fluorescence image can be measured with high efficiency. With the goal.

In order to achieve the above object, the digital holographic microscope of the present invention is a digital holographic microscope equipped with a fluorescence holographic optical system for acquiring a fluorescent image of a sample to be observed by fluorescence signal light, wherein the fluorescence holographic optical system is composed of polarized light. Separation means for separating the fluorescence signal light into two polarized light beams whose directions are orthogonal to each other, and optics for obtaining a fluorescence image by generating an off-axis hologram when recombining the light beams obtained by splitting the fluorescence signal light into two. Have a system.
According to the above configuration, a three-dimensional fluorescence distribution can be obtained by fluorescence holography in a highly efficient and simple system.
Here, the separating means uses a birefringent polarizing element capable of separating into two polarized light beams whose polarization directions are orthogonal to each other. Specifically, a polarizing prism utilizing birefringence is used to separate the fluorescence signal light into mutually orthogonal linearly polarized light. Polarizing prisms include Wollaston prisms, Rochon prisms, Nicol prisms, Glan-Thompson prisms, etc., and Wollaston prisms are particularly preferred.

The digital holographic microscope of the present invention may further include a phase image detection holographic optical system for acquiring a phase image by interference light in which the object light passing through the observation target sample and the reference light not passing through are superimposed. As a result, the fluorescence image and the phase image can be measured simultaneously, and the fluorescence image can be measured with high efficiency.
In the digital holographic microscope of the invention, the separating means are designed to act only on the fluorescence holographic optics.
A phase image detection holographic optical system that acquires a phase image has no sensitivity to the polarization of object light, and the separating means is designed so as not to cause lens action, while a fluorescence holographic optical system that acquires a fluorescent image. It is designed to have strong sensitivity to the polarization of the fluorescence of . Thereby, only the polarization of the fluorescent light of the fluorescent holographic optics is separated by the separation means, resulting in an off-axis hologram when recombining the light.

In the digital holographic microscope of the present invention, both the fluorescence holographic optical system and the phase image detection holographic optical system are preferably reflective.
In a fluorescence holographic optical system that acquires a fluorescence image of a sample to be observed by fluorescence excitation light, since the fluorescence signal is weak, it is very difficult to cut off only the strong fluorescence excitation light when using a transmissive system. Therefore, a reflective type is preferable. By making both the fluorescence holographic optical system and the phase image detection holographic optical system of a reflection type and using a common optical system, the apparatus can be made compact. If the fluorescence holographic optical system is to be a transmissive type instead of a reflective type, a dichroic mirror or a specific wavelength and phase of the fluorescence wavelength can be used to sufficiently cut off the fluorescence excitation light that excites the fluorescence signal and pass only the fluorescence signal. A bandpass filter must be used to pass the laser wavelength for measurement.

In the digital holographic microscope of the present invention, the imaging means of the fluorescence holographic optical system and the imaging means of the phase image detection holographic optical system are shared, and the imaging means simultaneously acquires the fluorescence image and the phase image as holograms. Then, the fluorescence image and the phase image are separated in the spatial frequency plane, and the object light and the fluorescence signal light are reconstructed from the respective interference intensity distributions.
A fluorescence image is acquired as an off-axis hologram when the two polarizations of the fluorescence signal light of the fluorescence holographic optics are combined. A phase image can also be acquired as an off-axis hologram. Both of them become the equi-angle interference pattern of the off-axis hologram, but it is possible to separate them in the spatial frequency plane. can be separated. By Fourier transforming the two hologram patterns, it is possible to extract equi-angle interference pattern components in the spatial frequency plane by means of a band-pass filter.

The digital holographic microscope of the present invention enables simultaneous high-speed imaging of fluorescence images and phase images 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 on cell nuclei from fluorescence images and spatiotemporal structures of cell nuclei and cell walls from phase images.

According to the digital holographic microscope of the present invention, it is possible to obtain three-dimensional fluorescence distribution by fluorescence holography with a highly efficient and simple system, and it is possible to solve the problem of phototoxicity in the field of bioimaging. Further, according to the digital holographic microscope of the present invention, a fluorescence digital holographic microscope and a phase image detection digital holographic microscope are combined, enabling simultaneous measurement of a fluorescence image and a phase image and highly efficient measurement of a fluorescence image. There is such an effect.
As in the digital holographic microscope already proposed by the present inventor, the method using a diffraction grating utilizes diffraction, so it is difficult to achieve sufficiently high diffraction efficiency for an image with a complex fluorescence distribution. Although difficult, the digital holographic microscope of the present invention does not use a diffractive optical element, so the light energy utilization efficiency can be increased.

1 is a configuration diagram of an optical system of a digital holographic microscope of Example 1. FIG. Illustration of Wollaston Prism Illustration of an off-axis hologram produced by a Wollaston prism Fluorescent hologram and its reconstructed image FIG. 2 is a configuration diagram of the optical system of the digital holographic microscope of Example 2; FIG. 3 is a configuration diagram of the optical system of the digital holographic microscope of Example 3; Explanatory diagram 1 of the principle of separating and reconstructing a fluorescent hologram and a phase hologram Explanatory diagram 2 of the principle of separating and reconstructing a fluorescent hologram and a phase hologram Intensity distribution graph for three reconstructed images at reconstruction propagation distance Diagram showing the results of fluorescence measurement and phase measurement of Physcomitrella patens Illustration of the optical system of a differential interference contrast microscope using a Wollaston prism

An example of an embodiment of the present invention will be described in detail below with reference to the drawings. The scope of the present invention is not limited to the following examples and illustrated examples, and many modifications and variations are possible.

FIG. 1 shows the configuration of the optical system of one embodiment of the digital holographic microscope of the present invention. The digital holographic microscope 101 shown in FIG. 1 is a digital holographic microscope equipped with a fluorescence holographic optical system that obtains a fluorescent image of a sample to be observed by fluorescent signal light. It is equipped with separating means for separating the fluorescence signal light into two polarized light beams, and an optical system for generating an off-axis hologram when recombining the light beams obtained by separating the fluorescence signal light into two beams to obtain a fluorescence image. be.

The optical system of the digital holographic microscope 101 will be described with reference to FIGS. 1-3. In the digital holographic microscope 101 , the laser light source 12 is used to irradiate the measurement object of the observation target sample 1 on the sample stage 11 with fluorescence excitation light, thereby exciting the fluorescent molecules of the observation target sample 1 . The digital holographic microscope 101 is a reflective microscope that receives illumination light from the same side as the objective lens 14 and observes an image of the sample by light reflected from the sample 1 to be observed.
The excited fluorescent molecules emit long-wavelength fluorescent signal light when irradiated with fluorescence excitation light, and enter the objective lens 14 . The dichroic mirror 13 is an optical element that reflects light of a specific wavelength and transmits light of other wavelengths, reflects fluorescence excitation light, and transmits fluorescence signal light.

After passing through the dichroic mirror 13 , the fluorescence signal light passes through the lens 17 a and then through the polarizer 15 . As shown in FIG. 3, the randomly polarized fluorescence signal light 31 is converted by the polarizer 15 into linearly polarized light 32 whose vibration direction (vibration plane) vibrates only in one specific direction. After passing through the polarizer 15, the fluorescence signal light passes through the Wollaston prism 16, is separated into two linearly polarized light beams (33, 34) whose vibration directions are orthogonal to each other, and travels at different angles.
Here, the Wollaston prism is a polarizing prism in which two calcite crystals (3a, 3b) are cemented so that their optical axes are perpendicular to each other, as shown in FIG. The light incident perpendicularly to the Wollaston prism 3 becomes two linearly polarized light beams perpendicular to each other by the two cemented prisms, and the light amounts of the light beams are substantially equal.

The fluorescence signal light that has passed through the Wollaston prism 16 passes through the lens 17b and is condensed, then passes through the polarizer 18 to align the polarization, and the two separated orthogonal linearly polarized lights are combined. As shown in FIG. 3, the two orthogonal linearly polarized light beams that are separated are at a slight angle when combined, resulting in off-axis interference and forming an equi-oblique patterned off-axis hologram 40. occur. Also, the imaging system spatially and temporally overlaps the two light waves.
Since the second-order phase term, which is depth information, remains due to the interference, the depth direction can be recorded. In addition, in a polarizing prism that utilizes birefringence, such as a Wollaston prism, the occurrence of a phase difference between the ordinary ray and the extraordinary ray also contributes to the residual second-order phase term, which is the depth information of the object. By forming an imaging relationship between a birefringence-based polarizing prism and an image sensor, the two orthogonal fluorescent linearly polarized light beams completely overlap to form an equi-oblique patterned off-axis hologram. Therefore, the hologram is highly efficient and spatio-temporally stable.

The fluorescence signal light multiplexed by the polarizer 18 then passes through a bandpass filter 19 and is imaged by an EM (Electron Multiplying)-CCD (Charge Coupled Device) camera 20 . The band-pass filter 19 passes wavelengths in the fluorescence wavelength band of the fluorescence signal light.
A bandpass filter improves the temporal coherence and improves the fringe contrast. As a result, noise is reduced in the reconstructed image, and a reconstructed image with high contrast and high resolution is obtained.

An EM-CCD camera 20, which is an imaging means, acquires a fluorescence image as an equi-angle pattern off-axis hologram, and a computer (not shown) converts the equi-angle interference pattern hologram into fluorescence signal light using the Fourier transform method. By extracting the amplitude distribution and phase distribution of , and back-propagating it to the position of the fluorescent molecule of the observation target sample by the off-axis method, the wavefront of the fluorescence signal light of the observation target sample is reproduced. Reconfigure. The EM-CCD camera 20 is equipped with a CCD image sensor having an electronic multiplication function, multiplies optical signals without increasing noise, and enables high-speed imaging even with weak light.

FIG. 4 shows a fluorescence hologram obtained by fluorescence measurement using the digital holographic microscope of Example 1 and its reconstructed image. In the fluorescence hologram shown in FIG. 4(1), a clean linear interference fringe was obtained, and the separability was improved.

FIG. 5 shows the configuration of the optical system of another embodiment of the digital holographic microscope of the present invention. The digital holographic microscope 102 shown in FIG. 5 is, like the digital holographic microscope 101 of the first embodiment, a digital holographic microscope equipped with a fluorescence holographic optical system for obtaining a fluorescence image of a sample to be observed using fluorescence signal light. The holographic optical system includes separating means for separating the fluorescence signal light into two polarized light beams whose polarization directions are orthogonal to each other, and generating an off-axis hologram when combining the two light beams obtained by separating the fluorescence signal light beams again. and an optical system for acquiring a fluorescence image. While the digital holographic microscope 101 of Example 1 is of a reflection type (incident-light type), the digital holographic microscope 102 of this example is of a transmission type.
When using a transmissive type, it is necessary to sufficiently cut off the fluorescence excitation light that excites the fluorescence signal light, so it is important to use a dichroic mirror that passes only the fluorescence signal light and a bandpass filter that passes a specific wavelength of the fluorescence wavelength light. become.

FIG. 6 shows the configuration of the optical system of another embodiment of the digital holographic microscope of the present invention. The digital holographic microscope 103 shown in FIG. 6 is the digital holographic microscope 101 of Embodiment 1 further provided with a reflection type phase image detection digital holographic optical system, and is a fluorescent holographic optical system and a phase image detection holographic optical system. In the two optical systems, as a common optical path, the object light and the fluorescence signal light are transmitted from the sample stage through the objective lens, and further coaxially superimposed to the imaging means of the image sensor. It is possible to acquire two holograms of the image simultaneously.
Each of the fluorescence holographic optical system and the phase image detection holographic optical system will be described below.

First, the fluorescence holographic optical system has the same configuration as that of the first embodiment. 1 fluorescent molecules are excited.
The excited fluorescent molecules emit long-wavelength fluorescent signal light when irradiated with fluorescence excitation light, and enter the objective lens 14 . The dichroic mirror 13 is an optical element that reflects light of a specific wavelength and transmits light of other wavelengths, reflects fluorescence excitation light, and transmits fluorescence signal light. The fluorescence excitation light emitted from the laser light source 12 is reflected by the dichroic mirror 13 and irradiated onto the observation target sample 1 on the sample stage 11 . Fluorescent molecules in the sample 1 to be observed emit fluorescence signal light with a long wavelength when irradiated with fluorescence excitation light. The fluorescence signal light travels to the dichroic mirror 13 together with the fluorescence excitation light reflected by the mirror 10 on the bottom surface of the glass substrate of the sample stage 11 . Since the dichroic mirror 13 reflects the fluorescence excitation light and transmits the fluorescence signal light, the fluorescence excitation light can be sufficiently attenuated and the fluorescence signal light can be emphasized.

The fluorescence signal light that has passed through the dichroic mirror 13 passes through the lens 17a, and then becomes linearly polarized light that oscillates in only one specific direction by the polarizer 15. FIG. After passing through the polarizer 15, the fluorescence signal light passes through the Wollaston prism 16 and is separated into two linearly polarized lights whose vibration directions are orthogonal to each other.
After passing through the Wollaston prism 16, the fluorescence signal light passes through the lens 17b and is condensed. When combined, off-axis holograms with equi-angled patterns are produced due to off-axis interference.
The fluorescent signal light combined by the polarizer 18 then passes through the bandpass filter 19 and is imaged by the EM-CCD camera 20 . The EM-CCD camera 20, which is an imaging means, acquires a fluorescence image as an equi-tilt pattern off-axis hologram. Extract the distribution and reconstruct the fluorescence signal light.

On the other hand, in the phase image detection holographic optical system, similarly to the fluorescence holographic optical system, the laser light source 12 is used to illuminate the measurement object of the observation target sample 1 on the sample stage 11 . However, the laser light is split by the beam splitter 21 into an object light path that passes through the measurement object and an empty reference light path. The object light transmitted through the object to be measured is reflected by the mirror 10 under the sample 1 to be observed, enters the objective lens 14 , and then travels to the dichroic mirror 13 . Utilizing the fact that the wavelength of the laser light that passes through the object to be measured is longer than the wavelength of the fluorescence excitation light and the fluorescence signal light that has a longer wavelength than that, the dichroic mirror 13 is used to reflect only the object light that passes through the object to be measured. , other light is transmitted. The object light passing through the measurement object interferes with the reference light by the beam splitter 24 . A slight angle between the object beam and the reference beam results in an off-axis hologram, ie, an equi-angled interference pattern hologram. This is captured by the EM-CCD camera 20 . The amplitude distribution and phase distribution of the object light are extracted from the hologram of the equi-angle interference pattern acquired by the EM-CCD camera 20 using the Fourier transform method. In the off-axis method, by propagating back to the original object position, the wavefront of the object light of the sample to be observed is reproduced, and the object light can be reconstructed.

Here, the principle of separating and reproducing the fluorescence hologram and the phase hologram will be described with reference to FIGS. In the digital holographic microscope of this embodiment, the fluorescence hologram and the phase hologram are acquired by the common EM-CCD camera 20. FIG. Both fluorescence holograms and phase holograms are equi-angled interference pattern holograms. However, by Fourier transforming the two patterns of the phase hologram and the fluorescence hologram, it is possible to extract the component of the equi-angle interference pattern of the signal existing at the position shifted from the origin in the spatial frequency plane by the window function filter. Specifically, a window function is used to separate two holograms, a fluorescence hologram appearing at different locations and a phase hologram existing at a position shifted from the origin, in the spatial frequency plane.

Using the digital holographic microscope of this embodiment, a sample in which fluorescent beads with a diameter of 10 μm are arranged on a slide glass is used, and by moving this in the height direction, recording and playback in the depth direction are possible. We proved that. Fig. 9 shows the reconstruction propagation distance when a focused reconstructed image is obtained when the height position of the sample is changed from -30 μm to +10 μm from the in-focus position (z = 0). is shown.
From FIG. 9, it can be seen that the digital holographic microscope of this embodiment can record and reproduce in the depth direction from −30 μm to +10 μm. It can be understood that the depth position can be obtained and three-dimensional measurement is possible.

Next, using Physcomitrella patens into which fluorescent proteins have been introduced, it was demonstrated that fluorescence three-dimensional imaging of living plant cells is possible. FIG. 10 shows the results of fluorescence measurement and phase measurement of Physcomitrella patens into which a fluorescent protein has been introduced using the digital holographic microscope of this example. FIG. 10(1) shows a reconstructed image of a fluorescence hologram from z=−10 μm to z=20 μm. Imaging of the cell nucleus that was in focus at the arrow is reproduced. FIG. 10(2) shows the phase distribution measured simultaneously with the measurement of the fluorescence hologram. When z=−5 μm and z=20 μm are compared, the edges are sharp at z=−5 μm, and the peripheral portion is in focus. At z=20 μm, the structure of the chloroplast near the center could be clearly observed. Since different structures such as cell nuclei, cell walls, and chloroplasts can be observed with fluorescence and phase, respectively, simultaneous measurement of fluorescence and phase images shows that more information can be obtained on the sample to be observed.

FIG. 11 is a schematic diagram of optical paths for explaining the principle of differential interference observation. In differential interference contrast observation, when light passes through an observation sample, the difference in the refractive index and thickness of the part that passes through the sample causes a difference in the distance traveled by the transmitted light (optical path difference). It is used to observe a transparent sample. Differential interference contrast microscopy can create contrast between light and dark by capturing minute differences in refractive index within cells. In the schematic diagram shown in FIG. 11, two Wollaston prisms (52, 57) are used to configure an optical system 50 for differential interference observation. First, randomly polarized light passes through the polarizer 51 and becomes linearly polarized light. At the Wollaston prism 52, it is split into two mutually orthogonal linearly polarized light beams traveling at different angles. It becomes parallel to the optical axis by the lens 53 and passes through the glass substrate 54 and the sample 55 to be observed. At this time, due to the action of the Wollaston prism 52, the two linearly polarized light beams pass through spatially separated positions. The two polarized lights become parallel to the optical axis by the lens 56 and the Wollaston prism 57, and pass through the polarizer 58 to be converted into interference intensity having a phase difference at a spatially separated position.

On the other hand, in the digital holographic microscope of the present invention, the Wollaston prism separates light into two mutually orthogonal polarized light beams having different angles, and the imaging lens spatially and temporally overlaps the two light waves. In addition, the light is passed through a polarizer to align the polarized light to form an equi-angle interference fringe. Compared to the differential interference microscope, the purpose (phase difference and polarization recording) and means are different.

INDUSTRIAL APPLICABILITY The present invention is useful for microscopes in the bioimaging field.

101, 102, 103 digital holographic microscope 1, 55 sample to be observed 11 sample stage 12 laser light source 13 dichroic mirror 14 objective lens 15, 18 polarizer 3, 16 Wollaston prism 19 bandpass filter 20 EM-CCD camera 17a to 17c Lens 10, 20, 23 Mirror 21, 24 Beam splitter 40 Off-axis hologram

Claims (9)

In a digital holographic microscope equipped with a fluorescence holographic optical system for acquiring a fluorescence image of a sample to be observed by fluorescence signal light,
The fluorescence holographic optical system includes a separation means for separating the fluorescence signal light into two polarized light beams whose polarization directions are orthogonal to each other, and an off-axis optical system for recombining the light beams obtained by separating the fluorescence signal light beams into two. A digital holographic microscope, comprising an optical system for producing a hologram and acquiring the fluorescent image. 2. The digital holographic microscope according to claim 1, wherein the optical system for obtaining the fluorescent image constitutes an imaging system. 3. The digital holographic microscope according to claim 1, wherein said separating means uses a polarizing prism utilizing birefringence to separate said fluorescence signal light into mutually orthogonal linearly polarized light. 4. The digital holographic microscope according to claim 3, wherein said polarizing prism and an image sensor for acquiring said fluorescent image are in an imaging relationship. 5. The digital holographic microscope according to claim 3, wherein said polarizing prism is a Wollaston prism, a Rochon prism, a Nicol prism, or a Glan-Thompson prism. 6. The system according to claim 1, further comprising a phase image detection holographic optical system for acquiring a phase image by interference light obtained by superimposing object light passing through said observation target sample and reference light not passing therethrough. A digital holographic microscope as described in . 7. A digital holographic microscope according to claim 6, wherein said separating means act only on said fluorescence holographic optical system. The imaging means of the fluorescence holographic optical system and the imaging means of the phase image detection holographic optical system are shared,
The imaging means simultaneously obtains a fluorescent image and a phase image as holograms, separates the fluorescent image of the off-axis hologram and the phase image of the off-axis hologram in the spatial frequency plane, and obtains the object light from each interference intensity distribution. 7. A digital holographic microscope according to claim 6, wherein the fluorescence signal light is reconstructed. 7. The digital holographic microscope according to claim 6, wherein both said fluorescence holographic optical system and said phase image detection holographic optical system are of a reflective type.

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